Introduction into biochemistry

Structure of proteins, methods of its determination. Physical-chemical properties of simple and complex proteins, precipitation reactions. Studing of the structure and physical-chemical properties of enzymes.Vitamins as nutrition components. Water and fat soluble vitamins.


Biochemistry can be defined as the science concerned with the chemical basis of life (Gk bios “life”). The cell is the structural unit of living systems. Thus, biochemistry can also be described as the science concerned with the chemical constituents of living cells and with the reactions and processes they undergo. By this definition, biochemistry encompasses large areas of cell biology, of molecular biology, and of molecular genetics.

The Aim of Biochemistry Is to Describe & Explain, in Molecular Terms, All Chemical Processes of Living Cells

The major objective of biochemistry is the complete understanding, at the molecular level, of all of the chemical processes associated with living cells. To achieve this objective, biochemists have sought to isolate the numerous molecules found in cells, determine their structures, and analyze how they function.

A Knowledge of Biochemistry Is Essential to All Life Sciences

The biochemistry of the nucleic acids lies at the heart of genetics; in turn, the use of genetic approaches has beencritical for elucidating many areas of biochemistry.

Physiology, the study of body function, overlaps with biochemistry almost completely. Immunology employs numerous biochemical techniques, and many immunologic approaches have found wide use by biochemists. Pharmacology and pharmacy rest on a sound knowledge of biochemistry and physiology; in particular, most drugs are metabolized by enzyme-catalyzed reactions.


Poisons act on biochemical reactions or processes; this is the subject matter of toxicology. Biochemical approaches are being used increasingly to study basic aspects of pathology (the study of disease), such as inflammation, cell injury, and cancer. Many workers in microbiology, zoology, and botany employ biochemical approaches almost exclusively. These relationships are not surprising, because life as we know it depends on biochemical reactions and processes. In fact, the old barriers among the life sciences are breaking down, and biochemistry is increasingly becoming their common language.


The World Health Organization (WHO) defines health as a state of “complete physical, mental and social well-being and not merely the absence of disease and infirmity.” From a strictly biochemical viewpoint, health may be considered that situation in which all of the many thousands of intra- and extracellular reactions that occur in the body are proceeding at rates commensurate with the organism’s maximal survival in the physiologic state. However, this is an extremely reductionist view, and it should be apparent that caring for the health of patients requires not only a wide knowledge of biologic principles but also of psychologic and social principles.


Protein is an important nutrient that builds muscles and bones and provides energy. Protein can help with weight control because it helps you feel full and satisfied from your meals.

The healthiest proteins are the leanest. This means that they have the least fat and calories. The best protein choices are fish or shellfish, skinless chicken or turkey, low-fat or fat-free dairy (skim milk, low-fat cheese), and egg whites or egg substitute. The best red meats are the leanest cuts (loin and tenderloin). Other healthy options are beans, legumes (lentils and peanut butter), and soy foods such as tofu or soymilk.

Protein is an important part of every diet and is found in many different foods. Lean protein, the best kind, can be found in fish, skinless chicken and turkey, pork tenderloin and certain cuts of beef, like the top round. Low-fat dairy products like milk, yogurt, ricotta and other cheeses supply both protein and calcium.

·                    Protein is crucial for tissue repair, building and preserving muscle, and making important enzymes and hormones.

·                    Lean meats and dairy contribute valuable minerals like calcium, iron, selenium and zinc. These are not only essential for building bones, and forming and maintaining nerve function, but also for fighting cancer, forming blood cells and keeping immune systems robust.

Structure and Function

The word protein was first coined in 1838 to emphasize the importance of this class of molecules. The word is derived from the Greek word proteios which means "of the first rank".

This chapter will provide a brief background into the structure of proteins and how this structure can determine the function and activity of proteins. It is not intended to substitute for the more detailed information provided in a biochemistry or cell biology course.

Proteins are the major components of living organisms and perform a wide range of essential functions in cells. While DNA is the information molecule, it is proteins that do the work of all cells - microbial, plant, animal. Proteins regulate metabolic activity, catalyze biochemical reactions and maintain structural integrity of cells and organisms. Proteins can be classified in a variety of ways, including their biological function (Table 2.1).

Table 2.1 Classification of Proteins According to biological function.



Enzymes- Catalyze biological reactions


Transport and Storage



And Myosin in muscles

Immune Protection


Regulatory Function within cells

Transeription Factors





How does one group of molecules perform such a diverse set of functions? The answer is found in the wide variety of possible structures for proteins.

In the English language, there are an enormous number of words with varied meaning that can be formed using only 26 letters as building blocks. A similar situation exists for proteins where an incredible variety of proteins can be formed using 20 different building blocks called amino acids. Each of these amino acid building blocks has a different chemical structure and different properties.

Each protein has a unique amino acid sequence that is genetically determined by the order of nucleotide bases in the DNA, the genetic code. Since each protein has different numbers and kinds of the twenty available amino acids, each protein has a unique chemical composition and structure. For example, two proteins may each have 37 amino acids but if the sequence of the amino acids is different, then the protein will be different. How many different proteins can be formed from the twenty different amino acids? Consider a protein containing 100 different amino acids linked into one chain. Since each of the 100 positions of this chain could be filled with any one of the 20 amino acids, there are 20100 possible combinations, more than enough to account for the 90-100 million different proteins that may be found in higher organisms.

A change in just one amino acid can change the structure and function of a protein. For example, sickle cell anemia is a disease that results from an altered structure of the protein hemoglobin, resulting from a change of the sixth amino acid from glutamic acid to valine. (This is the result of a single base pair change at the DNA level.) This single amino acid change is enough to change the conformation of hemoglobin so that this protein clumps at lower oxygen concentrations and causes the characteristic sickle shaped red blood cells of the disease.

The unique structure and chemical composition of each protein is important for its function; it is also important for separating proteins in a protein purification strategy. Each of these differences in properties can be used as a basis for the separation methods that are used to purify proteins. Because these differences in protein properties originate from differences in the chemical structure of the amino acids that make up the protein, we need to explore the structure of amino acids and their contribution to protein properties in more detail.

Chemical Composition of Proteins: (Protein Structure)

Amino acid structure:

Amino acids are composed of carbon, hydrogen, oxygen, and nitrogen. Two amino acids, cysteine and methionine, also contain sulfur. The generic form of an amino acid is shown in Figure 2.1. Atoms of these elements are arranged into 20 kinds of amino acids that are commonly found in proteins. All proteins in all species, from bacteria to humans, are constructed from the same set of twenty amino acids. All amino acids have an amino group (NH2) and a carboxyl group (COOH) bonded to the same carbon atom, known as the alpha carbon. Amino acids differ in the side chain or R group that is bonded to the alpha carbon. (Figure 2.2) Glycine, the simplest amino acid has a single hydrogen atom as its R group - Alanine has a methyl (-CH3) group.

picture of primary protein structure

Figure 2.1: A diagram showing a generic amino acid structure. All amino acids have an alpha carbon, an amino group, and a carboxyl group, but each of the 20 amino acids has a different side chain or R group. Amino acids are linked together to form a polypeptide chain, like beads on a string, the primary level of protein structure. Figure used with permission from National Human Genome Research Institute (NHGRI), artist Darryl Leja,


The chemical composition of the unique R groups is responsible for the important characteristics of amino acids such as chemical reactivity, ionic charge and relative hydrophobicity. In Figure 2.2, the amino acids are grouped according to their polarity and charge. They are divided into four categories, those with polar uncharged R groups, those with apolar (nonpolar) R groups, acidic (charged) and basic (charged) groups.

amino acid table

Figure 2.2. The twenty amino acids, grouped according to the character of their side chain or R group.


The polar amino acids are soluble in water because their R groups can form hydrogen bonds with water. For example, serine, threonine and tyrosine all have hydroxyl groups (OH). Amino acids that carry a net negative charge at neutral pH contain a second carboxyl group. These are the acidic amino acids, aspartic acid and glutamic acid, also called aspartate and glutamate, respectively. The basic amino acids have R groups with a net positive charge at pH 7.0. These include lysine, arginine and histidine. There are eight amino acids with nonpolar R groups. As a group, these amino acids are less soluble in water than the polar amino acids. If a protein has a greater percentage of nonpolar R groups, the protein will be more hydrophobic (water hating) in character.

peptide diagram

Figure 2.3. A diagram showing the formation of a peptide bond between 2 amino acids.

A protein is formed by amino acid subunits linked together in a chain. The bond between two amino acids is formed by the removal of a H20 molecule from two different amino acids, forming a dipeptide. (Figure 2.3) The bond between two amino acids is called a peptide bond and the chain of amino acids is called a peptide (20 amino acids or smaller) or a polypeptide.

Each protein consists of one or more unique polypeptide chains. Most proteins do not remain as linear sequences of amino acids; rather, the polypeptide chain undergoes a folding process. The process of protein folding is driven by thermodynamic considerations. This means that each protein folds into a configuration that is the most stable for its particular chemical structure and its particular environment. The final shape will vary but the majority of proteins assume a globular configuration. Many proteins such as myoglobin consist of a single polypeptide chain; others contain two or more chains. For example, hemoglobin is made up of two chains of one type (amino acid sequence) and two of another type.

Although the primary amino acid sequence determines how the protein folds, this process is not completely understood. Although certain amino acid sequences can be identified as more likely to form a particular conformation, it is still not possible to completely predict how a protein will fold based on its amino acid sequence alone, and this is an active area of biochemical research.

The final folded 3-D arrangement of the protein is referred to as its conformation. In order to maintain their function, proteins must maintain this conformation. To describe this complex conformation, scientists describe four levels of organization: primary, secondary, tertiary, and quaternary (Figure 2.4). The overall conformation of a protein is the combination of its primary, secondary, tertiary and quaternary elements.

Four levels of Organization of Protein Structure:

·                     Primary Structure refers to the linear sequence of amino acids that make up the polypeptide chain. This sequence is determined by the genetic code, the sequence of nucleotide bases in the DNA. The bond between two amino acids is a peptide bond. This bond is formed by the removal of a H20 molecule from two different amino acids, forming a dipeptide. The sequence of amino acids determines the positioning of the different R groups relative to each other. This positioning therefore determines the way that the protein folds and the final structure of the molecule.

·                     The secondary structure of protein molecules refers to the formation of a regular pattern of twists or kinks of the polypeptide chain. The regularity is due to hydrogen bonds forming between the atoms of the amino acid backbone of the polypeptide chain. The two most common types of secondary structure are called the alpha helix and ß pleated sheet. (Figure 2.4)

·                     Tertiary structure refers to the three dimensional globular structure formed by bending and twisting of the polypeptide chain. This process often means that the linear sequence of amino acids is folded into a compact globular structure. The folding of the polypeptide chain is stabilized by multiple weak, noncovalent interactions. These interactions include:

o        Hydrogen bonds that form when a Hydrogen atom is shared by two other atoms.

o        Electrostatic interactions that occur between charged amino acid side chains. Electrostatic interactions are attractions between positive and negative sites on macromolecules.

  • Hydrophobic interactions: During folding of the polypeptide chain, amino acids with a polar (water soluble) side chain are often found on the surface of the molecule while amino acids with non polar (water insoluble) side chain are buried in the interior. This means that the folded protein is soluble in water or aqueous solutions.

Covalent bonds may also contribute to tertiary structure. The amino acid, cysteine, has an SH group as part of its R group and therefore, the disulfide bond (S-S ) can form with an adjacent cysteine. For example, insulin has two polypeptide chains that are joined by two disulfide bonds.

·                     Quaternary structure refers to the fact that some proteins contain more than one polypeptide chain, adding an additional level of structural organization: the association of the polypeptide chains. Each polypeptide chain in the protein is called a subunit. The subunits can be the same polypeptide chain or different ones. For example, the enzyme ß-galactosidase is a tetramer, meaning that it is composed of four subunits, and, in this case, the subunits are identical - each polypeptide chain has the same sequence of amino acids. Hemoglobin, the oxygen carrying protein in the blood, is also a tetramer but it is composed of two polypeptide chains of one type (141 amino acids) and two of a different type (146 amino acids). In chemical shorthand, this is referred to as a2ß2 . For some proteins, quaternary structure is required for full activity (function) of the protein.


Levels of Protein Structure


The wide variety of 3-dimensional protein structures corresponds to the diversity of functions proteins fulfill.

Proteins fold in three dimensions. Protein structure is organized hierarchically from so-called primary structure to quaternary structure. Higher-level structures are motifs and domains.

Above all the wide variety of conformations is due to the huge amount of different sequences of amino acid residues. The primary structure is the sequence of residues in the polypedptide chain. The primary structure refers to amino acid linear sequence of the polypeptide chain. The primary structure is held together by covalent or peptide bonds, which are made during the process of protein biosynthesis or translation. The two ends of the polypeptide chainare referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Counting of residues always starts at the N-terminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond. The primary structure of a protein is determined by the gene corresponding to the protein. A specific sequence of nucleotides in DNA istranscribed into mRNA, which is read by the ribosome in a process called translation. The sequence of a protein is unique to that protein, and defines the structure and function of the protein. The sequence of a protein can be determined by methods such as Edman degradation or tandem mass spectrometry. Often however, it is read directly from the sequence of the gene using the genetic code. We know that there are over 10,000 proteins in our body which are composed of different arrangements of 20 types of amino acid residues (it is strictly recommended to use the word "amino acid residues" as when peptide bond is formed a water molecule is lost so, protein is made up of amino acid residues). Post-translational modifications such as disulfide formation, phosphorylations and glycosylations are usually also considered a part of the primary structure, and cannot be read from the gene.

Secondary structure is a local regulary occuring structure in proteins and is mainly formed through hydrogen bonds between backbone atoms. So-called random coils, loops or turns don't have a stable secondary structure. There are two types of stable secondary structures: Alpha helices and beta-sheets (see Figure 3 and Figure 4). Alpha-helices and beta-sheets are preferably located at the core of the protein, whereat loops prefer to reside in outer regions. 

Secondary structure refers to highly regular local sub-structures. Two main types of secondary structure, the alpha helix and the beta strand or beta sheets, were suggested in 1951 by Linus Pauling and coworkers. These secondary structures are defined by patterns of hydrogen bonds between the main-chain peptide groups. They have a regular geometry, being constrained to specific values of the dihedral angles ψ and φ on the Ramachandran plot. Both the alpha helix and the beta-sheet represent a way of saturating all the hydrogen bond donors and acceptors in the peptide backbone. Some parts of the protein are ordered but do not form any regular structures. They should not be confused with random coil, an unfolded polypeptide chain lacking any fixed three-dimensional structure. Several sequential secondary structures may form a "supersecondary unit".



Figure 3: An alpha helix:
The backbone is formed as a helix. 
An ideal alpha helix consists 
of 3.6 residues per complete turn. 
The side chains stick out.
There are hydrogen bonds 
between the carboxy group of amino acid n
and the amino group of another amino acid n+4 [1][2].
The mean phi angle is -62 degrees 
and the mean psi angle is -41 degrees.


Figure 4: An antiparallel beta sheet.
Beta sheets are created, 
when atoms of beta strands are hydrogen bound. 
Beta sheets may consist of parallel strands, 
antiparallel strands or out of a mixture 
of parallel and antiparallel strands


Tertiary structure describes the packing of alpha-helices, beta-sheets and random coils with respect to each other on the level of one whole polypeptide chain. Figure 5 shows the tertiary structure of Chain B of Protein Kinase C Interacting Protein. 

Tertiary structure refers to three-dimensional structure of a single protein molecule. The alpha-helices and beta-sheets are folded into a compact globule. The folding is driven by the non-specific hydrophobic interactions (the burial of hydrophobic residues from water), but the structure is stable only when the parts of a protein domain are locked into place by specific tertiary interactions, such as salt bridges, hydrogen bonds, and the tight packing of side chains and disulfide bonds. The disulfide bonds are extremely rare in cytosolic proteins, since the cytosol is generally a reducing environment.



Figure 5: Chain B of Protein Kinase C Interacting Protein. 
Helices are visualized as ribbons and 
extended strands of betasheets by broad arrows. 
(the figure was obtained by using rasmol 
and the PDB-file corresponding to PDB-ID 1AV5 
stored at PDB, the Brookhaven Protein Data Bank)

Quaternary structure only exists, if there is more than one polypeptide chain present in a complex protein. Then quaternary structure describes the spatial organization of the chains. Figure 6 shows both, Chain A and Chain B of Protein Kinase C Interacting Protein forming the quaternary structure.

Quaternary structure is the three-dimensional structure of a multi-subunit protein and how the subunits fit together. In this context, the quaternary structure is stabilized by the same non-covalent interactions and disulfide bonds as the tertiary structure. Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers. Specifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it contains four subunits. The subunits are frequently related to one another by symmetry operations, such as a 2-fold axis in a dimer. Multimers made up of identical subunits are referred to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits are referred to with a prefix of "hetero-" (e.g. a heterotetramer, such as the two alpha and two beta chains of hemoglobin).


Figure 6: Quaternary structure of 
Protein Kinase C Interacting Protein. 
(the figure was obtained by using rasmol 
and the PDB-file corresponding to PDB-ID 1AV5 
stored at PDB, the Brookhaven Protein Data Bank)


protein structure chart

Figure 2.4: The four levels of protein structure are illustrated in this diagram. Figure used with permission from National Human Genome Research Institute (NHGRI) , artist Darryl Leja:


The primary structure of proteins

Drawing the amino acids

In chemistry, if you were to draw the structure of a general 2-amino acid, you would probably draw it like this:,%20methods%20of%20its%20determination.files/image016.gif

However, for drawing the structures of proteins, we usually twist it so that the "R" group sticks out at the side. It is much easier to see what is happening if you do that.,%20methods%20of%20its%20determination.files/image018.gif

That means that the two simplest amino acids, glycine and alanine, would be shown as:,%20methods%20of%20its%20determination.files/image020.gif


Peptides and polypeptides

Glycine and alanine can combine together with the elimination of a molecule of water to produce a dipeptide. It is possible for this to happen in one of two different ways - so you might get two different dipeptides.



In each case, the linkage shown in blue in the structure of the dipeptide is known as a peptide link. In chemistry, this would also be known as an amide link, but since we are now in the realms of biochemistry and biology, we'll use their terms.

If you joined three amino acids together, you would get a tripeptide. If you joined lots and lots together (as in a protein chain), you get a polypeptide.

A protein chain will have somewhere in the range of 50 to 2000 amino acid residues. You have to use this term because strictly speaking a peptide chain isn't made up of amino acids. When the amino acids combine together, a water molecule is lost. The peptide chain is made up from what is left after the water is lost - in other words, is made up of amino acid residues.

By convention, when you are drawing peptide chains, the -NH2 group which hasn't been converted into a peptide link is written at the left-hand end. The unchanged -COOH group is written at the right-hand end.

The end of the peptide chain with the -NH2 group is known as the N-terminal, and the end with the -COOH group is the C-terminal.

A protein chain (with the N-terminal on the left) will therefore look like this:,%20methods%20of%20its%20determination.files/image025.gif


The "R" groups come from the 20 amino acids which occur in proteins. The peptide chain is known as the backbone, and the "R" groups are known as side chains.

 Note:  In the case where the "R" group comes from the amino acid proline, the pattern is broken. In this case, the hydrogen on the nitrogen nearest the "R" group is missing, and the "R" group loops around and is attached to that nitrogen as well as to the carbon atom in the chain.

I mention this for the sake of completeness - not because you would be expected to know about it in chemistry at this introductory level.


The primary structure of proteins

Now there's a problem! The term "primary structure" is used in two different ways.

At its simplest, the term is used to describe the order of the amino acids joined together to make the protein. In other words, if you replaced the "R" groups in the last diagram by real groups you would have the primary structure of a particular protein.

This primary structure is usually shown using abbreviations for the amino acid residues. These abbreviations commonly consist of three letters or one letter.

Using three letter abbreviations, a bit of a protein chain might be represented by, for example:,%20methods%20of%20its%20determination.files/image026.gif

If you look carefully, you will spot the abbreviations for glycine (Gly) and alanine (Ala) amongst the others.

If you followed the protein chain all the way to its left-hand end, you would find an amino acid residue with an unattached -NH2 group. The N-terminal is always written on the left of a diagram for a protein's primary structure - whether you draw it in full or use these abbreviations.

 The wider definition of primary structure includes all the features of a protein which are a result of covalent bonds. Obviously, all the peptide links are made of covalent bonds, so that isn't a problem.

But there is an additional feature in proteins which is also covalently bound. It involves the amino acid cysteine.,%20methods%20of%20its%20determination.files/image028.gif

If two cysteine side chains end up next to each other because of folding in the peptide chain, they can react to form a sulphur bridge. This is another covalent link and so some people count it as a part of the primary structure of the protein.,%20methods%20of%20its%20determination.files/image029.gif

Because of the way sulphur bridges affect the way the protein folds, other people count this as a part of the tertiary structure (see below). This is obviously a potential source of confusion!

 Important:  You need to know where your particular examiners are going to include sulphur bridges - as a part of the primary structure or as a part of the tertiary structure. You need to check your current syllabus and past papers. If you are studying a UK-based syllabus and haven't got these, follow this link to find out how to get hold of them.

The secondary structure of proteins

Within the long protein chains there are regions in which the chains are organised into regular structures known as alpha-helices (alpha-helixes) and beta-pleated sheets. These are the secondary structures in proteins.

These secondary structures are held together by hydrogen bonds. These form as shown in the diagram between one of the lone pairs on an oxygen atom and the hydrogen attached to a nitrogen atom:,%20methods%20of%20its%20determination.files/image030.gif


Important:  If you aren't happy about hydrogen bonding and are unsure about what this diagram means, follow this link before you go on. What follows is difficult enough to visualise anyway without having to worry about what hydrogen bonds are as well!

You must also find out exactly how much detail you need to know about this next bit. It may well be that all you need is to have heard of an alpha-helix and know that it is held together by hydrogen bonds between the C=O and N-H groups. Once again, you need to check your syllabus and past papers - particularly mark schemes for the past papers.

Hydrogen bonds

Notice that we are now talking about hydrogen bonds between side groups - not between groups actually in the backbone of the chain.

Lots of amino acids contain groups in the side chains which have a hydrogen atom attached to either an oxygen or a nitrogen atom. This is a classic situation where hydrogen bonding can occur.

For example, the amino acid serine contains an -OH group in the side chain. You could have a hydrogen bond set up between two serine residues in different parts of a folded chain.,%20methods%20of%20its%20determination.files/image038.gif

You could easily imagine similar hydrogen bonding involving -OH groups, or -COOH groups, or -CONH2 groups, or -NH2 groups in various combinations - although you would have to be careful to remember that a -COOH group and an -NH2 group would form a zwitterion and produce stronger ionic bonding instead of hydrogen bonds.


The alpha-helix

In an alpha-helix, the protein chain is coiled like a loosely-coiled spring. The "alpha" means that if you look down the length of the spring, the coiling is happening in a clockwise direction as it goes away from you.

 Note:  If your visual imagination is as hopeless as mine, the only way to really understand this is to get a bit of wire and coil it into a spring shape. The lead on your computer mouse is fine for doing this!

The next diagram shows how the alpha-helix is held together by hydrogen bonds. This is a very simplified diagram, missing out lots of atoms. We'll talk it through in some detail after you have had a look at it.,%20methods%20of%20its%20determination.files/image031.gif

What's wrong with the diagram? Two things:

First of all, only the atoms on the parts of the coils facing you are shown. If you try to show all the atoms, the whole thing gets so complicated that it is virtually impossible to understand what is going on.

Secondly, I have made no attempt whatsoever to get the bond angles right. I have deliberately drawn all of the bonds in the backbone of the chain as if they lie along the spiral. In truth they stick out all over the place. Again, if you draw it properly it is virtually impossible to see the spiral.

So, what do you need to notice?

Notice that all the "R" groups are sticking out sideways from the main helix.

Notice the regular arrangement of the hydrogen bonds. All the N-H groups are pointing upwards, and all the C=O groups pointing downwards. Each of them is involved in a hydrogen bond.

And finally, although you can't see it from this incomplete diagram, each complete turn of the spiral has 3.6 (approximately) amino acid residues in it.

If you had a whole number of amino acid residues per turn, each group would have an identical group underneath it on the turn below. Hydrogen bonding can't happen under those circumstances.

Each turn has 3 complete amino acid residues and two atoms from the next one. That means that each turn is offset from the ones above and below, such that the N-H and C=O groups are brought into line with each other.


Beta-pleated sheets

In a beta-pleated sheet, the chains are folded so that they lie alongside each other. The next diagram shows what is known as an "anti-parallel" sheet. All that means is that next-door chains are heading in opposite directions. Given the way this particular folding happens, that would seem to be inevitable.,%20methods%20of%20its%20determination.files/image032.gif

It isn't, in fact, inevitable! It is possible to have some much more complicated folding so that next-door chains are actually heading in the same direction. We are getting well beyond the demands of UK A level chemistry (and its equivalents) now.

The folded chains are again held together by hydrogen bonds involving exactly the same groups as in the alpha-helix.,%20methods%20of%20its%20determination.files/image033.gif

 Note:  Note that there is no reason why these sheets have to be made from four bits of folded chain alongside each other as shown in this diagram. That was an arbitrary choice which produced a diagram which fitted nicely on the screen!

The tertiary structure of proteins

What is tertiary structure?

The tertiary structure of a protein is a description of the way the whole chain (including the secondary structures) folds itself into its final 3-dimensional shape. This is often simplified into models like the following one for the enzyme dihydrofolate reductase. Enzymes are, of course, based on proteins.,%20methods%20of%20its%20determination.files/image035.jpg


Note:  This diagram was obtained from the RCSB Protein Data Bank. If you want to find more information about dihydrofolate reductase, their reference number for it is 7DFR.

There is nothing particularly special about this enzyme in terms of structure. I chose it because it contained only a single protein chain and had examples of both types of secondary structure in it.

The model shows the alpha-helices in the secondary structure as coils of "ribbon". The beta-pleated sheets are shown as flat bits of ribbon ending in an arrow head. The bits of the protein chain which are just random coils and loops are shown as bits of "string".

The colour coding in the model helps you to track your way around the structure - going through the spectrum from dark blue to end up at red.

You will also notice that this particular model has two other molecules locked into it (shown as ordinary molecular models). These are the two molecules whose reaction this enzyme catalyses.

What holds a protein into its tertiary structure?

The tertiary structure of a protein is held together by interactions between the the side chains - the "R" groups. There are several ways this can happen.

Ionic interactions

Some amino acids (such as aspartic acid and glutamic acid) contain an extra -COOH group. Some amino acids (such as lysine) contain an extra -NH2 group.

You can get a transfer of a hydrogen ion from the -COOH to the -NH2 group to form zwitterions just as in simple amino acids.

You could obviously get an ionic bond between the negative and the positive group if the chains folded in such a way that they were close to each other.,%20methods%20of%20its%20determination.files/image037.gif

van der Waals dispersion forces

Several amino acids have quite large hydrocarbon groups in their side chains. A few examples are shown below. Temporary fluctuating dipoles in one of these groups could induce opposite dipoles in another group on a nearby folded chain.

The dispersion forces set up would be enough to hold the folded structure together.

Conjugated Proteins

conjugated protein is a protein that functions in interaction with other chemical groups attached by covalent bonds or by weak interactions.

Many proteins contain only amino acids and no other chemical groups, and they are called simple proteins. However, other kind of proteins yield, on hydrolysis, some other chemical component in addition to amino acids and they are called conjugated proteins. The nonamino part of a conjugated protein is usually called its prosthetic group. Mostprosthetic groups are formed from vitamins. Conjugated proteins are classified on the basis of the chemical nature of their prosthetic groups.

Some examples of conjugated proteins are lipoproteinsglycoproteinsphosphoproteins,hemoproteinsflavoproteinsmetalloproteinsphytochromescytochromes and opsins.

Hemoglobin contains the prosthetic group containing iron, which is the heme. It is within the heme group that carries the oxygen molecule through the binding of the oxygen molecule to the iron ion (Fe2+) found in the heme group.

Glycoproteins are generally the largest and most abundant group of conjugated proteins. They range from glycoproteins in cell surface membranes that constitute the glycocalyx, to important antibodies produced by leukocytes.

Some proteins combine with other kinds of molecules such as carbohydrates, lipids, iron and other metals, or nucleic acids, to form glycoproteins, lipoproteins, hemoproteins, metalloproteins, and nucleoproteins respectively. The presence of these other biomolecules affects the protein properties. For example, a protein that is conjugated to carbohydrate, called a glycoprotein, would be more hydrophilic in character while a protein conjugated to a lipid would be more hydrophobic in character.

Protein Properties and Separation

Proteins are typically characterized by their size (molecular weight) and shape, amino acid composition and sequence, isolelectric point (pI), hydrophobicity, and biological affinity. Differences in these properties can be used as the basis for separation methods in a purification strategy (Chapter 4). The chemical composition of the unique R groups is responsible for the important characteristics of amino acids, chemical reactivity, ionic charge and relative hydrophobicity. Therefore protein properties relate back to number and type of amino acids that make up the protein.


Size of proteins is usually measured in molecular weight (mass) although occasionally the length or diameter of a protein is given in Angstroms. The molecular weight of a protein is the mass of one mole of protein, usually measured in units called daltons. One dalton is the atomic mass of one proton or neutron. The molecular weight can be estimated by a number of different methods including electrophoresis, gel filtration, and more recently by mass spectrometry. The molecular weight of proteins varies over a wide range. For example, insulin is 5,700 daltons while snail hemocyanin is 6,700,000 daltons. The average molecular weight of a protein is between 40,000 to 50,000 daltons. Molecular weights are commonly reported in kilodaltons or (kD), a unit of mass equal to 1000 daltons. Most proteins have a mass between 10 and 100 kD. A small protein consists of about 50 amino acids while larger proteins may contain 3,000 amino acids or more. One of the larger amino acid chains is myosin, found in muscles, which has 1,750 amino acids.

Separation methods that are based on size and shape include gel filtration chromatography (size exclusion chromatography) and polyacrylamide gel electrophoresis.

Amino Acid Composition and Sequence

The amino acid composition is the percentage of the constituent amino acids in a particular protein while the sequence is the order in which the amino acids are arranged.


Each protein has an amino group at one end and a carboxyl group at the other end as well as numerous amino acid side chains, some of which are charged. Therefore each protein carries a net charge. The net protein charge is strongly influenced by the pH of the solution. To explain this phenomenon, consider the hypothetical protein in Figure 2.5. At pH 6.8, this protein has an equal number of positive and negative charges and so there is no net charge on the protein. As the pH drops, more H+ ions are available in the solution. These hydrogen ions bind to negative sites on the amino acids. Therefore, as the pH drops, the protein as a whole becomes positively charged. Conversely, at a basic pH, the protein becomes negatively charged. pH 6.8 is called the pI, or isoelectric point, for this protein; that is, the pH at which there are an equal number of positive and negative charges. Different proteins have different numbers of each of the amino acid side chains and therefore have different isoelectric points. So, in a buffer solution at a particular pH, some proteins will be positively charged, some proteins will be negatively charged and some will have no charge.

Separation techniques that are based on charge include ion exchange chromatography, isoelectric focusing and chromatofocusing.

positive / negative charge chart

Figure 2.5. The pI is the pH at which there is no net charge on the protein. At lower pH readings, there are more positive charges in the environment and therefore, the protein has an increased cationic character. The reverse is true at pH readings above the pI.


Literally, hydrophobic means fear of water. In aqueous solutions, proteins tend to fold so that areas of the protein with hydrophobic regions are located in internal surfaces next to each other and away from the polar water molecules of the solution. Polar groups on the amino acid are called hydrophilic (water loving) because they will form hydrogen bonds with water molecules. The number, type and distribution of nonpolar amino acid residues within the protein determines its hydrophobic character. (Chart of hydrophobicity or hydropathy)

A separation method that is based on the hydrophobic character of proteins is hydrophobic interaction chromatography.


As the name implies, solubility is the amount of a solute that can be dissolved in a solvent. The 3-D structure of a protein affects its solubility properties. Cytoplasmic proteins have mostly hydrophilic (polar) amino acids on their surface and are therefore water soluble, with more hydrophobic groups located on the interior of the protein, sheltered from the aqueous environment. In contrast, proteins that reside in the lipid environment of the cell membrane have mostly hydrophobic amino acids (non polar) on their exterior surface and are not readily soluble in aqueous solutions.

Each protein has a distinct and characteristic solubility in a defined environment and any changes to those conditions (buffer or solvent type, pH, ionic strength, temperature, etc.) can cause proteins to lose the property of solubility and precipitate out of solution. The environment can be manipulated to bring about a separation of proteins- for example, the ionic strength of the solution can be increased or decreased, which will change the solubility of some proteins.,%20methods%20of%20its%20determination.files/image011.gif

Figure 2.6: Ionic Strength and Protein Folding. This figure shows the effect of ion concentration on protein folding.

Biological Affinity (Function):

Proteins often interact with other molecules in vivo in a specific way- in other words, they have a biological affinity for that molecule. These molecular counterparts, termed ligands, can be used as “bait” to “fish” out the target protein that you want to purify. For example, one such molecular pair is insulin and the insulin receptor. If you want to purify (or catch) the insulin receptor, you could couple many insulin molecules to a solid support and then run an extract (containing the receptor) over that column. The receptor would be “caught” by the insulin bait. These specific interactions are often exploited in protein purification procedures. Affinity chromatography is a very common method for purifying recombinant proteins (proteins produced by genetic engineering). Several histidine residues can be engineered at the end of a polypeptide chain. Since repeated histidines have an affinity for metals, a column of the metal can be used as bait to “catch” the recombinant protein.

Table 2.2: Methods Used for Protein Separation and Analysis


Protein Property Exploited

Bulk Methods

Ammonium sulfate precipitation




Chromatography Methods



Gel Filtration (Gel Permeation)

Size or molecular wt.

Hydrophobic Interaction



Biological Activity

Reversed Phase



pI (Charge)


Native Gel


Denaturing Gel (SDS-PAGE)

Mass (Molecular weight)


pI or charge

2D gels

Molecular weight and pI (charge)

 Working with proteins

How proteins lose their structure and function.

Although DNA can be isolated and amplified from thousand year old mummies, most proteins are more fragile biomolecules. Therefore, laboratory reagents and storage solutions must provide suitable conditions so that the normal structure and function of the protein is maintained. To understand how the structure of proteins is protected in laboratory solutions, it is necessary to understand how that structure can be destroyed.

·                    Proteins can denature, or unfold so that their three dimensional structure is altered but their primary structure remains intact.(Figure 2.7) Many of the interactions that stabilize the 3-D conformation of the protein are relatively weak and are sensitive to various environmental factors including high temperature, low or high pH and high ionic strength. Protein vary greatly in the degree of their sensitivity to these factors. Sometimes proteins can be renatured but often the denaturation is irreversible.,%20methods%20of%20its%20determination.files/image012.gif

Figure 2.7. A figure showing the process of denaturation. The polypeptide chain has lost its higher order structure and is now a random coil.

·                    Proteins can also be broken apart by enzymes, called proteases, that digest the covalent peptide bonds between amino acids that are responsible for the primary structure. This process is called proteolysis and is irreversible. Cells contain proteases that are found in lysosomes, membrane bound organelles inside the cell. When cells are disrupted, lysosomes break and release these proteases, which can damage the other proteins in the cell. In the laboratory, it is therefore necessary to minimize the activities of cellular proteases to protect proteins from proteolysis. Methods used to minimize proteolysis include working at lower temperatures (4°C), and adding chemicals that inhibit protease activity.

·                    Sulfur groups on cysteines may undergo oxidation to form disulfide bonds that are not normally present. Extra disulfide bonds can form when proteins are removed from their normal environment. Reducing agents such as dithiothreitol or ß-mercaptoethanol are often added to prevent undesirable disulfiate bond formation.

·                    Proteins readily adsorb (stick to) surfaces, thereby reducing their available activity. To prevent significant loss, do not store dilute solutions of proteins for prolonged periods of time. Always dilute them right before use.

The composition of the extraction buffer is important for maintaining structure and function of the target protein. To prevent denaturation, the buffering pH is based on the pH stability range of the protein. Other components such as ionic strength, divalent cations (Ca++ and Mg++), or reducing agents (dithiothreitol or ß-mercaptoethanol) may be needed to maintain activity. In making the extract, cells are lysed and proteases (enzymes that degrade proteins) are released from their intracellular compartments. To prevent proteases from digesting the target protein, two strategies are commonly followed: 1) The extract is kept cold. The activity of proteolytic enzymes is greatly reduced by cold temperatures. For this reason, the protein purification process is often conducted in cold rooms. At the very least, an effort is made to keep the extract at 4?C. 2) Protease inhibitors are sometimes added to the mixture to prevent degradation by proteases. The drawback to this strategy is that the inhibitors must eventually be removed, along with other contaminant proteins.

  Denaturation of proteins involves the disruption and possible destruction of both the secondary and tertiary structures. Since denaturation reactions are not strong enough to break the peptide bonds, the primary structure (sequence of amino acids) remains the same after a denaturation process. Denaturation disrupts the normal alpha-helix and beta sheets in a protein and uncoils it into a random shape.

Denaturation occurs because the bonding interactions responsible for the secondary structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are four types of bonding interactions between "side chains" including: hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions. which may be disrupted. Therefore, a variety of reagents and conditions can cause denaturation. The most common observation in the denaturation process is the precipitation or coagulation of the protein.


The natural or native structures of proteins may be altered, and their biological activity changed or destroyed by treatment that does not disrupt the primary structure. This denaturation is often done deliberately in the course of separating and purifying proteins. For example, many soluble globular proteins precipitate if the pH of the solution is set at the pI of the protein. Also, addition of trichloroacetic acid or the bis-amide urea (NH2CONH2) is commonly used to effect protein precipitation. Following denaturation, some proteins will return to their native structures under proper conditions; but extreme conditions, such as strong heating, usually cause irreversible change.
Some treatments known to denature proteins are listed in the following table.

Denaturing Action

Mechanism of Operation


hydrogen bonds are broken by increased translational and vibrational energy.
(coagulation of egg white albumin on frying.)

Ultraviolet Radiation

Similar to heat

Strong Acids or Bases

salt formation; disruption of hydrogen bonds.
(skin blisters and burns, protein precipitation.)

Urea Solution

competition for hydrogen bonds.
(precipitation of soluble proteins.)

Some Organic Solvents
(e.g. ethanol & acetone)

change in dielectric constant and hydration of ionic groups.
(disinfectant action and precipitation of protein.)


shearing of hydrogen bonds.
(beating egg white albumin into a meringue.)

Analytical Methods for amino Acid Separation and Identification

Separation and identification of amino acids are operations that must be performed frequently by biochemists. The 20 amino acids present in proteins have similar structures. However, each amino acid is unique in polarity and ionic characteristics. In this experiment, we will use a combination of ion exchange chromatography and paper chromatography to separate and identify the components of an unknown amino acid mixture.


Twenty amino acids are the fundamental building blocks of proteins. Amide bond linkages between a-amino acids construct all proteins found in nature. The amino acids isolated from proteins material all have common structural characteristics.

 The distinctive physical, chemical and biological properties associated with an amino acid are the result of the R group. There are 20 major amino acids that differ in their R-group.  The R-group can be hydrophobic or polar, aromatic or aliphatic, charged or uncharged.  The different R-groups are responsible for amino acids having different polarities, solubilities and chromatographic behavior (see below).

 The structure and biological function of a protein depend on its amino acid composition. It is a matter of basic importance to understand practical methods used for the separation and identification of the 20 common amino acids.


 Amino acids are amphitropic because they contain both an acidic group and basic group.  The COOH group is acidic with a pKa value of 1.7-2.4.  Thus at pH values below this, the group exists as COOH while at higher pH values, the group exists as COO-.  The NH2 group is basic with a pKa of 9-10.5, so below this it exists as NH3+ while above this pH it exists as NH2.  At neutral pH values, both groups are ionized and the amino acid exists in a dipolar form with no net charge.  This form is called a zwitterion.  The pH at which all the amino acid molecules are in this form is the isoionic point (pI) of the amino acid where (for amino acids with non-ionizable side chain chains)


Paper chromatography of amino acids

Paper chromatography can separate different amino acids based on their varying solubilities in two different solvents.  In this method, a sample of an amino acid (or mixture of amino acids) is applied as a small spot near one edge of a piece of chromatography paper.  The edge of the paper is then placed in a shallow layer of solvent mixture in a chromatography tank.

 The solvent mixture contains several components, one of which is usually water and another of which is a more non-polar solvent.  As the solvent mixture moves up the paper by capillary action, the water in the mixture binds to the hydrophilic paper (cellulose) and creates a liquid stationary phase of many small water droplets.  The non-polar solvent continues to move up the paper forming a liquid mobile phase.  Since amino acids have different R-groups, they also have different degrees of solubility in water vs. the non-polar solvent.  An amino acid with a polar R-group will be more soluble in water than in the non-polar solvent, so it will dissolve more in the stationary water phase and will move up the paper only slightly.  An amino acid with a hydrophobic R-group will be more soluble in the mobile non-polar solvent than in water, so it will continue to move up the paper.  Different amino acids will move different distances up the paper depending upon their relative solubilities in the two solvents, allowing for separation of amino acid mixtures.

 The movement of amino acids can be defined by a quantity known as Rf value, which measures the movement of an amino acid compared to the movement of the solvent.  At the start of the chromatography, the amino acid is spotted at what is called the origin.  The chromatography is then performed, and the procedure is stopped before the solvent runs all the way up the paper.  The level to which the solvent has risen is called the solvent front.  The Rf value of an amino acid is the ratio of the distance traveled by the amino acid from the origin to the distance traveled by the solvent from the origin.

 Since Rf value for an amino acid is constant for a given chromatography system, an unknown amino acid can be identified by comparing its Rf value to those of known amino acids.

 Certain technical aspects are important when performing paper chromatography.  First, it is necessary to keep the applied amino acid spot very small.  The spot tends to spread out as it moves up the paper, so starting with a big spot will produce a large smear by the end of the procedure, making it difficult to measure an accurate Rf value.  Second, the chromatogram paper must be kept very clean.  Fingerprints or other types of contamination will interfere with the chromatography and give poor results.  Finally, since amino acids are colorless, something must be done to detect the amino acids at the completion of the chromatography.  One of the simplest methods for this involves spraying the paper with ninhydrin.  When heated, ninhydrin reacts with amino acids to produce a blue-purple color (yellow in the case of proline), making the amino acids spots visible for analysis.


In this experiment, paper chromatography will be performed using an unknown amino acid along with known standards.  Through a comparison of Rf values, the unknown amino acid will be identified.


 Spectrophotometry is widely used in biochemistry.   Many biochemical compounds absorb light in the ultraviolet (200-400 nm), visible (400-700 nm), or near infrared (700-900 nm) regions of the spectrum.   Even if a particular compound does not absorb light itself, it can often be reacted with another compound to produce a light-absorbing substance.   Thus spectrophotometry allows for the qualitative and quantitative determination of biochemical compounds.   In addition, such techniques are often simple, fast, and clean.   Because of their sensitivity, these methods are frequently employed by biochemists.

 When white light is passed through a solution containing a colored compound, certain wavelengths of light are absorbed.   Which wavelengths (energies) of light are absorbed depends upon the chemical structure of the compound.   The absorption of a particular wavelength of light indicates the absorption of photons possessing particular energies, and the absorption of these photons increases various types of molecular energy (electronic, rotational, vibrational, etc.) of the compound.   Those wavelengths of light that are not absorbed by the compound are reflected or transmitted, and are responsible for the appearance of the compound.   Since different types of compounds have characteristic wavelengths at which they absorb light, it is possible to measure the absorbance of a substance at many different wavelengths to obtain its absorption spectrum.   A compound can often be qualitatively identified in this manner.

Protein Analysis

 The preceding discussion applies to both inorganic and biochemical spectrophotometry.   However, in biochemistry, only a few important compounds are highly colored and so can be studied directly.   Many biochemical molecules absorb UV light, but the amount of absorption is often too small for an accurate analysis if one is dealing with a limited amount of the compound to be analyzed.   To circumvent this difficulty, various reactions have been developed in which a particular type of biochemical compound is converted into a highly colored substance.   In performing such quantitative determinations, a series of solutions of the compound (or a similar one) are made, the concentrations of which are known.   Under defined conditions, the compound in these solutions is reacted with an excess of the color-forming reagents.   The absorbances of the solutions are measured, and a standard Beer's law plot  showing the variation of absorbance with concentration can be drawn.   In addition, a blank is prepared which contains all of the color-forming reagents, but none of the compound being assayed.   The absorbance of the blank serves as a control.   Then, the color-forming reaction can be performed with the sample where the concentration of the compound is unknown, and a quantitative determination can be made.

 Proteins in particular are a biochemical compound that must often be measured.   Proteins absorb UV light at 280 nm due to the presence of aromatic amino acids, allowing for a direct determination of protein.   Most pure protein solutions containing 1 mg/mL of protein have an absorbance of about 1.0 when the light path is 1 cm.   This method is simple, rapid, and allows for full recovery of the protein.   However, many other biochemicals absorb near this wavelength, making an accurate quantitation difficult.   Furthermore, different proteins absorb to different extents depending upon their aromatic amino acid content. To avoid these problems, the Biuret test was developed.  Compounds containing 2 or more peptide bonds take on a purple color when treated with dilute copper sulfate in an alkaline solution.   This reaction is reproducible from protein to protein, but it requires a relatively large amount of protein (1-20 mg).   The Folin-Ciocalteu (Lowry) test depends upon the reaction of protein with alkaline copper (as in the Biuret test) and the reduction of phosphomolybdate-phosphotungstate salts by tyrosine and tryptophan residues to yield a blue-green color.   This test is very sensitive, requiring as little as 5 µg of protein.   However, variations from protein to protein can be substantial.

Protein Molecular Weight Determination

The purpose of this experiment is to determine the molecular weight of a protein using gel filtration and SDS-gel electrophoresis.

I. Gel Filtration

 Gel filtration is a chromatographic technique that separates different molecules on the basis of size.  It is commonly used during protein purification to remove unwanted proteins from the protein being purified.  It can also be used to determine the molecular weight of a protein.

 In gel filtration, a dextran, polyacrylamide, or agarose gel is suspended in buffer and packed in a glass or plastic column.  The sample to be analyzed is applied to the top of the column and is allowed to run down into the gel.  A continuous supply of buffer is then provided at the top of the column, and, as the buffer runs through the column, the components in the sample are carried down the gel and separated.  The buffer is collected at the bottom of the column in fractions of constant volume (i.e. 1.0 mL), and all the fractions are analyzed for the presence of the various components in the sample.  The separation of the components is caused by cross-linking in the gel which creates pores.  Small molecules can penetrate the pores and so are slowed down and retained as they pass down the column.  Large molecules cannot penetrate the pores and so run down the column quickly.  Gels with different degrees of cross-linking (and therefore different sized pores) are commercially available to separate molecules in different molecular weight ranges.  In this experiment, Sephadex G-75 will be used.  This gel is a dextran capable of separating proteins with molecular weights between 3000 and 70,000.

 For a Sephadex column, the total volume, Vt, is equal to the sum of the volume of the gel matrix, the volume inside the gel matrix, and the volume outside the matrix.  The total volume is also , in most cases, equal to the amount of the buffer required to run a substance through the column (also known as eluting a substance) when the substance is small enough to completely penetrate the pores of the gel.  Such a substance is said to be completely included by the gel.  For Sephadex G-75, compounds with molecular weights less than 3000 are completely included.  The volume outside the gel matrix is known as the void volume, Vo.  This is the volume required to elute a substance so large that it cannot penetrate the pores at all.  Such a substance is said to be completely excluded by the gel.  For Sephadex G-75, proteins with molecular weights greater than 70,000 are completely excluded.  Compounds with intermediate molecular sizes that can partially penetrate the pores elute between the void volume and the total volume, and are said to be partially included by the gel.  The volume of buffer required to elute any given substance is known as the elution volume, Ve, of the compound.  Thus on Sephadex G-75, a protein with a molecular weight of 60,000 will be less included than a protein with a molecular weight of 30,000.  The larger protein will have a smaller elution volume and run through the column more quickly than the smaller protein.

 During protein purification, a mixture of many proteins can be subjected to gel filtration, and all proteins that have molecular weights different from the one being purified can be separated out.  Thus gel filtration is a powerful technique for purifying a protein.  Gel filtration can also be used to determine the molecular weight of a protein.  To do this, several proteins with known molecular weights are run on the column and their elution volumes determined.  If the elution volumes are then plotted against the log molecular weight of the corresponding proteins, a straight line is obtained for the separation range of the gel being used.  If the elution volume of a protein of unknown molecular weight is then found, it can be compared to the calibration curve and the molecular weight determined.

 Gel filtration has many advantages as a biochemical technique.  It is relatively simple to perform, and the mild conditions used tend to prevent denaturation of proteins, unlike some other techniques.  The protein that runs off the column can be collected and used for further analysis, so no protein is consumed in gel filtration.  However, there are also disadvantages as well.  The column must be carefully prepared to obtain optimal separation.  Any cracks or discontinuities in the column will interfere.  The size of the sample and the rate of buffer flow must be strictly controlled.  If a column is run several times, each run must be done under the exact same conditions in order to compare the different runs.  finally, some substances stick to Sephadex and do not elute properly.

SDS-gel electrophoresis

The second method used to find the molecular weight of a protein will be
SDS-gel electrophoresis.  When a charged protein is placed in an electric field, it will migrate toward the oppositely charged region, and this is the basis of electrophoresis.  In most electrophoresis methods, the molecules being analyzed are placed on a solid support and then allowed to migrate.  For proteins, a polyacrylamide gel support is commonly used.  The proteins are applied to the gel, and the gel is contained in an electrophoresis cell, which in turn is connected to a power supply which creates a positive electrode and a negative electrode in the cell.  Buffer is used to complete the circuit in the cell between the gel and the electrode wires.  The buffer in the cell and contained in the gel is important, since its pH determines the charge on the protein molecules.

 Usually the determining factor in the separation of the molecules is their charge.  The more highly charged the molecule, the faster and farther it will move during electrophoresis.  With proteins, however, a second effect is seen, namely the size of the protein.  As a protein moves through the gel, it must overcome frictional forces which oppose its movement.  The larger the protein, the greater the frictional force.  Thus in most gels, the exact rate of movement of a particular protein depends on both its charge and its size.


 One type of electrophoresis is SDS-gel electrophoresis.  In this method, the proteins to be separated are denatured (usually in urea) and then mixed with the detergent SDS (sodium dodecyl sulfate).  SDS binds along the length of the protein, obscuring the protein’s own charges and giving all proteins the same negative charge per unit length.  Thus charge is essentially removed as a factor in the separation and size alone becomes important.  All proteins will move toward the positive electrode, but large proteins will move more slowly than small proteins.  The distance moved is inversely proportional to the log of the molecular weight. It is therefore possible to run several proteins of known molecular weight in an SDS-gel electrophoresis procedure, measure their migration distances, and construct a calibration curve.  The distance moved by a protein of unknown molecular weight can be compared to the standards and its size determined.

 Some proteins are colored and can be seen directly on a gel, but most are colorless.  To visualize most proteins, a staining procedure is needed.  Coomassie blue is a general protein stain, causing the protein to be come visible as blue bands within the gel.  Silver stain can detect very small amounts of proteins, causing them to turn brown-black

Structure-Property Relationships

The compounds we call proteins exhibit a broad range of physical and biological properties. Two general categories of simple proteins are commonly recognized.


Fibrous Proteins


As the name implies, these substances have fiber-like structures, and serve as the chief structural material in various tissues. Corresponding to this structural function, they are relatively insoluble in water and unaffected by moderate changes in temperature and pH. Subgroups within this category include:
      Collagens & Elastins, the proteins of connective tissues. tendons and ligaments.
      Keratins, proteins that are major components of skin, hair, feathers and horn.
      Fibrin, a protein formed when blood clots.

Globular Proteins


Members of this class serve regulatory, maintenance and catalytic roles in living organisms. 
They include hormones, antibodies and enzymes. and either dissolve or form colloidal suspensions in water. 
Such proteins are generally more sensitive to temperature and pH change than their fibrous counterparts. 

Fibrous proteins such as keratins, collagens and elastins are robust, relatively insoluble, quaternary structured proteins that play important roles in the physical structure of organisms. Secondary structures such as the α-helix and β-sheet take on a dominant role in the architecture and aggregation of keratins. In addition to the intra- and intermolecular hydrogen bonds of these structures, keratins have large amounts of the sulfur-containing amino acid Cys, resulting in disulfide bridges that confer additional strength and rigidity. The more flexible and elastic keratins of hair have fewer interchain disulfide bridges than the keratins in mammalian fingernails, hooves and claws. Keratins have a high proportion of the smallest amino acid, Gly, as well as the next smallest, Ala. In the case of β-sheets, Gly allows sterically-unhindered hydrogen bonding between the amino and carboxyl groups of peptide bonds on adjacent protein chains, facilitating their close alignment and strong binding. Fibrous keratin chains then twist around each other to form helical filaments. 

Elastin, the connective tissue protein, also has a high percentage of both glycine and alanine. An insoluble rubber-like protein, elastin confers elasticity on tissues and organs. Elastin is a macromolecular polymer formed from tropoelastin, its soluble precursor. The secondary structure is roughly 30% β-sheets, 20% α-helices and 50% unordered. The elastic properties of natural elastin are attributed to polypentapeptide sequences (Val-Pro-Gly-Val-Gly) in a cross-linked network of randomly coiled chains. Water is believed to act as a "plasticizer", assisting elasticity. 

Collagen is a major component of the extracellular matrix that supports most tissues and gives cells structure. It has great tensile strength, and is the main component of fascia, cartilage, ligaments, tendons, bone and skin. Collagen contains more Gly (33%) and proline derivatives (20 to 24%) than do other proteins, but very little Cys. The primary structure of collagen has a frequent repetitive pattern, Gly-Pro-X (where X is a hydroxyl bearing Pro or Lys). This kind of regular repetition and high glycine content is found in only a few other fibrous proteins, such as silk fibroin (75-80% Gly and Ala + 10% Ser). Collagen chains are approximately 1000 units long, and assume an extended left-handed helical conformation due to the influence of proline rings. Three such chains are wound about each other with a right-handed twist forming a rope-like superhelical quaternary structure, stabilized by interchain hydrogen bonding.

Globular proteins are more soluble in aqueous solutions, and are generally more sensitive to temperature and pH change than are their fibrous counterparts; furthermore, they do not have the high glycine content or the repetitious sequences of the fibrous proteins. Globular proteins incorporate a variety of amino acids, many with large side chains and reactive functional groups. The interactions of these substituents, both polar and nonpolar, often causes the protein to fold into spherical conformations which gives this class its name. In contrast to the structural function played by the fibrous proteins, the globular proteins are chemically reactive, serving as enzymes (catalysts), transport agents and regulatory messengers. 

Although globular proteins are generally sensitive to denaturation (structural unfolding), some can be remarkably stable. One example is the small enzyme ribonuclease A, which serves to digest RNA in our food by cleaving the ribose phosphate bond. Ribonuclease A is remarkably stable. One procedure for purifying it involves treatment with a hot sulfuric acid solution, which denatures and partially decomposes most proteins other than ribonuclease A. This stability reflects the fact that this enzyme functions in the inhospitable environment of the digestive tract. Ribonuclease A was the first enzyme synthesized by R. Bruce Merrifield, demonstrating that biological molecules are simply chemical entities that may be constructed artificially. By clicking the cartoon image on the left, an interactive model of ribonuclease A will be displayed.


Chromatographic methods are applicable not only to sepa­ration, identification, and quantitative analysis of amino acid mixtures but also of peptides, proteins, nucleotides, nucleic acids, lipids, and carbohydrates.

Partition Chromatography. When a solute is allowed to distribute itself between equal volumes of two immiscible liquids, the ratio of the concentrations of the solute in the two phases is called the partition coefficient. Amino acids can be partitioned in this manner between two liquid phases, e.g., the pairs phenol-water or n-butanol-water. Each amino acid has a distinctive partition coefficient for any given pair of immiscible solvents.

Partition chromatography is the chromatographic separation of mixtures essentially by the countercurrent-partition principle. The separation is achieved in a huge number of separate partition steps, which take place on microscopic granules of a hydrated insoluble inert substance, such as starch or silica gel, packed in a column about 10 to 100 cm long. Starch or silica gel granules are hydrophilic and are surrounded by a layer of a tightly bound water, which serve as a stationary phase, past which flows a moving phase of an immiscible solvent containing the mixture to be separated. The mixture of solutes undergoes a microscopic partition process between the fixed water layer and the flowing solvent.

The total number of partition steps in the column is so great that the different amino acids in the mixture move down the column at different rates as the moving liquid phase flows through it. The liquid appearing at the bottom of the column, called the eluate, is caught in small fractions with an automatic fraction collector and analyzed by means of the quantitative ninhydrin reaction.

Precisely the same principle is involved in filter-paper chromatography of amino acids. The cellulose of the filter-paper is hydrated. As a solvent containing an amino acid mixture ascends in the vertically held paper by capillary action (or descends, in descending chromatography), many microscopic distributions of the amino acids occur between the flowing phase and the stationary water phase bound to the paper fibers. At the end of the process, the different amino acids have moved different distances from the origin. The paper is dried, sprayed with ninhydrin solution, and heated in order to locate the amino acids. In the important refinement of two-dimensional paper chromatography, the mixture of amino acids is chromatographed in one direction; then the paper is dried and subjected to chromatography with a different solvent system in a direction at right angles to the first. A two-dimensional map of the different amino acids results.

Ion-Exchange Chromatography. The partition principle has been further refined in ion-exchange chromatography. In this method solute molecules are sorted out by the differences in their acid-base behavior. A column is filled with granules of synthetic resins: cation exchangers and anion exchangers. Amino acids are usually separated on cation exchange columns.

Amino acids can also be separated by thin-layer chromatography, a refinement of partition chromatography.

Molecular-Exclusion Chromatography. One of the most useful and powerful tools for separating pro­teins from each other on the basis of size is molecular-exclusion chromatography, also known as gel-filtration or molecular-sieve chromatography. It differs from ion-exchange chromatography, which separates solutes on the basis of their electric charge and acid-base properties. In molecular-exclusion chromatography the mixture of pro­teins is allowed to flow by gravity down a column packed with beads of an inert, highly hydrated polymeric material that has previously been washed and equilibrated with the buffer alone. Common column materials are Sephadex, the commercial name of a polysaccharide derivative; Bio-Gel, a commercialpolyacryl-amide derivative; and agarose, another polysaccharide — all of which can be prepared with different degrees of internal porosity. In the column proteins of different molecular size penetrate into the internal pores of the beads to different degrees and thus travel down the column at different rates. Very large protein molecules cannot enter the pores of the beads; they are said to be excluded and thus remain in the excluded volume of the column, denned as the volume of the aqueous phase outside the beads. On the other hand, very small proteins can enter the pores of the beads freely. Small proteins are retarded by the column while large proteins pass through rapidly, since they cannot enter the hydrated polymer particles. Proteins of inter­mediate size will be excluded from the beads to a degree that depends on their size. From measurements of the protein concentration in small fractions of the eluate an elution curve can be con­structed.

Molecular-exclusion chromatography can also be used to separate mixtures of other kinds of macromolecules, as well as very large biostructures, e.g., viruses, ribosomes, cell nu­clei, or even bacteria, simply by using beads or gels with dif­ferent degrees of internal porosity. The resolving power of molecular-exclusion chromatography is so great that this simple method is now widely used as a way of determining the molecular weight of proteins.

Selective Adsorption. Proteins can be adsorbed to, and selectively eluted from, columns of finely divided, relatively inert materials with a very large surface area in relation to particle size. They include nonpolar substances, e.g., charcoal, and polar sub­stances, e.g., silica gel or alumina. The precise nature of the forces binding the protein to such adsorbents is not known, but presumably van der Waals and hydrophobic interactions prevail with nonpolar adsorbents, whereas ionic attractions and/or hydrogen bonding are the main forces with polar ad­sorbents.

Affinity Chromatography. Some proteins can be isolated from a very complex mixture and brought to a high degree of purification, often in a single step, by affinity chromatography. This method is based on a biological property of some proteins, namely, their capacity for specific, noncovalent binding of another molecule, called the ligand. For example, some enzymes bind their specific coenzymes very tightly through noncovalent forces. In order to separate such an enzyme from other pro­teins by affinity chromatography, its specific coenzyrne is covalently attached, by means of an appropriate chemical reaction, to a functional group on the surface of large hydrated particles of a porous column material, e.g., the poly-saccharide agarose, which otherwise allows protein mole­cules to pass freely. When a mixture of proteins containing the enzyme to be isolated is added to such a column, the enzyme molecule, which is capable of binding tightly and specifically to the immobilized ligand molecule, adheres to the ligand-derivatized agarose particles, whereas all the other proteins, which lack a specific binding site for that particularligand molecule, will pass through. This method thus depends on the biologi­cal affinity of the protein for its characteristic ligand. The protein specifically bound to the column particles in this manner can then be eluted, often with a solution of the free ligand molecule.

Diagnostic significance of blood and urine chromatographic analysis. Hypo- and hyperaminoacidemia, hypo- and hyperaminoaciduria.

The measurement of amino acids level in organism is important for studing of protein metabolism in organism. There are approximately 21,2 mmol/l amino acids in blood plasma in normal conditions. Hyperaminoacidemia – the increasing of amino acid level in blood plasma. The possible causes of such state are liver diseases, diabetus mellitus, acute and chronical kidney failure, congenital enzymopathy.

Hypoaminoacidemia is observed during the protein starvation, fever, kidney diseases, hyperfunction of adrenal cortex.

1 g of amino acids is excreted per day via the kidneys. Hyperaminoaciduria occurs in liver diseases (because in this case the disturbances of protein synthesis take place), infectious diseases, diabetus mellitus, hyperthyroidism, malignant tumor, traumas.

Acid-Base Properties of Peptides. Since none of the a-carboxyl groups and none of the a-amino groups that are combined in peptide linkages can ionize in the pH zone 0 to 14, the acid-base behavior of peptides is contributed by the free a-amino group of the N-terminal residue, the free a-carboxyl group of the carboxy-terminal (abbreviated C-terminal) residue, and those R groups of the residues in inter­mediate positions which can ionize. In long poly­peptide chains the ionizing R groups necessarily greatly outnumber the terminal ionizing groups.

Optical Properties of Peptides. If partial hydrolysis of a protein is carried out under sufficiently mild conditions, the peptides formed are optically active, since they contain only L-amino acid residues. In relatively short peptides, the total observed optical activity is approximately an additive function of the optical activities of the component amino acid residues. However, the optical activity of long polypeptide chains of proteins in their native conformation is much less than additive, a fact of great significance with regard to the secondary and tertiary structure of proteins.

Chemical Properties of Peptides. The free N-terminal amino groups of peptides undergo the same kinds of chemical reactions as those given by the a-amino groups of free amino acids, such as acylation and carbamoylation. The N-terminal amino acid residue of peptides also reacts quantitatively with ninhydrin to form colored derivatives; the ninhydrin reaction is widely used for detection and quantitative estimation of peptides in electrophoretic and chromatographic procedures. Similarly, the C-terminal carboxyl group of a peptide may be esterified or reduced. Moreover, the various R groups of the different amino acid residues found in peptides usually yield the same characteristic reactions as free amino acids.

One widely employed color reaction of peptides and pro­teins that is not given by free amino acids is the biuret reac­tion. Treatment of a peptide or protein with Cu2+ and alkali yields a purple Cu2+-peptide complex, which can be mea­sured quantitatively in a spectrophotometer.

The mo­lecular weight of proteins and its determination.

The mo­lecular weights of proteins ranges from about 5000, which is the lower limit, to 1 mil­lion or more.

Many proteins having molecular weights above 40000 contain two or more polypeptide chains. The individual polypeptide chains of most proteins of known structure contain from 100 to 300 amino acid resi­dues. However, some proteins have much longer chains, such as serum albumin (approx­imately 550 residues) and myosin (approximately 1800 resi­dues).

Determination of the Molecular Weight from Osmotic-Pressure Measurements

When a semipermeable membrane separates a solution of a protein from pure water, the water moves across the mem­brane into the compartment containing the solute, a process called osmosis. The molecular weight of a protein can be determined from measurements of the os­motic pressure of a solution of a known concentration of ­pro­tein.

Determination of Molecular Weight by Sedimentation Analysis

The ultracentrifuge can yield centrifugal fields exceeding 250 000 times the force of gravity. Such a high centrifugal field causes protein sedimentation, opposing the force of diffusion, which normally keeps them evenly dispersed in solution. If the centrifugal force exerted on protein molecules in a solution greatly exceeds the opposing diffusion force, the molecules will sediment down. The rate of sedimentation is observed by optical mea­surements and depend on molecular weight of proteins.

Determining Molecular Weight by Light Scattering

When a beam of light is passed through a protein solution in a darkened room, the path of the beam can be seen because the light is scattered by the protein molecules. This is called theTyndal effect. From the wavelength of the incident radi­ation, the intensity of the scattered light, the refractive index of the solvent and solute, and the concentration of the solute, the molecular weight of the protein can be calculated.

Determining Molecular Weight by Molecular-Exclusion Chromatography

Protein mixtures can be sorted out on the basis of molecular weight by molecular-exclusion chromatography. This simple method, which requires no complex equipment, can yield accurate deter­minations of the molecular weight of a protein. Molecular-exclusion columns measure not the true molecu­lar weight of an unknown protein but its Stokes radius, which is most simply defined as the radius of a perfect unhydrated sphere having the same rate of passage through the column as the unknown protein in question. If the unknowm and marker proteins are spherical, the method yields the molecular weight directly.

Proteins Solubility. Factors Determining the Solubility.

Proteins in solution show profound changes in solubility as a function of (1) pH, (2) ionic strength, (3) the dielectric properties of the solvent (hydrated shell), and (4) temperature.

The solubility of most globular proteins is profoundly in­fluenced by the pH of the system because the electric charge of protein molecule results from pH. When the protein molecule has no net electric charge there is no electrostatic repul­sion between neighboring protein molecules and they tend to coalesce and precipitate. When all the protein molecules have a net charge of the same sign they repel each other, preventing coalescence of single molecules into insoluble aggregates.

Electric charge of proteins and hence the availability of hydrated shell and solubility of proteins depend also on the ionic composition of the medium, since proteins can bind certain anions and/or cations.

Methods of protein precipitation.

There are two methods of protein precipitation: reversible (salting-out) and inreversible (denaturation).

Reversible coagulation of proteins. Salting-in and Salting-out of Proteins.

Reversible coagulation of proteins - precipitation without the loss of native structure. If optimal conditions will be created for proteins (for example, the adding of solvent) they can be dis­solved again.

Neutral salts have pronounced effects on the solubility of globular proteins. In low concentration, salts increase the solubility of many proteins, a phenomenon called salting-in. Salts of divalent ions, such as MgCI2 are far more effective at salting-in than salts of monovalent ions, such as NaCl and KCl. The ability of neutral salts to influence the solubility of pro­teins is a function of their ionic strength, a measure of both the concentration and the number of electric charges on the cations and anions contributed by the salt. Salting-in effects are caused by changes in the tendency of dissociable R groups on the protein to ionize.

On the other hand, as the ionic strength is increased further, the solubility of a protein begins to decrease. At sufficiently high ionic strength a protein may be al­most completely precipitated from solution, an effect called salting-out. The physicochemical basis of salting-out is rather complex; one factor is that the high concentration of salt may remove water of hydration from the protein mole­cules, thus reducing their solubility, but other factors are also involved. Proteins precipitated by salting-out retain their native conformation and can be dis­solved again, usually withoutdenaturation. Ammonium sulfate is preferred for salting out proteins because it is so solu­ble in water that very high ionic strengths can be attained.

Separation, Purification and Characterization of Proteins

Each type of cell may contain thousands of different proteins. The isolation in pure form of a given protein from a given cell or tissue may appear to be a difficult task, particu­larly since any given protein may exist in only a very low concentration in the cell, along with thousands of others.

Separation Procedures Based on Molecular Size.

Dialysis and Ultrafiltration. Globular proteins in solution can easily be separated from low-molecular-weight solutes by dialysis, which utilizes a semipermeable membrane to retain protein mole­cules and allow small solute molecules and water to pass through.

Another way of separating proteins from small molecules is by ultrafiltration, in which pressure or centrifugal force is used to filter the aqueous medium and small solute molecules through asemipermeable membrane, which retains the protein molecules. Cellophane and other synthetic materials are commonly used as the membrane in such procedures.

Density-Gradient (Zonal) Centrifugation. Because proteins in solution tend to sediment at high cen­trifugal fields, thus overcoming the opposing tendency of diffusion, it is possible to separate mixtures of proteins by centrifugal methods.

Molecular-Exclusion Chromatography. One of the most useful and powerful tools for separating pro­teins from each other on the basis of size is molecular-exclusion chromatography, also known as gel-filtration. In molecular-exclusion chromatography the mixture of pro­teins, dissolved in a suitable buffer, is allowed to flow by gravity down a column packed with beads of an inert, highly hydrated polymeric material. Common column materials are Sephadex, the commercial name of a polysaccharide derivative, which can be prepared with different degrees of internal porosity. In the column proteins of different molecular size penetrate into the internal pores of the beads to different degrees and thus travel down the column at different rates. Very large protein molecules cannot enter the pores of the beads, very small proteins can enter the pores of the beads freely. Small proteins are retarded by the column while large proteins pass through rapidly, since they cannot enter the polymer particles. Proteins of inter­mediate size will be excluded from the beads to a degree that depends on their size. From measurements of the protein concentration in small fractions of the eluate an elution curve can be con­structed.

Separation Procedures Based on Solubility Differences.

Isoelectric Precipitation. The solubility of most globular proteins is profoundly in­fluenced by the pH of the system. Since different proteins have different isoelectric pH val­ues, because their content of amino acids with ionizable R groups differs, they can often be separated from each other by isoelectric precipitation. When the pH of a protein mix­ture is adjusted to theisoelectric pH of one of its com­ponents, much or that entire component will precipitate, leaving behind in solution proteins with isoelectric pH val­ues above or below that pH. The precipitatedisoelectric protein remains in its native conformation and can be redissolved in a medium having an appropriate pH and salt concentration.

Salting-out of Proteins. A protein may be al­most completely precipitated from solution adding to it neutral salts. This effect is called salting-out. The physicochemical basis of salting-out is rather complex; one factor is that the high concentration of salt may remove water of hydration from the protein mole­cules, thus reducing their solubility.

Solvent Fractionation. The addition of water-miscible neutral organic solvents, par­ticularly ethanol or acetone, decreases the solubility of most globular proteins in water to such an extent that they precip­itate out of solution. Quantitative study of this effect shows that protein solubility at a fixed pH and ionic strength is a function of the dielectric constant of the medium. Since ethanol has a lower dielectric constant than water, its addition to an aqueous protein solution in­creases the attractive force between opposite charges, thus decreasing the degree of ionization of the R groups of the protein. As a result, the protein molecules tend to aggregate and precipitate. Mixtures of proteins can be sepa­rated on the basis of quantitative differences in their solu­bility in cold ethanol-water or acetone-water mixtures. A disadvantage of this method is that since such solvents can denature proteins at higher temperatures, the temperature must be kept rather low.

Effect of Temperature on Solubility of Proteins.

Within a limited range, from about 0 to about 40 °C, most globular proteins increase in solubility with increasing tem­perature, although there are some exceptions. Above 40 to 50 °C, most proteins become increasingly unstable and begin to denature, ordi­narily with a loss of solubility at the neutral pH zone.

Separation Procedures Based on Electric Charge.

Electrophoretic Methods. This method can separate a protein mix­ture on the basis of both electric charge and molecular size. For this purpose, special paper, gels of potato starch orpolyacrylamide are commonly used. By this technique the protein components of blood plasma can be resolved into 15 or more bands.

Ion-Exchange Chromatography. Columns of ion-exchange resins are successfully applied to the separation of protein mixtures. The most com­monly used materials for chromatography of proteins are synthetically prepared derivatives of cellulose. Protein mixtures are resolved and the individual components successively eluted from DEAE-cellulose columns by passing a series of buffers of decreasing pH or a series of salt solutions of increasing ionic strength, which have the effect of de­creasing the binding of anionic proteins. The protein concentration in the eluate, which is collected in small fractions, is estimated op­tically by its capacity to absorb light in the ultraviolet region.

Separation of Proteins by Selective Adsorption.

Proteins can be adsorbed to, and selectively eluted from, columns of finely divided, relatively inert materials with a very large surface area in relation to particle size. They include nonpolarsubstances, e.g., charcoal, and polar sub­stances, e.g., silica gel or alumina. The precise nature of the forces binding the protein to such adsorbents is not known, but presumably van der Waals and hydrophobic interactions prevail with nonpolar adsorbents, whereas ionic attractions and/or hydrogen bonding are the main forces with polar ad­sorbents.

Separations Based on Ligand Specificity: Affinity Chromatography.

This method is based on a biological property of some proteins, namely, their capacity for specific, noncovalent binding of another molecule, called the ligand. For example, some enzymes bind their specific coenzymes very tightly through noncovalent forces. In order to separate such an enzyme from other pro­teins by affinity chromatography, its specific coenzyrne is covalently attached, by means of an appropriate chemical reaction, to a functional group on the surface of large hydrated particles of a porous column material, which otherwise allows protein mole­cules to pass freely. When a mixture of proteins containing the enzyme to be isolated is added to such a column, the enzyme molecule, which is capable of binding tightly and specifically to the immobilized ligand molecule, adheres to the ligand-derivatized agarose particles, whereas all the other proteins, which lack a specific binding site for that particular ligand molecule, will pass through.


Biuret test. The protein is warmed gently with 10 % solution of sodium hydroxide and then а drop of very dilute copper sulphate solution is added, the formation of reddish - violet colour indicates the presence of peptide link, – СО – NH – . The test is given by all proteins, peptones and peptides. Its name is derived from the fact that the test is also positive for the compound biuret, Н2N –CONH – CONH2 obtained from urea by heating.

It should be noted that dipeptides do not give the biuret test, while all other polypeptides do so. Hence biuret test is important to know whether hydrolysis of proteins is complete or not. If the biuret test is negative, hydrolysis is complete, at least to the dipeptide stage.

Xanthoproteic test. On treatment with concentrated nitric acid, certain proteins give yellow colour. This yellow colour is the same that is formed on the skin when the latter comes in contact with the concentrated nitric acid. The test is given only by the proteins having at least one mole of aromatic amino acid, such as tryptophan, phenylalanine, and tyrosine which are actually nitrated during treatment with concentrated nitric acid.

Millon's test. Protein on adding Millon's reagent (а solution of mercuric and mercurous nitrates in nitric acid containing а little nitrous acid) followed by heating the solution give а red precipitate or colour. The test is responded by the proteins having tyrosine. The hydroxyphenyl group of tyrosine is the structure responsible for this test. Moreover, the non-proteinous material having phenolic group also responds the test.

Foll reaction. This reaction reveals the sulfur containing amino acids (cysteine, cystine). Treatment of the sulfur containing amino acids with salt of lead and alkali yields a black sediment.

Adamkevich reaction. This reaction detects the amino acid tryptophan containing indol ring. The addition of the concentrated acetic and sulfuric acids to the solution of tryptophan results in the formation of red-violet ring appearing on the boundary of different liquids.

Ninhydrin test. The ninhydrin colour reaction is the most commonly test used for the detection of amino acids. This is an extremely delicate test, to which proteins, their hydrolytic products, and α-amino acids react. Although the test is positive for all free amino groups in amino acids, peptides, or proteins, the test is much weaker for peptides or proteins because not as many free groups are available as in amino acids. For certain amino acids the test is positive in dilutions as high as 1 part in 100,000 parts of water.

When ninhydrin is added to а protein solution and the mixture is heated to boil, blue to violet colour appears on cooling. The colour is due to the formation of а complex compound.

The test is also given by ammonia, ammonium salts, and certain amines. Ninhydrin is also used as а reagent for the quantitative determination of free carboxyl groups in solutions of amino acids.

Nitroprusside test. Proteins containing free -SH groups (of cysteine) give а reddish colour with sodium nitroprusside in ammonical solution.

Proteins are polypeptides that contain more than 50 amino acid units. The dividing line between а polypeptide and а protein is arbitrary. The important point is that proteins are polymers containing а large number of amino acid units linked by peptide bonds. Polypeptides are shorter chains of amino acids. Some proteins have molecular masses in the millions. Some proteins also contain more than one polypeptide chain.

To aid us in describing protein structure, we will consider four levels of substructure: primary, secondary, tertiary, and quaternary. Even though we consider these structure levels one by one, remember that it is the combination of all four levels of structure that controls protein function.

Biochemistry – subject and main tasks. Investigation of structure and physical-chemical properties of proteins-enzymes. Mechanism of enzyme action, kinetic of enzymatic catalysis, role of cofactors and coenzyme vitamins in catalytic action of enzymes.

Biochemistry is the study of the chemical processes and transformations in living organisms. It deals with the structure and function of cellular components, such as proteins, carbohydrates, lipids, nucleic acids, and other biomolecules. Chemical biology aims to answer many questions arising from biochemistry by using tools developed within synthetic chemistry.

This article only discusses terrestrial biochemistry (carbon- and water-based), as all the life forms we know are on Earth. Since life forms alive today are hypothesized by most to have descended from the same common ancestor, they would naturally have similar biochemistries, even for matters that seem to be essentially arbitrary, such as handedness of various biomolecules. It is unknown whether alternative biochemistries are possible or practical.

History of biochemistry 

In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms. He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells."

In 1878 German physiologist Wilhelm  Kühne (1837–1900) coined the term enzyme, which comes from Greek ενζυμον "in leaven", to describe this process. The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment used to refer to chemical activity produced by living organisms.

In 1897 Eduard Buchner began to study the ability of yeast extracts to ferment sugar despite the absence of living yeast cells. In a series of experiments at the University of Berlin, he found that the sugar was fermented even when there were no living yeast cells in the mixture. He named the enzyme that brought about the fermentation of sucrose "zymase". In 1907 he received the Nobel Prize in Chemistry "for his biochemical research and his discovery of cell-free fermentation". Following Buchner's example; enzymes are usually named according to the reaction they carry out. Typically the suffix -ase is added to the name of the substrate (e.g., lactase is the enzyme that cleaves lactose) or the type of reaction (e.g., DNA polymerase forms DNA polymers).

Having shown that enzymes could function outside a living cell, the next step was to determine their biochemical nature. Many early workers noted that enzymatic activity was associated with proteins, but several scientists (such as Nobel laureate Richard Willstätter) argued that proteins were merely carriers for the true enzymes and that proteins per se were incapable of catalysis. However, in 1926, James B. Sumner showed that the enzyme urease was a pure protein and crystallized it; Sumner did likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes was definitively proved by Northrop and Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel Prize in Chemistry.

This discovery that enzymes could be crystallized eventually allowed their structures to be solved by x-ray crystallography. This was first done for lysozyme, an enzyme found in tears, saliva and egg whites that digests the coating of some bacteria; the structure was solved by a group led by David Chilton Phillips and published in 1965. This high-resolution structure of lysozyme marked the beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail.

Today, the findings of biochemistry are used in many areas, from genetics to molecular biology and from agriculture to medicine.

Characteristics of Enzymes

Enzymes are proteins that catalyze (i.e. accelerate) chemical reactions. In these reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. Almost all processes in a biological cell need enzymes in order to occur at significant rates. Since enzymes are extremely selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.

Like all catalysts, enzymes work by lowering the activation energy (ΔG‡) for a reaction, thus dramatically accelerating the rate of the reaction. Most enzyme reaction rates are millions of times faster than those of comparable uncatalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific. Enzymes are known to catalyze about 4,000 biochemical reactions. Not all biochemical catalysts are proteins, since some RNA molecules called ribozymes also catalyze reactions.


Enzymes are protein molecules that are tailored to recognize and bind specific reactants and speed their conversion into products. These proteins are responsible for increasing the rates of all of the many thousand of reaction taking place inside cells.

All enzymatic proteins have several characteristics in common table 1.

Table 1: Characteristics of enzymes proteins



Enzymes combine briefly with reactants during an enzyme-catalyzed reaction.


Enzymes are released unchanged after catalyzing the conversion of reactants to Product


Enzymes are specific in their activity; each enzyme catalyzes the reaction of a single type of molecules or a group of closely related molecules.


Enzymes are saturated by high substrate concentrations.


Many enzymes contain nonproteins groups called cofactors, which contribute to their activity. Inorganic cofactors are all metallic ions. Organic cofactors, called coenzymes, are complex groups derived from vitamins.


Many enzymes are pH and temperature sensitive

The rate of combination and release, known as the turnover number, lies near 1000 per second for most enzymes. Some enzymes have turnover numbers as small as 100 per second or as large as 10 million per second. As a result of enzyme turnover, a relatively small number of enzyme molecules can catalyze a large number of reactant molecules.

The part of an enzyme that combines with substrate molecule is the active site. In most enzymes the active site is located in a cavity or pocket on the enzyme surface, frequently within a cleft marking the boundary between two or more major domains. Within the cleft or pocket, amino acid side groups are situated to fit and bind parts of substrate molecules that are critical to the reaction catalyzed by the enzyme. The active site also separates substrate molecules from the surrounding solutions and place them in environments with unique characteristics, including partial or complete exclusion of water.

How Enzymes Lower the Energy of Activation

The mechanisms by which enzymes lower the energy of activation are still not totally understood. However, the mechanisms are believed to be directly or indirectly related to achievement of what is known as the transition state for a reaction. During any chemical interaction the reactants briefly enter a state in which old chemical bonds are incompletely broken and new ones are incompletely formed. In this transition state electron orbital assume intermediate positions between their locations in the reactants and their positions in the products. The transition state is highly unstable and can easily move in either direction with little change in energy - forward toward products ore back ward toward reactants. In effect, achievement of the transition state places a reacting system in a poised and precariously balanced position at the top of the activation energy barrier.

For example, in the transfer of a phosphate group from one molecule to another, a transition state is set up in which both molecules (shown as X and Y in Figure 2) link to the phosphate group a fraction of a second via transitory bonds (dotted lines). This unstable state can change readily in the direction of either products or unchanged reactants.

Enzymes as Biological Catalysts

In cells and organisms most reactions are catalyzed by enzymes, which are regenerated during the course of a reaction. These biological catalysts are physiologically important because they speed up the rates of reactions that would otherwise be too slow to support life. Enzymes increase reaction rates--- sometimes by as much as one millionfold, but more typically by about one thousand fold. Catalysts speed up the forward and reverse reactions proportionately so that, although the magnitude of the rate constants of the forward and reverse reactions is are increased, the ratio of the rate constants remains the same in the presence or absence of enzyme. Since the equilibrium constant is equal to a ratio of rate constants, it is apparent that enzymes and other catalysts have no effect on the equilibrium constant of the reactions they catalyze.

Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity; activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, pH, and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins, making the meat easier to chew).



Most enzymes are much larger than the substrates they act on, and only a very small portion of the enzyme (around 3–4 amino acids) is directly involved in catalysis.


The region that contains these catalytic residues, binds the substrate, and then carries out the reaction is known as the active site. Enzymes can also contain sites that bind cofactors, which are needed for catalysis. Some enzymes also have binding sites for small molecules, which are often direct or indirect products or substrates of the reaction catalyzed.

Ribbon-diagram showing carbonic anhydrase II. The grey sphere is the zinc cofactor in the active site. Diagram drawn from PDB 1MOO.The activities of enzymes are determined by their three-dimensional structure. 

This binding can serve to increase or decrease the enzyme's activity, providing a means for feedback regulation. Enzymes are catalysts. Most are proteins. (A few ribonucleoprotein enzymes have been discovered and, for some of these, the catalytic activity is in the RNA part rather than the protein part. Link to discussion of these ribozymes.)

Enzymes bind temporarily to one or more of the reactants of the reaction they catalyze. In doing so, they lower the amount of activation energy needed and thus speed up the reaction.


·   Catalase. It catalyzes the decomposition of hydrogen peroxide into water and oxygen.

2H2O2 -> 2H2O + O2

One molecule of catalase can break 40 million molecules of hydrogen peroxide each second.

·   Carbonic anhydrase. It is found in red blood cells where it catalyzes the reaction

CO2 + H2O <-> H2CO3

It enables red blood cells to transport carbon dioxide from the tissues to the lungs. One molecule of carbonic anhydrase can process one million molecules of CO2 each second.

Like all proteins, enzymes are made as long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a unique structure, which has unique properties. Individual protein chains may sometimes group together to form a protein complex. Most enzymes can be denatured—that is, unfolded and inactivated—by heating, which destroys the three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible.


Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Enzymes can also show impressive levels of stereospecificity, regioselectivity and chemoselectivity.


Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyses a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases. Similar proofreading mechanisms are also found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes.

Some enzymes that produce secondary metabolites are described as promiscuous, as they can act on a relatively broad range of different substrates. It has been suggested that this broad substrate specificity is important for the evolution of new biosynthetic pathways.

 "Lock and key" model

Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve.

Induced fit model

 Diagrams to show the induced fit hypothesis of enzyme action.In 1958 Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site can be reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid active site, the amino acid side chains which make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site.

Providing an alternative pathway (e.g. temporarily reacting with the substrate to form an intermediate ES Complex which would be impossible in the absence of the enzyme).

Reducing the reaction entropy change by bringing substrates together in the correct orientation to react. Considering ΔH‡ alone overlooks this effect.

  Dynamics and function

Recent investigations have provided new insights into the connection between internal dynamics of enzymes and their mechanism of catalysis. An enzyme's internal dynamics are described as the movement of internal parts (e.g. amino acids, a group of amino acids, a loop region, an alpha helix, neighboring beta-sheets or even entire domain) of these biomolecules, which can occur at various time-scales ranging from femtoseconds to seconds. Networks of protein residues throughout an enzyme's structure can contribute to catalysis through dynamic motions. Protein motions are vital to many enzymes, but whether small and fast vibrations or larger and slower conformational movements are more important depends on the type of reaction involved. These new insights also have implications in understanding allosteric effects, producing designer enzymes and developing new drugs.



Cofactors and coenzymes

Role of Coenzymes

The functional role of coenzymes is to act as transporters of chemical groups from one reactant to another. The chemical groups carried can be as simple as the hydride ion (H+ + 2e-) carried by NAD or the mole of hydrogen carried by FAD; or they can be even more complex than the amine (-NH2) carried by pyridoxal phosphate.

Since coenzymes are chemically changed as a consequence of enzyme action, it is often useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different holoenzymes. In all cases, the coenzymes donate the carried chemical grouping to an acceptor molecule and are thus regenerated to their original form. This regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme as a chemical catalyst, since (unlike the usual substrates, which are used up during the course of a reaction) coenzymes are generally regenerated.

 Enzyme Relative to Substrate Type

Enzymes also are generally specific for a particular steric configuration (optical isomer) of a substrate. Enzymes that attack D sugars will not attack the corresponding L isomer. Enzymes that act on L amino acids will not employ the corresponding D optical isomer as a substrate. The enzymes known as racemases provide a striking exception to these generalities; in fact, the role of racemases is to convert D isomers to L isomers and vice versa. Thus racemases attack both D and L forms of their substrate.

Enzyme cofactors

Many enzymes require the presence of an additional, nonprotein, cofactor.

·   Some of these are metal ions such as Zn2+ (the cofactor for carbonic anhydrase), Cu2+, Mn2+, K+, and Na+.

·   Some cofactors are small organic molecules called coenzymes. The B vitamins

o                          thiamine (B1)

o                          riboflavin (B2) and

o                          nicotinamide

are precursors of coenzymes.

Coenzymes may be covalently bound to the protein part (called the apoenzyme) of enzymes as a prosthetic group. Others bind more loosely and, in fact, may bind only transiently to the enzyme as it performs its catalytic act.

LysozymeActionLysozyme: a model of enzyme action

A number of lysozymes are found in nature; in human tears and egg white, for examples. The enzyme is antibacterial because it degrades the polysaccharide that is found in the cell walls of many bacteria. It does this by catalyzing the insertion of a water molecule at the position indicated by the red arrow. This hydrolysis breaks the chain at that point.

The bacterial polysaccharide consists of long chains of alternating amino sugars:

·   N-acetylglucosamine (NAG)

·   N-acetylmuramic acid (NAM)

These hexose units resemble glucose except for the presence of the side chains containing amino groups.
Lysozyme is a globular protein with a deep cleft across part of its surface. Six hexoses of the substrate fit into this cleft.

·   With so many oxygen atoms in sugars, as many as 14 hydrogen bonds form between the six amino sugars and certain amino acid R groups such as Arg-114, Asn-37, Asn-44, Trp-62, Trp-63, and Asp-101.

·   Some hydrogen bonds also form with the C=O groups of several peptide bonds.

·   In addition, hydrophobic interactions may help hold the substrate in position.

As for lysozyme itself, binding of the substrate induces a small (~0.75Å) movement of certain amino acid residues so the cleft closes slightly over its substrate. So the "lock" as well as the "key" changes shape as the two are brought together. (This is sometimes called "induced fit".)

The amino acid residues in the vicinity of rings 4 and 5 provide a plausible mechanism for completing the catalytic act. Residue 35, glutamic acid (Glu-35), is about 3Å from the -O- bridge that is to be broken. The free carboxyl group of glutamic acid is a hydrogen ion donor and available to transfer H+ to the oxygen atom. This would break the already-strained bond between the oxygen atom and the carbon atom of ring 4.

Now having lost an electron, the carbon atom acquires a positive charge. Ionized carbon is normally very unstable, but the attraction of the negatively-charged carboxyl ion of Asp-52 could stabilize it long enough for an -OH ion (from a spontaneously dissociated water molecule) to unite with the carbon. Even at pH 7, water spontaneously dissociates to produce H+ and OH- ions. The hydrogen ion (H+) left over can replace that lost by Glu-35.

In either case, the chain is broken, the two fragments separate from the enzyme, and the enzyme is free to attach to a new location on the bacterial cell wall and continue its work of digesting it.


Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules to be bound for activity. Cofactors can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds, (e.g., flavin and heme). Organic cofactors (coenzymes) are usually prosthetic groups, which are tightly bound to the enzymes that they assist. These tightly-bound cofactors are distinguished from other coenzymes, such as NADH, since they are not released from the active site during the reaction.

 An example of an enzyme that contains a cofactor is carbonic anhydrase, and is shown in the ribbon diagram above with a zinc cofactor bound in its active site. These tightly-bound molecules are usually found in the active site and are involved in catalysis. For example, flavin and heme cofactors are often involved in redox reactions.

 Enzymes that require a cofactor but do not have one bound are called apoenzymes. An apoenzyme together with its cofactor(s) is called a holoenzyme (i.e., the active form). Most cofactors are not covalently attached to an enzyme, but are very tightly bound. However, organic prosthetic groups can be covalently bound (e.g., thiamine pyrophosphate in the enzyme pyruvate dehydrogenase).


 Space-filling model of the coenzyme NADH Coenzymes are small molecules that transport chemical groups from one enzyme to another. Some of these chemicals such as riboflavin, thiamine and folic acid are vitamins, this is when these compounds cannot be made in the body and must be acquired from the diet. The chemical groups carried include the hydride ion (H-) carried by NAD or NADP+, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosylmethionine.

 Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, about 700 enzymes are known to use the coenzyme NADH.



Coenzymes are usually regenerated and their concentrations maintained at a steady level inside the cell: for example, NADPH is regenerated through the pentose phosphate pathway and S-adenosylmethionine by methionine adenosyltransferase.

Factors Affecting Enzyme Action

The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH (left graph) and temperature (right graph), its activity decreasing at values above and below that point. This is not surprising considering the importance of

·   tertiary structure (i.e. shape) in enzyme function and

·   noncovalent forces, e.g., ionic interactions and hydrogen bonds, in determining that shape.


·   the protease pepsin works best as a pH of 1-2 (found in the stomach) while

·   the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small intestine as the bicarbonate of the pancreatic fluid neutralizes the arriving stomach contents).

Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Without the unionized -COOH group of Glu-35 and the ionized -COO- of Asp-52, the catalytic action of lysozyme would cease.



Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve (red arrow in right-hand graph above) reflects the general effect of increasing temperature on the rate of chemical reactions (graph at left). The descending portion of the curve above (blue arrow) reflects the loss of catalytic activity as the enzyme molecules become denatured at high temperatures.


EnzymePathControlRegulation of Enzyme Activity

Several mechanisms work to make enzyme activity within the cell efficient and well-coordinated.


 Anchoring enzymes in membranes

Many enzymes are inserted into cell membranes, for examples,

·   the plasma membrane

·   the membranes of mitochondria and chloroplasts

·   the endoplasmic reticulum

·   the nuclear envelope

These are locked into spatial relationships that enable them to interact efficiently.

Inactive precursors

Enzymes, such as proteases, that can attack the cell itself are inhibited while within the cell that synthesizes them. For example, pepsin is synthesized within the chief cells (in gastric glands) as an inactive precursor, pepsinogen. Only when exposed to the low pH outside the cell is the inhibiting portion of the molecule removed and active pepsin produced.

Feedback Inhibition


 If the product of a series of enzymatic reactions, e.g., an amino acid, begins to accumulate within the cell, it may specifically inhibit the action of the first enzyme involved in its synthesis (red bar). Thus further production of the enzyme is halted.

Precursor Activation

The accumulation of a substance within a cell may specifically activate (blue arrow) an enzyme that sets in motion a sequence of reactions for which that substance is the initial substrate. This reduces the concentration of the initial substrate.

In the case if feedback inhibition and precursor activation, the activity of the enzyme is being regulated by a molecule which is not its substrate. In these cases, the regulator molecule binds to the enzyme at a different site than the one to which the substrate binds. When the regulator binds to its site, it alters the shape of the enzyme so that its activity is changed. This is called an allosteric effect.

·   In feedback inhibition, the allosteric effect lowers the affinity of the enzyme for its substrate.

·   In precursor activation, the regulator molecule increases the affinity of the enzyme in the series for its substrate.If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down.

Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase - by transcribing and translating the necessary gene(s) - and so can begin to metabolize the sugar.

Е. coli also has a mechanism which regulates enzyme synthesis by controlling translation of a needed messenger RNA..

Factors Affecting Enzymes


Main aticles: Activation energy, Thermodynamic equilibrium, and Chemical equilibrium.

Diagram of a catalytic reaction, showing the energy niveau at each stage of the reaction. The substrates usually need a large amount of energy to reach the transition state, which then decays into the end product. The enzyme stabilizes the transition state, reducing the energy needed to form this species and thus reducing the energy required to form products.As all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. Usually, in the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly. However, in the absence of the enzyme, other possible uncatalyzed, "spontaneous" reactions might lead to different products, because in those conditions this different product is formed faster.

Furthermore, enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a thermodynamically unfavorable one. For example, the hydrolysis of ATP is often used to drive other chemical reactions.

Enzymes catalyze the forward and backward reactions equally. They do not alter the equilibrium itself, but only the speed at which it is reached. For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its reactants.

 (in tissues; high CO2 concentration)

 (in lungs; low CO2 concentration)

Nevertheless, if the equilibrium is greatly displaced in one direction, that is, in a very exergonic reaction, the reaction is effectively irreversible. Under these conditions the enzyme will, in fact, only catalyze the reaction in the thermodynamically allowed direction.


 Mechanism for a single substrate enzyme catalyzed reaction. The enzyme (E) binds a substrate (S) and produces a product (P).Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are obtained from enzyme assays. In 1913 Leonor Michaelis and Maud Menten proposed a quantitative theory of enzyme kinetics, which is referred to as Michaelis-Menten kinetics. Their work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely used today.

 Michaelis-Menton Kinetics

In typical enzyme-catalyzed reactions, reactant and product concentrations are usually hundreds or thousands of times greater than the enzyme concentration. Consequently, each enzyme molecule catalyzes the conversion to product of many reactant molecules. In biochemical reactions, reactants are commonly known as substrates. The catalytic event that converts substrate to product involves the formation of a transition state, and it occurs most easily at a specific binding site on the enzyme. This site, called the catalytic site of the enzyme, has been evolutionarily structured to provide specific, high-affinity binding of substrate(s) and to provide an environment that favors the catalytic events. The complex that forms, when substrate(s) and enzyme combine, is called the enzyme substrate (ES) complex. Reaction products arise when the ES complex breaks down releasing free enzyme.

 Between the binding of substrate to enzyme, and the reappearance of free enzyme and product, a series of complex events must take place. At a minimum an ES complex must be formed; this complex must pass to the transition state (ES*); and the transition state complex must advance to an enzyme product complex (EP). The latter is finally competent to dissociate to product and free enzyme. The series of events can be shown thus:

E + S <---> ES <---> ES* <---> EP <---> E + P

The kinetics of simple reactions like that above were first characterized by biochemists Michaelis and Menten. The concepts underlying their analysis of enzyme kinetics continue to provide the cornerstone for understanding metabolism today, and for the development and clinical use of drugs aimed at selectively altering rate constants and interfering with the progress of disease states. The Michaelis-Menten equation is a quantitative description of the relationship among the rate of an enzyme- catalyzed reaction [v1], the concentration of substrate [S] and two constants, Vmax and Km (which are set by the particular equation). The symbols used in the Michaelis-Menton equation refer to the reaction rate [v1], maximum reaction rate (Vmax), substrate concentration [S] and the Michaelis-Menton constant (Km).

  The Michaelis-Menten equation can be used to demonstrate that at the substrate concentration that produces exactly half of the maximum reaction rate, i.e.,1/2 Vmax, the substrate concentration is numerically equal to Km. This fact provides a simple yet powerful bioanalytical tool that has been used to characterize both normal and altered enzymes, such as those that produce the symptoms of genetic diseases. Rearranging the Michaelis-Menton equation leads to:

From this equation it should be apparent that when the substrate concentration is half that required to support the maximum rate of reaction, the observed rate, v1, will, be equal to Vmax divided by 2; in other words, v1 = [Vmax/2]. At this substrate concentration Vmax/v1 will be exactly equal to 2, with the result that:

[S](1) = Km

The latter is an algebraic statement of the fact that, for enzymes of the Michaelis-Menten type, when the observed reaction rate is half of the maximum possible reaction rate, the substrate concentration is numerically equal to the Michaelis-Menten constant. In this derivation, the units of Km are those used to specify the concentration of S, usually Molarity.

 The Michaelis-Menten equation has the same form as the equation for a rectangular hyperbola; graphical analysis of reaction rate (v) versus substrate concentration [S] produces a hyperbolic rate plot.

  Plot of substrate concentration versus reaction velocity


 The key features of the plot are marked by points A, B and C. At high substrate concentrations the rate represented by point C the rate of the reaction is almost equal to Vmax, and the difference in rate at nearby concentrations of substrate is almost negligible. If the Michaelis-Menten plot is extrapolated to infinitely high substrate concentrations, the extrapolated rate is equal to Vmax. When the reaction rate becomes independent of substrate concentration, or nearly so, the rate is said to be zero order. (Note that the reaction is zero order only with respect to this substrate. If the reaction has two substrates, it may or may not be zero order with respect to the second substrate). The very small differences in reaction velocity at substrate concentrations around point C (near Vmax) reflect the fact that at these concentrations almost all of the enzyme molecules are bound to substrate and the rate is virtually independent of substrate, hence zero order. At lower substrate concentrations, such as at points A and B, the lower reaction velocities indicate that at any moment only a portion of the enzyme molecules are bound to the substrate. In fact, at the substrate concentration denoted by point B, exactly half the enzyme molecules are in an ES complex at any instant and the rate is exactly one half of Vmax. At substrate concentrations near point A the rate appears to be directly proportional to substrate concentration, and the reaction rate is said to be first order.

 Inhibition of Enzyme Catalyzed Reactions

 To avoid dealing with curvilinear plots of enzyme catalyzed reactions, biochemists Lineweaver and Burk introduced an analysis of enzyme kinetics based on the following rearrangement of the Michaelis-Menten equation:

[1/v] = [Km (1)/ Vmax[S] + (1)/Vmax]

 Plots of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax and an intercept on the ordinate at 1/Vmax.


A Lineweaver-Burk Plot

 An alternative linear transformation of the Michaelis-Menten equation is the Eadie-Hofstee transformation:

v/[S] = -v [1/Km] + [Vmax/Km]

 and when v/[S] is plotted on the y-axis versus v on the x-axis, the result is a linear plot with a slope of -1/Km and the value Vmax/Km as the intercept on the y-axis and Vmax as the intercept on the x-axis.

Both the Lineweaver-Burk and Eadie-Hofstee transformation of the Michaelis-Menton equation are useful in the analysis of enzyme inhibition. Since most clinical drug therapy is based on inhibiting the activity of enzymes, analysis of enzyme reactions using the tools described above has been fundamental to the modern design of pharmaceuticals. Well- known examples of such therapy include the use of methotrexate in cancer chemotherapy to semi-selectively inhibit DNA synthesis of malignant cells, the use of aspirin to inhibit the synthesis of prostaglandins which are at least partly responsible for the aches and pains of arthritis, and the use of sulfa drugs to inhibit the folic acid synthesis that is essential for the metabolism and growth of disease-causing bacteria. In addition, many poisons, such as cyanide, carbon monoxide and polychlorinated biphenols (PCBs). produce their life- threatening effects by means of enzyme inhibition.

 The major contribution of Michaelis and Menten was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis-Menten complex in their honor. The enzyme then catalyzes the chemical step in the reaction and releases the product.

Saturation curve for an enzyme reaction showing the relation between the substrate concentration (S) and rate (v).Enzymes can catalyze up to several million reactions per second. For example, the reaction catalyzed by orotidine 5'-phosphate decarboxylase will consume half of its substrate in 78 million years if no enzyme is present. However, when the decarboxylase is added, the same process takes just 25 milliseconds. Enzyme rates depend on solution conditions and substrate concentration. Conditions that denature the protein abolish enzyme activity, such as high temperatures, extremes of pH or high salt concentrations, while raising substrate concentration tends to increase activity. To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen. This is shown in the saturation curve, shown on the right. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form. At the maximum velocity (Vmax) of the enzyme, all enzyme active sites are saturated with substrate, and the amount of ES complex is the same as the total amount of enzyme.


Inhibitor Type

Binding Site on Enzyme

Kinetic effect

Competitive Inhibitor

Specifically at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate.

Vmax is unchanged; Km, as defined by [S] required for 1/2 maximal activity, is increased.

Noncompetitive Inhibitor

Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate.

Km appears unaltered; Vmax is decreased proportionately to inhibitor concentration.

Uncompetitive Inhibitor

Binds only to ES complexes at locations other than the catalytic site. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate.

Apparent Vmax decreased; Km, as defined by [S] required for 1/2 maximal activity, is decreased.


Inhibitor Type

Specifically at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate.

 Vmax is unchanged; Km, as defined by [S] required for 1/2 maximal activity, is increased.

Noncompetitive Inhibitor

 Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate.

 Km appears unaltered; Vmax is decreased proportionately to inhibitor concentration.

Uncompetitive Inhibitor

 Binds only to ES complexes at locations other than the catalytic site. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate.

 Apparent Vmax decreased; Km, as defined by [S] required for 1/2 maximal activity, is decreased.

 The hallmark of all the reversible inhibitors is that when the inhibitor concentration drops, enzyme activity is regenerated. Usually these inhibitors bind to enzymes by non-covalent forces and the inhibitor maintains a reversible equilibrium with the enzyme. The equilibrium constant for the dissociation of enzyme inhibitor complexes is known as KI:

KI = [E][I]/[E--I--complex]

The importance of KI is that in all enzyme reactions where substrate, inhibitor and enzyme interact, the normal Km and or Vmax for substrate enzyme interaction appear to be altered. These changes are a consequence of the influence of KI on the overall rate equation for the reaction. The effects of KI are best observed in Lineweaver-Burk plots.

Probably the best known reversible inhibitors are competitive inhibitors, which always bind at the catalytic or active site of the enzyme. Most drugs that alter enzyme activity are of this type. Competitive inhibitors are especially attractive as clinical modulators of enzyme activity because they offer two routes for the reversal of enzyme inhibition, while other reversible inhibitors offer only one. First, as with all kinds of reversible inhibitors, a decreasing concentration of the inhibitor reverses the equilibrium regenerating active free enzyme. Second, since substrate and competitive inhibitors both bind at the same site they compete with one another for binding .Raising the concentration of substrate (S), while holding the concentration of inhibitor constant, provides the second route for reversal of competitive inhibition. The greater the proportion of substrate, the greater the proportion of enzyme present in competent ES complexes.

As noted earlier, high concentrations of substrate can displace virtually all competitive inhibitor bound to active sites. Thus, it is apparent that Vmax should be unchanged by competitive inhibitors. This characteristic of competitive inhibitors is reflected in the identical vertical-axis intercepts of Lineweaver-Burk plots, with and without inhibitor.

Since attaining Vmax requires appreciably higher substrate concentrations in the presence of competitive inhibitor, Km (the substrate concentration at half maximal velocity) is also higher, as demonstrated by the differing negative intercepts on the horizontal axis in panel B.

 Analogously, panel C illustrates that noncompetitive inhibitors appear to have no effect on the intercept at the x-axis implying that noncompetitive inhibitors have no effect on the Km of the enzymes they inhibit. Since noncompetitive inhibitors do not interfere in the equilibration of enzyme, substrate and ES complexes, the Km's of Michaelis-Menten type enzymes are not expected to be affected by noncompetitive inhibitors, as demonstrated by x-axis intercepts in panel C. However, because complexes that contain inhibitor (ESI) are incapable of progressing to reaction products, the effect of a noncompetitive inhibitor is to reduce the concentration of ES complexes that can advance to product. Since Vmax = k2[Etotal], and the concentration of competent Etotal is diminished by the amount of ESI formed, noncompetitive inhibitors are expected to decrease Vmax, as illustrated by the y-axis intercepts in panel C.

 A corresponding analysis of uncompetitive inhibition leads to the expectation that these inhibitors should change the apparent values of Km as well as Vmax. Changing both constants leads to double reciprocal plots, in which intercepts on the x and y axes are proportionately changed; this leads to the production of parallel lines in inhibited and uninhibited reactions.


Competitive inhibitors bind reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor. Substrate and inhibitor compete for the enzyme.Main article: Enzyme inhibitor

Enzyme reaction rates can be decreased by various types of enzyme inhibitors.

Reversible inhibitors

In competitive inhibition the inhibitor binds to the substrate binding site (figure right, top, thus preventing substrate from binding (EI complex). Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of folic acid and this drug are shown in the figure to the right bottom.

Non-competitive inhibition

In order to do its work, an enzyme must unite - even if ever so briefly - with at least one of the reactants. In most cases, the forces that hold the enzyme and its substrate are noncovalent, an assortment of:

·   hydrogen bonds

·   ionic interactions and hydrophobic interactions


Most of these interactions are weak and especially so if the atoms involved are farther than about one angstrom from each other. So successful binding of enzyme and substrate requires that the two molecules be able to approach each other closely over a fairly broad surface. Thus the analogy that a substrate molecule binds its enzyme like a key in a lock.




This requirement for complementarity in the configuration of substrate and enzyme explains the remarkable specificity of most enzymes. Generally, a given enzyme is able to catalyze only a single chemical reaction or, at most, a few reactions involving substrates sharing the same general structure.

The necessity for a close, if brief, fit between enzyme and substrate explains the phenomenon of competitive inhibition.

It catalyzes the oxidation (by the removal of two hydrogen atoms) of succinic acid (a). If one adds malonic acid to cells, or to a test tube mixture of succinic acid and the enzyme, the action of the enzyme is strongly inhibited. This is because the structure of malonic acid allows it to bind to the same site on the enzyme (b). But there is no oxidation so no speedy release of products. The inhibition is called competitive because if you increase the ratio of succinic to malonic acid in the mixture, you will gradually restore the rate of catalysis. At a 50:1 ratio, the two molecules compete on roughly equal terms for the binding (=catalytic) site on the enzyme.

Non-competitive inhibitors can bind either to the active site, or to other parts of the enzyme far away from the substrate-binding site. Moreover, non-competitive inhibitors bind to the enzyme-substrate (ES) complex and to the free enzyme. Their binding to this site changes the shape of the enzyme and stops the active site binding substrate(s). Consequently, since there is no direct competition between the substrate and inhibitor for the enzyme, the extent of inhibition depends only on the inhibitor concentration and will not be affected by the substrate concentration.

Control activity

There are five main ways that enzyme activity is controlled in the cell.

Regulation of Enzyme Activity

While it is clear that enzymes are responsible for the catalysis of almost all biochemical reactions, it is important to also recognize that rarely, if ever, do enzymatic reactions proceed in isolation. The most common scenario is that enzymes catalyze individual steps of multi-step metabolic pathways, as is the case with glycolysis, gluconeogenesis or the synthesis of fatty acids. As a consequence of these lock- step sequences of reactions, any given enzyme is dependent on the activity of preceding reaction steps for its substrate.

In humans, substrate concentration is dependent on food supply and is not usually a physiologically important mechanism for the routine regulation of enzyme activity. Enzyme concentration, by contrast, is continually modulated in response to physiological needs. Three principal mechanisms are known to regulate the concentration of active enzyme in tissues:

 1. Regulation of gene expression controls the quantity and rate of enzyme synthesis.

  2. Proteolytic enzyme activity determines the rate of enzyme degradation.

3. Covalent modification of preexisting pools of inactive proenzymes produces active enzymes.

Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished by a cell in response to changes in the cell's environment. This form of gene regulation is called enzyme induction and inhibition. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam ring within the penicillin molecule. Another example are enzymes in the liver called cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of these enzymes can cause drug interactions.

Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular compartments. For example, fatty acids are synthesized by one set of enzymes in the cytosol, endoplasmic reticulum and the Golgi apparatus and used by a different set of enzymes as a source of energy in the mitochondrion, through β-oxidation.

Enzymes can be regulated by inhibitors and activators. For example, the end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because the amount of the end product produced is regulated by its own concentration. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps allocate materials and energy economically, and prevents the manufacture of excess end products. Like other homeostatic devices, the control of enzymatic action helps to maintain a stable internal environment in living organisms.

Enzymes can be regulated through post-translational modification. This can include phosphorylation, myristoylation and glycosylation. For example, in the response to insulin, the phosphorylation of multiple enzymes, including glycogen synthase, helps control the synthesis or degradation of glycogen and allows the cell to respond to changes in blood sugar. Another example of post-translational modification is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut. This type of inactive precursor to an enzyme is known as a zymogen.

Some enzymes may become activated when localized to a different environment (eg. from a reducing (cytoplasm) to an oxidising (periplasm) environment, high pH to low pH etc). For example, hemagglutinin of the influenza virus undergoes a conformational change once it encounters the acidic environment of the host cell vesicle causing its activation.

Allosteric modulation

Allosteric enzymes change their structure in response to binding of effectors. Modulation can be direct, where the effector binds directly to binding sites in the enzyme, or indirect, where the effector binds to other proteins or protein subunits that interact with the allosteric enzyme and thus influence catalytic activity.

In addition to simple enzymes that interact only with substrates and inhibitors, there is a class of enzymes that bind small, physiologically important molecules and modulate activity in ways other than those described above. These are known as allosteric enzymes; the small regulatory molecules to which they bind are known as effectors. Allosteric effectors bring about catalytic modification by binding to the enzyme at distinct allosteric sites, well removed from the catalytic site, and causing conformational changes that are transmitted through the bulk of the protein to the catalytically active site(s).

The hallmark of effectors is that when they bind to enzymes, they alter the catalytic properties of an enzyme's active site. Those that increase catalytic activity are known as positive effectors. Effectors that reduce or inhibit catalytic activity are negative effectors.

Most allosteric enzymes are oligomeric (consisting of multiple subunits); generally they are located at or near branch points in metabolic pathways, where they are influential in directing substrates along one or another of the available metabolic paths. The effectors that modulate the activity of these allosteric enzymes are of two types. Those activating and inhibiting effectors that bind at allosteric sites are called heterotropic effectors. (Thus there exist both positive and negative heterotropic effectors.) These effectors can assume a vast diversity of chemical forms, ranging from simple inorganic molecules to complex nucleotides such as cyclic adenosine monophosphate (cAMP). Their single defining feature is that they are not identical to the substrate.

In many cases the substrate itself induces distant allosteric effects when it binds to the catalytic site. Substrates acting as effectors are said to be homotropic effectors. When the substrate is the effector, it can act as such, either by binding to the substrate-binding site, or to an allosteric effector site. When the substrate binds to the catalytic site it transmits an activity-modulating effect to other subunits of the molecule. Often used as the model of a homotropic effector is hemoglobin, although it is not a branch-point enzyme and thus does not fit the definition on all counts.

There are two ways that enzymatic activity can be altered by effectors: the Vmax can be increased or decreased, or the Km can be raised or lowered. Enzymes whose Km is altered by effectors are said to be K-type enzymes and the effector a K-type effector. If Vmax is altered, the enzyme and effector are said to be V-type. Many allosteric enzymes respond to multiple effectors with V-type and K-type behavior. Here again, hemoglobin is often used as a model to study allosteric interactions, although it is not strictly an enzyme.

In the preceding discussion we assumed that allosteric sites and catalytic sites were homogeneously present on every subunit of an allosteric enzyme. While this is often the case, there is another class of allosteric enzymes that are comprised of separate catalytic and regulatory subunits. The archetype of this class of enzymes is cAMP-dependent protein kinase (PKA), whose mechanism of activation is illustrated in the Figure below. The enzyme is tetrameric, containing two catalytic subunits and two regulatory subunits, and enzymatically inactive. When intracellular cAMP levels rise, one molecule of cAMP binds to each regulatory subunit, causing the tetramer to dissociate into one regulatory dimer and two catalytic monomers. In the dissociated form, the catalytic subunits are fully active; they catalyze the phosphorylation of a number of other enzymes, such as those involved in regulating glycogen metabolism. The regulatory subunits have no catalytic activity.


Representative pathway for the activation of cAMP-dependent protein kinase (PKA). In this example glucagon binds to its' cell-surface receptor, thereby activating the receptor. Activation of the receptor is coupled to the activation of a receptor-coupled G-protein (GTP-binding and hydrolyzing protein) composed of 3 subunits. Upon activation the a-subunit dissociates and binds to and activates adenylate cyclase. Adenylate cylcase then converts ATP to cyclic-AMP (cAMP). The cAMP thus produced then binds to the regulatory subunits of PKA leading to dissociation of the associated catalytic subunits. The catalytic subunits are inactive until dissociated from the regulatory subunits. Once released the catalytic subunits of PKA phosphorylate numerous substrate using ATP as the phosphate donor.

Factors Affecting Enzyme Action

The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH (left graph) and temperature (right graph), its activity decreasing at values above and below that point. This is not surprising considering the importance of

·                     tertiary structure (i.e. shape) in enzyme function and noncovalent forces, e.g., ionic interactions and hydrogen bonds, in determining that shape.


·                     the protease pepsin works best as a pH of 1-2 (found in the stomach) while

·                     the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small intestine as the bicarbonate of the pancreatic fluid neutralizes the arriving stomach contents).

Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Without the unionized -COOH group of Glu-35 and the ionized -COO- of Asp-52, the catalytic action of lysozyme would cease.

Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve (red arrow in right-hand graph above) reflects the general effect of increasing temperature on the rate of chemical reactions (graph at left). The descending portion of the curve above (blue arrow) reflects the loss of catalytic activity as the enzyme molecules become denatured at high temperatures.

Enzyme and Activation Energy

Activation energy is the energy barrier over which the molecules in a system must be raised for a reaction to take place (Figure 1).

This condition is analogous to a rock resting in a depression at the top of a hill. As long as the rock remains undisturbed, it will not spontaneously roll downhill unless activation energy is applied to the rock. Spontaneous movement over the barrier occurs because molecules, unlike the rock are in constant motion at temperatures above absolute zero. Although the average amount of movement, or kinetic energy, is below the amount required for activation, some molecular collision may raise a number of molecules to the energy level required for the reaction to proceed. The higher the activating barrier, the fewer the molecules that will proceed over the energy barrier per unit time.

Factors Affecting Enzyme Activity

A number of external factors affect the activity of enzymes in speeding conversion of reactants to products. These factors, including variations in the concentration for substrate molecules, temperature, and pH, speed or slow enzymatic activity in highly characteristic patterns.

Lock and key

Substrate Concentration

Enzymes react distinctively to alteration in the concentration of reacting molecules. At very low substrate concentration, collisions between enzyme and substrate molecules are infrequent and reaction proceeds slowly. As the substrate concentration increases, there reaction rate initially increases proportionately as collisions between enzyme molecules and reactants become more frequent Figure 3. When the enzymes begin to approach the maximum rate at which they can combine with reactants and release products, the effects of increasing substrate concentration diminish. At the point at which the enzymes are cycling as rapidly as possible, further increases in substrate concentration have no effect on there reaction rate. At this point the enzyme is saturated and the reaction remains at the saturation level.

Concentration and rate

If the reaction reaches a point at which further increases in reactants have no effect in increasing the rate of the reaction, then there is a good chance that the reaction is catalyzed by an enzyme. Uncatalyzed reactions, in contrast, increase the rate rate of the reaction almost indefinitely as the concentration of reactants increases.

The fact that enzymes combine briefly with their reactants makes them susceptible to inhibition by unreactive molecules that resemble the substrate. The inhibiting molecules can combine with the active site of the enzyme but tend to remain bound without change, blocking access by the normal substrate. As a result, the rate of there reaction slows. If the concentration of the inhibitor becomes high enough, the reaction may stop completely. Inhibition of this type is called competitive because the inhibitor competes with the normal substrate for binding to the active site.
Some inhibitors interfere with enzyme-catalyzed reactions by combining with enzymes at locations outside the active site. These inhibitors, rather than reducing accessibility of the active site to the substrate, cause changes in folding conformation that reduce the ability of the enzyme to lower the activation energy. Because such inhibitors do no directly compete for binding to the active site, their pattern of inhibition is called noncompetitive. Some poisons or toxins exert their damaging effects by acting as enzyme inhibitors. For example, the action of cyanide and carbon monoxide as poisons depends on their ability to inhibit enzyme important the utilization of oxygen in cellular respiration. Poisons and toxins typically act irreversibly by combining so strongly with enzymes, either covalently or nocovalently, that the inhibition is essentially permanent. Some irreversible poisons, rather than combining with the enzyme, destroy enzyme activity by chemically modifying critical amino acid side groups.

Allosteric Inhibition

The cell has built in mechanisms to control directly both enzyme concentration and activity. First cells are able to regulate whether an enzyme is present at all. This type of control regulates protein synthesis and will be discussed in a later chapter. Cells also have ways to control the level of activity of enzymes that have already been synthesized and are present in the cell.

In noncompetitive inhibition, a molecule binds to an enzyme but not at the active site. The other binding site is called the allosteric site (allo - other and steric -structure or space). The molecule that binds to the allosteric site is an inhibitor because it causes a change in the 3-dimensional structure of the enzyme that prevents the substrate from binding to the active site. In cells inhibition usually reversible; that is the inhibitor isn't permanently bound to the enzyme. Irreversible inhibition of enzymes also occurs, due to the presence of a poison. For example, penicillin cause the death of bacteria due to irreversible inhibition of an enzyme needed to form the bacterial cell wall. In humans, hydrogen cyanide irreversibly bind to a very important enzyme (cytochrome oxidase) present in all cells, and this accounts for its lethal effect on the body.

The activity of almost every enzyme is a cell is regulated by feedback inhibition. Feedback inhibition is an example of common biological control mechanism called negative feedback. Just as high temperature will cause furnace to shut off, in a similar manner the product of an enzyme can inhibit a enzyme reaction. When the product is in abundance, it binds competitively with its enzyme's active site; as the product is used up, inhibition is reduced and more product can be produced. In this way the concentration of the product is always controlled within a certain range.

Most enzymatic pathways are also regulated by feedback inhibition, but in these cases the end product of the pathway binds at an allosteric site on the first enzyme of the pathway. This binding shuts down the pathway, and not more product id produced. The reaction series converting theronine to isoleucine is a classic example of allosteric regulation. Five enzymes acting in sequence catalyze the pathway. The final product of the sequence, isoleucine, acts as an inhibitor of the first enzyme of the pathway, threonine deaminase. As the pathway produces isoleucine, any molecules made in excess of cell requirements combine reversibly with threonine deaminase at a location outside the active site. The combination converts threonine deaminase to the T state and inhibits its ability to combine with threonine. The pathway is then turned off. If the concentration of isoleucine later falls as a result of its use in cell synthesis, isoleucine releases from the threonine deaminase enzymes, converting them to the R state in which they have high affinity of the substrate, conversion of threonine to isoleucine takes place.

 Activation of an allosteric enzyme by an activator is another form of feedback inhibition. Combination of the activator and the allosteric site cause a conformational change in the active site permitting substrate binding and the reaction will be caltalyzed.

Feedback inhibition

 Enzyme Regulation


Energy releasing processes, ones that "generate" energy, are termed exergonic reactions. Reactions that require energy to initiate the reaction are known as endergonic reactions. All natural processes tend to proceed in such a direction that the disorder or randomness of the universe increases (the second law of thermodynamics).

Time-energy graphs of an exergonic reaction (top) and endergonic reaction (bottom). Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.


Biochemical reactions in living organisms are essentially energy transfers. Often they occur together, "linked", in what are referred to as oxidation/reduction reactions. Reduction is the gain of an electron. Sometimes we also have H ions along for the ride, so reduction also becomes the gain of H. Oxidation is the loss of an electron (or hydrogen). In oxidation/reduction reactions, one chemical is oxidized, and its electrons are passed (like a hot potato) to another (reduced, then) chemical. Such coupled reactions are referred to as redox reactions. The metabolic processes glycolysis, Kreb's Cycle, and Electron Transport Phosphorylation involve the transfer of electrons (at varying energy states) by redox reactions.

Passage of electrons from compound A to compound B. When A loses its electrons it is oxidized; when B gains the electrons it is reduced.

Oxidation/reduction via an intermediary (energy carrier) compound, in this case NAD+. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Anabolism is the total series of chemical reactions involved in synthesis of organic compounds. Autotrophs must be able to manufacture (synthesize) all the organic compounds they need. Heterotrophs can obtain some of their compounds in their diet (along with their energy). For example humans can synthesize 12 of the 20 amino acids, we must obtain the other 8 in our diet. Catabolism is the series of chemical reactions that breakdown larger molecules. Energy is released this way, some of it can be utilized for anabolism. Products of catabolism can be reassembled by anabolic processes into new anabolic molecules.

Enzymes allow many chemical reactions to occur within the homeostasis constraints of a living system. Enzymes function as organic catalysts. A catalyst is a chemical involved in, but not changed by, a chemical reaction. Many enzymes function by lowering the activation energy of reactions. By bringing the reactants closer together, chemical bonds may be weakened and reactions will proceed faster than without the catalyst.

The use of enzymes can lower the activation energy of a reaction (Ea). Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Enzymes can act rapidly, as in the case of carbonic anhydrase (enzymes typically end in the -ase suffix), which causes the chemicals to react 107 times faster than without the enzyme present. Carbonic anhydrase speeds up the transfer of carbon dioxide from cells to the blood. There are over 2000 known enzymes, each of which is involved with one specific chemical reaction. Enzymes are substrate specific. The enzyme peptidase (which breaks peptide bonds in proteins) will not work on starch (which is broken down by human-produced amylase in the mouth).

Enzymes are proteins. The functioning of the enzyme is determined by the shape of the protein. The arrangement of molecules on the enzyme produces an area known as the active site within which the specific substrate(s) will "fit". It recognizes, confines and orients the substrate in a particular direction.

Space filling model of an enzyme working on glucose. Note the shape change in the enzyme (indicated by the red arrows) after glucose has fit into the binding or active site.

The induced fit hypothesis suggests that the binding of the substrate to the enzyme alters the structure of the enzyme, placing some strain on the substrate and further facilitating the reaction. Cofactors are nonproteins essential for enzyme activity. Ions such as K+ and Ca+2 are cofactors. Coenzymes are nonprotein organic molecules bound to enzymes near the active site. NAD (nicotinamide adenine dinucleotide).

A cartoonish view of the formation of an enzyme-substrate complex. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Enzymatic pathways form as a result of the common occurrence of a series of dependent chemical reactions. In one example, the end product depends on the successful completion of five reactions, each mediated by a specific enzyme. The enzymes in a series can be located adjacent to each other (in an organelle or in the membrane of an organelle), thus speeding the reaction process. Also, intermediate products tend not to accumulate, making the process more efficient. By removing intermediates (and by inference end products) from the reactive pathway, equilibrium (the tendency of reactions to reverse when concentrations of the products build up to a certain level) effects are minimized, since equilibrium is not attained, and so the reactions will proceed in the "preferred" direction.

Negative feedback and a metabolic pathway. The production of the end product (G) in sufficient quantity to fill the square feedback slot in the enzyme will turn off this pathway between step C and D. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Temperature: Increases in temperature will speed up the rate of nonenzyme mediated reactions, and so temperature increase speeds up enzyme mediated reactions, but only to a point. When heated too much, enzymes (since they are proteins dependent on their shape) become denatured. When the temperature drops, the enzyme regains its shape. Thermolabile enzymes, such as those responsible for the color distribution in Siamese cats and color camouflage of the Arctic fox, work better (or work at all) at lower temperatures.

Concentration of substrate and product also control the rate of reaction, providing a biofeedback mechanism.

Activation, as in the case of chymotrypsin, protects a cell from the hazards or damage the enzyme might cause.

Changes in pH will also denature the enzyme by changing the shape of the enzyme. Enzymes are also adapted to operate at a specific pH or pH range.

Plot of enzyme activity as a function of pH for several enzymes. Note that each enzyme has a range of pH at which it is active as well as an optimal pH at which it is most active. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Allosteric Interactions may allow an enzyme to be temporarily inactivated. Binding of an allosteric effector changes the shape of the enzyme, inactivating it while the effector is still bound. Such a mechanism is commonly employed in feedback inhibition. Often one of the products, either an end or near-end product act as an allosteric effector, blocking or shunting the pathway.

Action of an allosteric inhibitor as a negative control on the action of an enzyme.

Competitive Inhibition works by the competition of the regulatory compound and substrate for the binding site. If enough regulatory compound molecules bind to enough enzymes, the pathway is shut down or at least slowed down. PABA, a chemical essential to a bacteria that infects animals, resembles a drug, sulfanilamide, that competes with PABA, shutting down an essential bacterial (but not animal) pathway.


Top: general diagram showing competitor in the active site normally occupied by the natural substrate; Bottom: specific case of succinate dehydrogenase and its natural substrate (succinate) and competitors (oxalate et al.).

 Noncompetitive Inhibition occurs when the inhibitory chemical, which does not have to resemble the substrate, binds to the enzyme other than at the active site. Lead binds to SH groups in this fashion. Irreversible Inhibition occurs when the chemical either permanently binds to or massively denatures the enzyme so that the tertiary structure cannot be restored. Nerve gas permanently blocks pathways involved in nerve message transmission, resulting in death. Penicillin, the first of the "wonder drug" antibiotics, permanently blocks the pathways certain bacteria use to assemble their cell wall components.

The four mechanisms described above regulate the activity of enzymes already present within the cell.

What about enzymes that are not needed or are needed but not present?

Here, too, control mechanisms are at work that regulate the rate at which new enzymes are synthesized. Most of these controls work by turning on — or off — the transcription of genes.

If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down.

Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase — by transcribing and translating the necessary gene(s) — and so can begin to metabolize the sugar.



 The principle of international classification and nomenclature of enzymes.


The first general principle of these 'Recommendations' is that names purporting to be names of enzymes, especially those ending in -ase, should be used only for single enzymes, i.e. single catalytic entities. They should not be applied to systems containing more than one enzyme. When it is desired to name such a system on the basis of the overall reaction catalysed by it, the word system should be included in the name. For example, the system catalysing the oxidation of succinate by molecular oxygen, consisting of succinate dehydrogenase, cytochrome oxidase, and several intermediate carriers, should not be named succinate oxidase, but it may be called the succinate oxidase system. Other examples of systems consisting of several structurally and functionally linked enzymes (and cofactors) are the pyruvate dehydrogenase system, the similar 2-oxoglutarate dehydrogenase system, and the fatty acid synthase system.

To classify an enzyme according to the type of reaction catalysed, it is occasionally necessary to choose between alternative ways of regarding a given reaction. In general, that alternative should be selected which fits in best with the general system of classification and reduces the number of exceptions.

 Common and Systematic Names

The first Enzyme Commission gave much thought to the question of a systematic and logical nomenclature for enzymes, and finally recommended that there should be two nomenclatures for enzymes, one systematic, and one working or trivial. The systematic name of an enzyme, formed in accordance with definite rules, showed the action of an enzyme as exactly as possible, thus identifying the enzyme precisely. The trivial name was sufficiently short for general use, but not necessarily very systematic; in a great many cases it was a name already in current use. The introduction of (often cumbersome) systematic names was strongly criticised. In many cases the reaction catalysed is not much longer than the systematic name and can serve just as well for identification, especially in conjunction with the code number.

The Commission for Revision of Enzyme Nomenclature discussed this problem at length, and a change in emphasis was made. It was decided to give the trivial names more prominence in the Enzyme List; they now follow immediately after the code number, and are described as Common Name. Also, in the index the common names are indicated by an asterisk. Nevertheless, it was decided to retain the systematic names as the basis for classification for the following reasons:

(i) the code number alone is only useful for identification of an enzyme when a copy of the Enzyme List is at hand, whereas the systematic name is self-explanatory;

(ii) the systematic name stresses the type of reaction, the reaction equation does not;

(iii) systematic names can be formed for new enzymes by the discoverer, by application of the rules, but code numbers should not be assigned by individuals;

(iv) common names for new enzymes are frequently formed as a condensed version of the systematic name; therefore, the systematic names are helpful in finding common names that are in accordance with the general pattern.

 Scheme for the classification of enzymes and the generation of EC numbers

The first Enzyme Commission, in its report in 1961, devised a system for classification of enzymes that also serves as a basis for assigning code numbers to them. These code numbers, prefixed by EC, which are now widely in use, contain four elements separated by points, with the following meaning:

(i) the first number shows to which of the six main divisions (classes) the enzyme belongs,

(ii) the second figure indicates the subclass,

(iii) the third figure gives the sub-subclass,

(iv) the fourth figure is the serial number of the enzyme in its sub-subclass.


Enzyme Classifications

Currently enzymes are grouped into six functional classes by the International Union of Biochemists (I.U.B.).



Biochemical Properties



Act on many chemical groupings to add or remove hydrogen atoms.



Transfer functional groups between donor and acceptor molecules. Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules.



Add water across a bond, hydrolyzing it.



Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds.



Carry out many kinds of isomerization: L to D isomerizations, mutase reactions (shifts of chemical groups) and others.



Catalyze reactions in which two chemical groups are joined (or ligated) with the use of energy from ATP.


These rules give each enzyme a unique number. The I.U.B. system also specifies a textual name for each enzyme. The enzyme's name is comprised of the names of the substrate(s), the product(s) and the enzyme's functional class. Because many enzymes, such as alcohol dehydrogenase, are widely known in the scientific community by their common names, the change to I.U.B.-approved nomenclature has been slow. In everyday usage, most enzymes are still called by their common name.

Enzymes are also classified on the basis of their composition. Enzymes composed wholly of protein are known as simple enzymes in contrast to complex enzymes, which are composed of protein plus a relatively small organic molecule. Complex enzymes are also known as holoenzymes. In this terminology the protein component is known as the apoenzyme, while the non-protein component is known as the coenzyme or prosthetic group where prosthetic group describes a complex in which the small organic molecule is bound to the apoenzyme by covalent bonds; when the binding between the apoenzyme and non-protein components is non-covalent, the small organic molecule is called a coenzyme. Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins. It should be noted that the main clinical symptoms of dietary vitamin insufficiency generally arise from the malfunction of enzymes, which lack sufficient cofactors derived from vitamins to maintain homeostasis.

The non-protein component of an enzyme may be as simple as a metal ion or as complex as a small non-protein organic molecule. Enzymes that require a metal in their composition are known as metalloenzymes if they bind and retain their metal atom(s) under all conditions with very high affinity. Those which have a lower affinity for metal ion, but still require the metal ion for activity, are known as metal-activated enzymes.

Hydrolases. Mechanism of action of esterases, peptidases, glycosidases. Examples.


These enzymes catalyse the hydrolytic cleavage of C-O, C-N, C-C and some other bonds, including phosphoric anhydride bonds. Although the systematic name always includes hydrolase, the common name is, in many cases, formed by the name of the substrate with the suffix -ase. It is understood that the name of the substrate with this suffix means a hydrolytic enzyme.

A number of hydrolases acting on ester, glycosyl, peptide, amide or other bonds are known to catalyse not only hydrolytic removal of a particular group from their substrates, but likewise the transfer of this group to suitable acceptor molecules. In principle, all hydrolytic enzymes might be classified as transferases, since hydrolysis itself can be regarded as transfer of a specific group to water as the acceptor. Yet, in most cases, the reaction with water as the acceptor was discovered earlier and is considered as the main physiological function of the enzyme. This is why such enzymes are classified as hydrolases rather than as transferases.

Some hydrolases (especially some of the esterases and glycosidases) pose problems because they have a very wide specificity and it is not easy to decide if two preparations described by different authors (perhaps from different sources) have the same catalytic properties, or if they should be listed under separate entries. An example is vitamin A esterase (formerly EC, now believed to be identical with EC To some extent the choice must be arbitrary; however, separate entries should be given only when the specificities are sufficiently different.

Another problem is that proteinases have 'esterolytic' action; they usually hydrolyse ester bonds in appropriate substrates even more rapidly than natural peptide bonds. In this case, classification among the peptide hydrolases is based on historical priority and presumed physiological function.

The second figure in the code number of the hydrolases indicates the nature of the bond hydrolysed; EC 3.1 are the esterases; EC 3.2 the glycosylases, and so on.

The third figure normally specifies the nature of the substrate, e.g. in the esterases the carboxylic ester hydrolases (EC 3.1.1), thiolester hydrolases (EC 3.1.2), phosphoric monoester hydrolases (EC 3.1.3); in the glycosylases the O-glycosidases (EC 3.2.1), N-glycosylases (EC 3.2.2), etc. Exceptionally, in the case of the peptidyl-peptide hydrolases the third figure is based on the catalytic mechanism as shown by active centre studies or the effect of pH.

Esterases - enzymes that hydrolyse esters, i.e. cleave the ester linkage to form free acid and alcohol. Those that hydrolyse the ester linkages of fats are generally known as lipases, and those that hydrolyse phospholipids as phospholipases. Any enzymes catalyses the hydrolysis of an ester.

Peptidase, also called protease or proteinase, is a type of enzyme that helps to break down proteins in the body. This type of enzyme occurs naturally in the living things and forms part of many metabolic processes. They form part of the larger systems in the body, including the digestive, immune, and blood circulation systems. These enzymes are classified into five different groups: aspartic proteinases, cysteine proteinases, metalloproteinases, serine proteinases, and threonine proteases.

In the digestive system, peptidases break down proteins by destroying the chains between their amino acids, and many can usually be found in the digestive tract. When protein enters the body, it needs to be digested and broken down into smaller molecules so that it can be used. This type of enzyme is responsible for this catabolic process.

Aspartic proteinases can usually be found in an acidic environment like the stomach. They are responsible for the breakdown of food and are also called pepsins. Other places that aspartic proteinases can be found are in blood plasma and in the immune system.

Glycoside hydrolases (also called glycosidases or glycosyl hydrolases) catalyze the hydrolysis of the glycosidic linkage to release smaller sugars. They are extremely common enzymes with roles in nature including degradation of biomass such as cellulose and hemicellulose, in anti-bacterial defense strategies (e.g., lysozyme), in pathogenesis mechanisms (e.g., viral neuraminidases) and in normal cellular function (e.g., trimming mannosidases involved in N-linked glycoprotein biosynthesis). Together with glycosyltransferases, glycosidases form the major catalytic machinery for the synthesis and breakage of glycosidic bonds.

The subclasses and sub-subclasses are formed according to principles indicated below. he main divisions and subclasses are:


Oxidoreductases are a class of enzymes that catalyze oxidoreduction reactions.  Oxidoreductases catalyze the transfer of electrons from one molecule (the oxidant) to another molecule (the reductant).  Oxidoreductases catalyze reactions similar to the following, A + B → A + B where A is the oxidant and B is the reductant.  Oxidorecuctases can be oxidases or dehydrogenasesOxidases are enzymes involved when molecular oxygen acts as an acceptor of hydrogen or electrons.  Whereas, dehydrogenases are enzymes that oxidize a substrate by transferring hydrogen to an acceptor that is either NAD+/NADP+ or a flavin enzyme.  Other oxidoreductases include peroxidases, hydroxylases, oxygenases, and reductases. Peroxidases are localized in peroxisomes, and catalyzes the reduction of hydrogen peroxide. Hydroxylases add hydroxyl groups to its substrates.

 Oxygenases incorporate oxygen from molecular oxygen into organic substrates. Reductases catalyze reductions, in most cases reductases can act like an oxidases.

Oxidoreductase enzymes play an important role in both aerobic and anaerobic metabolism.  They can be found in glycolysis, TCA cycle, oxidative phosphorylation, and in amino acid metabolism.  In glycolysis, the enzyme glyceraldehydes-3-phosphate dehydrogenase catalyzes the reduction of NAD+ to NADH.  In order to maintain the re-dox state of the cell, this NADH must be re-oxidized to NAD+, which occurs in the oxidative phosphorylation pathway.  Additional NADH molecules are generated in the TCA cycle.  The product of glycolysis, pyruvate enters the TCA cycle in the form of acetyl-CoA.  During anaerobic glycolysis, the oxidation of NADH occurs through the reduction of pyruvate to lactate.  The lactate is then oxidized to pyruvate in muscle and liver cells, and the pyruvate is further oxidized in the TCA cycle.  All twenty of the amino acids, except leucine and lysine, can be degraded to TCA cycle intermediates.  This allows the carbon skeletons of the amino acids to be converted into oxaloacetate and subsequently into pyruvate.  The gluconeogenic pathway can then utilize the pyruvate forme


Transferases are enzymes transferring a group, e.g. a methyl group or a glycosyl group, from one compound (generally regarded as donor) to another compound (generally regarded as acceptor). The systematic names are formed according to the scheme donor:acceptor grouptransferase. The common names are normally formed according to acceptor grouptransferase or donor grouptransferase. In many cases, the donor is a cofactor (coenzyme) charged with the group to be transferred. A special case is that of the transaminases (see below).

Some transferase reactions can be viewed in different ways. For example, the enzyme-catalysed reaction   X-Y + Z = X + Z-Y may be regarded either as a transfer of the group Y from X to Z, or as a breaking of the X-Y bond by the introduction of Z. Where Z represents phosphate or arsenate, the process is often spoken of as 'phosphorolysis' or 'arsenolysis', respectively, and a number of enzyme names based on the pattern of phosphorylase have come into use. These names are not suitable for a systematic nomenclature, because there is no reason to single out these particular enzymes from the other transferases, and it is better to regard them simply as Y-transferases.

In the above reaction, the group transferred is usually exchanged, at least formally, for hydrogen, so that the equation could more strictly be written as:

X-Y + Z-H = X-H + Z-Y

Another problem is posed in enzyme-catalysed transaminations, where the -NH2 group and -H are transferred to a compound containing a carbonyl group in exchange for the =O of that group, according to the general equation:

R1-CH(-NH2)-R2 + R3-CO-R4 [arrow right]R1-CO-R2 + R3-CH(-NH2)-R4

The reaction can be considered formally as oxidative deamination of the donor (e.g. amino acid) linked with reductive amination of the acceptor (e.g. oxo acid), and the transaminating enzymes (pyridoxal-phosphate proteins) might be classified as oxidoreductases. However, the unique distinctive feature of the reaction is the transfer of the amino group (by a well-established mechanism involving covalent substrate-coenzyme intermediates), which justified allocation of these enzymes among the transferases as a special subclass (EC 2.6.1, transaminases).


Lyases are enzymes cleaving C-C, C-O, C-N, and other bonds by elimination, leaving double bonds or rings, or conversely adding groups to double bonds. The systematic name is formed according to the pattern substrate group-lyase. The hyphen is an important part of the name, and to avoid confusion should not be omitted, e.g. hydro-lyase not 'hydrolyase'. In the common names, expressions like decarboxylase, aldolase, dehydratase (in case of elimination of CO2, aldehyde, or water) are used. In cases where the reverse reaction is much more important, or the only one demonstrated, synthase (not synthetase) may be used in the name. Various subclasses of the lyases include pyridoxal-phosphate enzymes that catalyse the elimination of a β- or γ-substituent from an α-amino acid followed by a replacement of this substituent by some other group. In the overall replacement reaction, no unsaturated end-product is formed; therefore, these enzymes might formally be classified as alkyl-transferases (EC 2.5.1...). However, there is ample evidence that the replacement is a two-step reaction involving the transient formation of enzyme-bound α,β(or β,γ)-unsaturated amino acids. According to the rule that the first reaction is indicative for classification, these enzymes are correctly classified as lyases. Examples are tryptophan synthase (EC and cystathionine β-synthase (EC

The second figure in the code number indicates the bond broken: EC 4.1 are carbon-carbon lyases, EC 4.2 carbon-oxygen lyases and so on.

The third figure gives further information on the group eliminated (e.g. CO2 in EC 4.1.1, H2O in EC 4.2.1).


These enzymes catalyse geometric or structural changes within one molecule. According to the type of isomerism, they may be called racemases, epimerases, cis-trans-isomerases, isomerases, tautomerases, mutases or cycloisomerases.

In some cases, the interconversion in the substrate is brought about by an intramolecular oxidoreduction (EC 5.3); since hydrogen donor and acceptor are the same molecule, and no oxidized product appears, they are not classified as oxidoreductases, even though they may contain firmly bound NAD(P)+.

The subclasses are formed according to the type of isomerism, the sub-subclasses to the type of substrates.


Ligases are enzymes catalysing the joining together of two molecules coupled with the hydrolysis of a diphosphate bond in ATP or a similar triphosphate. The systematic names are formed on the system X:Y ligase (ADP-forming). In earlier editions of the list the term synthetase has been used for the common names. Many authors have been confused by the use of the terms synthetase (used only for Group 6) and synthase (used throughout the list when it is desired to emphasis the synthetic nature of the reaction).

Consequently NC-IUB decided in 1983 to abandon the use of synthetase for common names, and to replace them with names of the type X-Y ligase. In a few cases in Group 6, where the reaction is more complex or there is a common name for the product, a synthase name is used (e.g. EC and EC

It is recommended that if the term synthetase is used by authors, it should continue to be restricted to the ligase group.

The second figure in the code number indicates the bond formed: EC 6.1 for C-O bonds (enzymes acylating tRNA), EC 6.2 for C-S bonds (acyl-CoA derivatives), etc. Sub-subclasses are only in use in the C-N ligases.

In a few cases it is necessary to use the word other in the description of subclasses and sub-subclasses. They have been provisionally given the figure 99, in order to leave space for new subdivisions.


Enzymes, specific to different organs. Localization of enzymes in cell’s organells


An adaptive enzyme or inducible enzyme is an enzyme that is expressed only under conditions in which it is clear of adaptive value, as opposed to a constitutive enzyme which is produced all the time. The Inducible enzyme is used for the breaking-down of things in the cell. It is also a part of the Operon Model, which illustrates a way for genes to turn "on" and "off". The Inducer causes the gene to turn on (controlled by the amount of reactant which turns the gene on). Then there's the repressor protein that turns genes off.

The inducer can remove this repressor, turning genes back on. The operator is a section of DNA where the repressor binds to shut off certain genes; the promoter is the section of DNA where the RNA polymerase binds. Lastly, the regulatory gene is the gene for the repressor protein. An example of inducible enzyme is COX-2 which is synthesized in macrophages to produce Prostaglandin E2 while the constitutive enzyme COX-1 (another isozyme in COX family) is always produced in variety of organisms in body (like stomach).

Constitutive enzymes are produced constitutively by the cell under all physiological conditions. Therefore, they are not controlled by induction or repression.

Constitutive enzymes are produced in constant amounts without regard to the physiological demand or the concentration of the substrate. They are continuously synthesized because their role in maintaining cell processes or structure is indispensable.


The methods of separation and purification of enzymes


Analysis of the biological properties Understand its structure.

Study interactions. No single procedure can be used to isolate every protein

Exploit specific characteristics (structure or function) of the protein. Different steps should exploit a different characteristic. Ensure method has little/no effect on function.

Ammonium sulphate precipitation (40%) exploits changes in the solubility of proteins as consequence of a change in ionic strength (salt conc.) of the solution

At low salt, the solubility of a protein increases with salt concentration,



But as salt conc. (ionic strength) is increased further, the solubility of the protein begins to decrease, until a point where the protein is precipitated from solution,


 Ion Exchange Chromatography (IEC)

Separates molecules based on their charge.

The side-chain groups of some amino acids are ionizable, e.g., lysine, arginine, histidine, glutamic acid, aspartic acid as are the N-terminal amino and C-terminal carboxyl groups. Thus proteins are charge molecule s and can have a different charge at a given pH because they have different compositions of ionizable amino acids.

For any given amphoteric protein, there will be a pH at which its overall charge is 0

(No. of negative charges equals the No. of positive charges).

This is referred to as the ISOELECTRIC POINT (pI) or ISOTONIC POINT of the protein. At a pH above its pI a protein will have a net negative charge while.

At a pH below its pI a protein will have a net positive charge.


Gel Filtration Chromatography (GFC)

GFC (also Size Exclusion Chromatography, Molecular Sieve Chromatography or Molecular Exclusion Chromatography)

Separates molecules based on their size (& shape)

It can also be used to determine the size and molecular weight of a protein

Separation occurs due to the differential diffusion of various molecules into gel pores in a porous matrix. For protein purification, the matrix typically consists of porous beads (with pores of a specific size distribution) of an inert, highly hydrated gel.


Affinity Chromatography:

Separates molecules based on specific interactions between the protein of interest and the column matrix E.g. Antibodies which bind Protein. Enzyme which binds a co-enzyme or inhibitor.

A ligand is covalently bound to a solid matrix (usually agarose) which is then packed into a chromatography column When a mixture containing the protein of interest is applied to the column, the desired protein is bound by the immobilised

ligands, while all other proteins in the mixture, which should have no affinity for the ligand pass through and are discarded

Affinity chromatography (with HIS-tagged proteins).

Affinity chromatography can be performed using a number of  different protein tags. poly-hisitidine.

The histidine tag is very short (6 His residues).

Should not alter the conformation of the tagged protein.

Should not be involved in artificial interactions.

The poly-his tag binds to a nickel chelate resin.

Eluted by 1.0 M imidazole.

 One international unit is the amount of enzyme that will convert one micromole of substrate per minute per litre of sample and is abbreviated as U/L. The SI Unit (System Internationale) expression is more scientific, where or Katal (catalytic activity) is defined as the number of mole of substrate transformed per second per litre of sample. Katal is abbreviated as kat or k (60 U = 1 μkat and 1 nk = 0.06 U).

A major disadvantage in the use of enzymes for the diagnosis of tissue damage is their lack of specificity to a particular tissue or cell type. Many enzymes are common to more than one tissue, with the result that an increase in the plasma activity of a particular enzyme could reflect damage to any one of these tissues. This problem may be obviated to some extent in two ways:

first, different tissues may contain (and thus release when they are damaged) two or more enzymes in different proportions; thus alanine and aspartate aminotransferases are both present in cardiac and skeletal muscle and hepatocytes, but there is only a very little alanine aminotransferase in either type of muscle;

second, some enzymes exist in different forms (isoforms), colloquially termed isoenzymes (although, strictly, the term 'isoenzyme' refers only to a genetically determined isoform). Individual isoforms are often characteristic of a particular tissue: although they may have similar catalytic activities, they often differ in some other measurable property, such as heat stability or sensitivity to inhibitors.

After a single insult to a tissue, the activity of intracellular enzymes in the plasma rises as they are released from the damaged cells, and then falls as the enzymes are cleared. It is thus important to consider the time at which the blood sample is taken in relation to the insult. If taken too soon, there may have been insufficient time for the enzyme to reach the blood- stream and if too late, it may have been completely cleared. As with all diagnostic techniques, data acquired from measurements of enzymes in plasma must always be assessed in the light of whatever clinical and other information is available, and their limitations borne in mind.



Most catalases exist as tetramers of 60 or 75 kDa, each subunit containing an active site haem group buried deep within the structure, but which is accessible from the surface through hydrophobic channels.  The very rigid, stable structure of catalases is resistant to unfolding, which makes them uniquely stable enzymes that are more resistant to pH, thermal denaturation and proteolysis than most other enzymes.  Their stability and resistance to proteolysis is an evolutionary advantage, especially since they are produced during the stationary phase of cell growth when levels of proteases are high and there is a rapid rate of protein turnover.

Haem-containing catalases break down hydrogen peroxide by a two-stage mechanism in which hydrogen peroxide alternately oxidises and reduces the haem iron at the active site.  In the first step, one hydrogen peroxide molecule oxidises the haem to an oxyferryl species.  In the second step, a second hydrogen peroxide molecule is used as a reductant to regenerate the enzyme, producing water and oxygen.  Some catalases contain NADPH as a cofactor, which functions to prevent the formation of an inactive compound.  Catalases may have another role: the generation of ROS, possibly hydroperoxides, upon UVB irradiation.  In this way, UVB light can be detoxified through the generation of hydrogen peroxide, which can then be degraded by the catalase.  NADPH may play a role in providing the electrons needed to reduce molecular oxygen in the production of ROS.

Much of the hydrogen peroxide that is produced during oxidative cellular metabolism comes from the breakdown of one of the most damaging ROS, namely the superoxide anion radical (O2-).  Superoxide is broken down by superoxide dismutases into hydrogen peroxide and oxygen.  Superoxide is so damaging to cells that mutations in the superoxide dismutase enzyme can lead to ALS, which is characterised by the loss of motoneurons in the spinal cord and brain stem, possibly involving the activation of caspase-12 and the apoptosis cascade via oxidative stress. 

Regulation of Antioxidant Enzymes


Antioxidant enzymes, including catalase, form the first line of defence against free radicals, therefore their regulation depends mainly upon the oxidant status of the cell. 

However, there are other factors involved in their regulation, including the enzyme-modulating action of various hormones such as growth hormone, prolactin and melatonin.  Melatonin is a derivative of the amino acid tryptophan that acts as a neurohormone in mammals, but is also synthesized by many other species, including plants, algae and bacteria.  Melatonin has been shown to markedly protect both membrane lipids and nuclear DNA from oxidative damage.  Melatonin can directly neutralise several ROS, including hydrogen peroxide.  It can also stimulate various antioxidant enzymes, including catalase, either by increasing their activity or by stimulating gene expression for these enzymes.  The decrease in melatonin levels observed with age correlates with an increase in neurogenerative disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and stroke, all of which may involve oxidative stress.  In general, the production of ROS increases with aging and is associated with DNA damage to the tissues.

         By contrast, growth hormone, and possibly prolactin, was found to decrease catalase and other antioxidant enzymes in various tissues in mice, suggesting that this hormone acts as a suppressor of key antioxidant components.


The origin of blood enzymes

Do you mean a blood enzymes test, or more generally, enzymes in the blood?

Enzymes are proteins that carry out chemical reactions (as opposed to structural enzymes). Most of the detectable enzymes in the blood come from the various tissues and organs of the body. Abnormal levels may reflect problems with a particular organ.

The most common blood enzymes test is for liver enzymes. When the cells of the liver are damaged, such as from a viral infection, their enzymes can leak out and be detected in the blood. Another common test measures enzymes from heart damage, such as from a heart attack.

The measurement of the serum levels of numerous enzymes has been shown to be of diagnostic significance. This is because the presence of these enzymes in the serum indicates that tissue or cellular damage has occurred resulting in the release of intracellular components into the blood. Hence, when a physician indicates that he/she is going to assay for liver enzymes, the purpose is to ascertain the potential for liver cell damage.

Commonly assayed enzymes are the amino transferases: alanine transaminase, ALT (sometimes still referred to as serum glutamate-pyruvate aminotransferase, SGPT) and aspartate aminotransferase, AST (also referred to as serum glutamate-oxaloacetate aminotransferase, SGOT); lactate dehydrogenase, LDH; creatine kinase, CK (also called creatine phosphokinase, CPK); gamma-glutamyl transpeptidase, GGT. Other enzymes are assayed under a variety of different clinical situations but they will not be covered here.

The typical liver enzymes measured are AST and ALT. ALT is particularly diagnostic of liver involvement as this enzyme is found predominantly in hepatocytes. When assaying for both ALT and AST the ratio of the level of these two enzymes can also be diagnostic. Normally in liver disease or damage that is not of viral origin the ratio of ALT/AST is less than 1. However, with viral hepatitis the ALT/AST ratio will be greater than 1. Measurement of AST is useful not only for liver involvement but also for heart disease or damage. The level of AST elevation in the serum is directly proportional to the number of cells involved as well as on the time following injury that the AST assay was performed. Following injury, levels of AST rise within 8 hours and peak 24-36 hours later. Within 3-7 days the level of AST should return to pre-injury levels, provided a continuous insult is not present or further injury occurs. Although measurement of AST is not, in and of itself, diagnostic for myocardial infarction, taken together with LDH and CK measurements (see below) the level of AST is useful for timing of the infarct.

The measurement of LDH is especially diagnostic for myocardial infarction because this enzyme exist in 5 closely related, but slightly different forms (isozymes). The 5 types and their normal distribution and levels in non-disease/injury are listed below.


LDH 1 - Found in heart and red-blood cells and is 17% - 27% of the normal serum total.

LDH 2 - Found in heart and red-blood cells and is 27% - 37% of the normal serum total.

LDH 3 - Found in a variety of organs and is 18% - 25% of the normal serum total.

LDH 4 - Found in a variety of organs and is 3% - 8% of the normal serum total.

LDH 5 - Found in liver and skeletal muscle and is 0% - 5% of the normal serum total.

Following a myocardial infarct the serum levels of LDH rise within 24-48 hours reaching a peak by 2-3 days and return to normal in 5-10 days. Especially diagnostic is a comparison of the LDH-1/LDH-2 ratio. Normally, this ration is less than 1. A reversal of this ration is referred to as a "flipped LDH.". Following an acute myocardial infart the flipped LDH ratio will appear in 12-24 hours and is definitely present by 48 hours in over 80% of patients. Also important is the fact that persons suffering chest pain due to angina only will not likely have altered LDH levels.

CPK is found primarily in heart and skeletal muscle as well as the brain. Therefore, measurement of serum CPK levels is a good diagnostic for injury to these tissues. The levels of CPK will rise within 6 hours of injury and peak by around 18 hours. If the injury is not persistent the level of CK returns to normal within 2-3 days. Like LDH, there are tissue-specific isozymes of CPK and there designations are described below.

CPK3 (CPK-MM) is the predominant isozyme in muscle and is 100% of the normal serum total.

CPK2 (CPK-MB) accounts for about 35% of the CPK activity in cardiac muscle, but less than 5% in skeletal muscle and is 0% of the normal serum total.

CPK1 (CPK-BB) is the characteristic isozyme in brain and is in significant amounts in smooth muscle and is 0% of the normal serum total.

Since most of the released CPK after a myocardial infarction is CPK-MB, an increased ratio of CPK-MB to total CPK may help in diagnosis of an acute infarction, but an increase of total CPK in itself may not. CPK-MB levels rise 3-6 hours after a myocardial infarct and peak 12-24 hours later if no further damage occurs and returns to normal 12-48 hours after the infarct.


Inhibitors are often used as drugs, but they can also act as poisons. However, the difference between a drug and a poison is usually only a matter of amount, since most drugs are toxic at some level, as Paracelsus wrote, "In all things there is a poison, and there is nothing without a poison." Equally, antibiotics and other anti-infective drugs are just specific poisons that can kill a pathogen but not its host.

An example of an inhibitor being used as a drug is aspirin, which inhibits the COX-1 and COX-2 enzymes that produce the inflammation messenger prostaglandin, thus suppressing pain and inflammation. The poison cyanide is an irreversible enzyme inhibitor that combines with the copper and iron in the active site of the enzyme cytochrome c oxidase and blocks cellular respiration.

In many organisms inhibitors may act as part of a feedback mechanism. If an enzyme produces too much of one substance in the organism, that substance may act as an inhibitor for the enzyme that produces it, causing production of the substance to slow down or stop when there is sufficient amount. This is a form of negative feedback.

Diacarb – inhibitor of carbohydrase. This enzyme regulates the metabolism of Na+ and K+ in kidney canaliculus and diuresis.

Inhibitors of proteolytic enzymes (kontrical, trasilol) are used in acute pancreatitis for inhibition trypsin, chemotrypsin activity. Irreversible inhibitors

Some enzyme inhibitors react with the enzyme and form a covalent adduct with the protein. The inactivation produced by this type of inhibitor is irreversible. A class of these compounds called suicide inhibitors includes eflornithine a drug used to treat the parasitic disease sleeping sickness. Penicillin and its derivatives also act in this manner. With these drugs, the compound is bound in the active site and the enzyme then converts the inhibitor into an activated form that reacts irreversibly with one or more with amino acid residues.

Regulatory Enzymes

All enzymes exhibit various features that could conceivably be elements in the regulation of their activity in living cells. All have a characteristic optimum pH, which makes possible alteration of their catalytic rates with changes in intracellular pH. The rates of all enzymatic reactions also depend on the substrate concentration, which may vary significantly under intercellular conditions. Moreover, many enzymes require either metal ions, such as Mg2+ or K+, or coenzymes for activity, suggesting that fluctuations in the concen­tration of these metals or coenzymes in the cell can regulate enzyme activity. However, over and above these properties of all enzymes, some enzymes possess other properties that specifically endow them with regulatory roles in metabolism. Such more highly specialized forms are called reguiatory enzymes. There are two major types of regulatory enzymes: (1) allosteric enzymes, whose catalytic activity is modulated through the noncovalent binding of a specific metabolite at a site on the protein other than the catalytic site, and (2) covalently modulated enzymes, which are interconverted between active and inactive forms by the action of other en­zymes. Some of the enzymes in the second class also respond to noncovalent allosteric modulators. These two types of regulatory enzymes are responsive to alterations in the metabolic state of a cell or tissue on a relatively short time scale — allosteric enzymes within seconds and covalently regulated enzymes within minutes.

Allosteric Enzymes

In many multienzyme systems the end product of the reac­tion sequence may act as a specific inhibitor of an enzyme at or near the beginning of the sequence, with the result that the rate of the entire sequence of reactions is determined by the steady-state concentration of the end product. The clas­sical example is the multienzyme sequence catalyzing the conversion of L-threonine to L-isoleucine, which occurs in five enzyme-catalyzed steps. The first enzyme of the sequence, L-threonine dehydratose, is strongly inhib­ited by L-isoleucine, the end product, but not by any other intermediate in the sequence. The kinetic characteristics of the inhibition by isoleucine are atypical; the inhibition is neither competitive with the substrate L-threonine, nor is it noncompetitive. Isoleucine is quite specific as an inhibitor; other amino acids or related compounds do not inhibit. This type of inhibition is variously called end-product inhibition or feedback inhibition. The first enzyme in this sequence, that which is inhibited by the end product, is called an allosteric enzyme. The term allosteric denotes "another space" or "another structure"; allosteric enzymes possess, in addition to the catalytic site, the "other space," to which the specific effector оr modulator is reversibly and noncovalently bound. In general, the allosteric site is as specific for binding the modulator as the catalytic site is for binding the substrate. Some modulators, e.g., L-isoleucine for threonine dehydratase, are inhibitory and therefore called inhibi­tory or negative modulators. Other allosteric enzymes may have stimulatory, or positive, modulators. When an allosteric enzyme has only one specific modulator, it is said to be monovlent. Some allosteric enzymes respond to two or more specific modulators, each bound to a specific site on the enzyme; they are polyvalent. Moreover, a given allosteric enzyme may have both positive and negative modulators. Two or more multienzyme systems may be connected by one or more polyvalent enzymes in a control network.

The first step in a multienzyme reaction sequence, i.e., the step catalyzed by the allosteric enzyme, is usually irrever­sible under intracellular conditions. It is often called the committing reaction; once it occurs, all the ensuing reactions of the sequence take place. Clearly, it is good strategy for the cell to regulate a metabolic pathway at its first step, to achieve maximum economy of metabolites.

Allosteric enzymes are usually much larger in molecular weight, more complex, and often more difficult to purify than ordinary enzymes because nearly all known allosteric enzymes are oligomeric and thus have two or more polypeptide chain subunits, usually in an even number; some con­tain many chains. Allosteric enzymes show a number of anomalous properties. Some are unstable at 0°C but stable at room or body temperature, unlike single-chain enzymes, which do not show cold lability.

Allosteric enzymes show two different types of control, heterotropic and homotropic, depending on the nature of the modulating molecule. Heterotropic enzymes are stimulated or inhibited by an effector or modulator molecule other than their substrates. For the heterotropic enzyme threonine dehydratase  the substrate is threonine and the mod­ulator is L-isoleucine. In homotropic enzymes, on the other hand, the substrate also functions as the modulator. Homotropic enzymes contain two or more binding sites for the substrate; modulation of these enzymes depends on how many of the substrate sites are occupied. However, a great many (if not most) allosteric enzymes are of mixed homotropic-heterotropic type, in which both the substrate and some other metabolite(s) may function as modulators.



Vitamins are nutrients required in tiny amounts for essential metabolic reactions in the body. The term vitamin does not include other essential nutrients such as dietary minerals, essential fatty acids, or essential amino acids, nor does it encompass the large number of other nutrients that promote health but that are not essential for life.

Image:La Boqueria.JPG

Vitamins are bio-molecules that act both as catalysts and substrates in chemical reactions. When acting as a catalyst, vitamins are bound to enzymes and are called cofactors. (For example, vitamin K forms part of the proteases involved in blood clotting.) Vitamins also act as coenzymes to carry chemical groups between enzymes. (For example, folic acid carries various forms of carbon groups–methyl, formyl or methylene–in the cell.).



Until the 1900s, vitamins were obtained solely through food intake. Many food sources contain different ratios of vitamins. Therefore, if the only source of vitamins is food, changes in diet will alter the types and amounts of vitamins ingested. However, as many vitamins can be stored by the body, short-term deficiencies (which, for example, could occur during a particular growing season) do not usually cause disease.

Vitamins have been produced as commodity chemicals and made widely available as inexpensive pills for several decades, allowing supplementation of the dietary intake.


Difference from water soluble vitamins: water soluble vitamins are included into coenzymes, don't have provitamins, are not included into the membranes, and hypervitaminoses are not peculiar for them.

With exception of vitamin B6 and B12, they are readily excreted in urine without appreciable storage, so frequent consumption becomes necessary. They are generally nontoxic when present in excess of needs, although symptoms may be reported in people taking megadoses of niacin, vitamin C, or pyridoxine (vitamin B6). All the B vitamins function as coenzymes or cofactors, assisting in the activity of important enzymes and allowing energy-producing reactions to proceed normally. As a result, any lack of water-soluble vitamins mostly affects growing or rapidly metabolizing tissues such as skin, blood, the digestive tract, and the nervous system. Water-soluble vitamins are easily lost with overcooking.

Thiamin (Vitamin B1)



Thiamin (also spelled thiamine) is a water-soluble B vitamin, previously known as vitamin B1 or aneurine. Isolated and characterized in the 1930s, thiamin was one of the first organic compounds to be recognized as a vitamin. Thiamin occurs in the human body as free thiamin and as various  phosphorylated forms: thiamin monophosphate (TMP), thiamin triphosphate (TTP), and thiamin pyrophosphate (TPP), which is also known as thiamin diphosphate.



 Coenzyme function


Thiamin pyrophosphate (TPP) is a required  coenzyme for a small number of very important enzymes. The  synthesis of TPP from free thiamin requires magnesium, adenosine triphosphate (ATP), and the enzyme, thiamin pyrophosphokinase.

 Pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched chain ketoacid (BCKA) dehydrogenase each comprise a different enzyme complex found within cellular organelles called mitochondria. They  catalyze the  decarboxylation of pyruvate, α-ketoglutarate, and branched-chain amino acids to form acetyl-coenzyme A, succinyl-coenzyme A, and derivatives of branched chain amino acids, respectively; all products play critical roles in the production of energy from food. In addition to the thiamin coenzyme (TPP), each dehydrogenase complex requires a niacin-containing coenzyme (NAD), a riboflavin-containing coenzyme (FAD), and  lipoic acid.

 Transketolase catalyzes critical reactions in another metabolic pathway known as the pentose phosphate pathway. One of the most important intermediates of this pathway is ribose-5-phosphate, a phosphorylated 5-carbon sugar required for the synthesis of the high-energy ribonucleotides,  ATP and guanosine triphosphate (GTP). It is also required for the synthesis of the  nucleic acids,  DNA and RNA, and the niacin-containing coenzyme NADPH, which is essential for a number of biosynthetic reactions. Because transketolase decreases early in thiamin deficiency, measurement of its activity in red blood cells has been used to assess thiamin nutritional status.



 Beriberi, the disease resulting from severe thiamin deficiency, was described in Chinese literature as early. Thiamin deficiency affects the cardiovascular, nervous, muscular, and  gastrointestinal systems. Beriberi has been termed dry, wet, or cerebral, depending on the systems affected by severe thiamin deficiency.


Dry beriberi

The main feature of dry (paralytic or nervous) beriberi is peripheral neuropathy. Early in the course of the neuropathy, "burning feet syndrome" may occur. Other symptoms include abnormal (exaggerated) reflexes as well as diminished sensation and weakness in the legs and arms. Muscle pain and tenderness and difficulty rising from a squatting position have also been observed. Severely thiamin deficient individuals may experience seizures.


Wet beriberi

 In addition to neurologic symptoms, wet (cardiac) beriberi is characterized by cardiovascular manifestations of thiamin deficiency, which include rapid heart rate, enlargement of the heart, severe swelling (edema), difficulty breathing, and ultimately  congestive heart failure.


Cerebral beriberi

 Cerebral beriberi may lead to Wernicke's encephalopathy and Korsakoff's psychosis, especially in people who abuse alcohol. The diagnosis of Wernicke's encephalopathy is based on a "triad" of signs, which include abnormal eye movements, stance and gait abnormalities, and abnormalities in mental function that may include a confused apathetic state or a profound memory disorder termed Korsakoff's amnesia or Korsakoff's psychosis. Thiamin deficiency affecting the central nervous system is referred to as Wernicke's disease when the amnesic state is not present and Wernicke-Korsakoff syndrome (WKS) when the amnesic symptoms are present along with the eye movement and gait disorders. Most WKS sufferers are alcoholics, although it has been observed in other disorders of gross malnutrition, including stomach cancer and AIDS. Administration of intravenous thiamin to WKS patients generally results in prompt improvement of the eye symptoms, but improvements in motor coordination and memory may be less, depending on how long the symptoms have been present. Recent evidence of increased immune cell activation and increased free radical production in the areas of the brain that are selectively damaged suggests that  oxidative stress plays an important role in the neurologic pathology of thiamin deficiency.


Causes of thiamin deficiency

 Thiamin deficiency may result from inadequate thiamin intake, increased requirement for thiamin, excessive loss of thiamin from the body, consumption of anti-thiamin factors in food, or a combination of these factors.


Inadequate intake

 Inadequate consumption of thiamin is the main cause of thiamin deficiency in underdeveloped countries. Thiamin deficiency is common in low-income populations whose diets are high in  carbohydrate and low in thiamin (e.g., milled or polished rice). Breast-fed infants whose mothers are thiamin deficient are vulnerable to developing infantile beriberi. Alcoholism, which is associated with low intake of thiamin among other nutrients, is the primary cause of thiamin deficiency in industrialized countries.


Increased requirement


Conditions resulting in an increased requirement for thiamin include strenuous physical exertion, fever, pregnancy, breast-feeding, and adolescent growth. Such conditions place individuals with marginal thiamin intake at risk for developing symptomatic thiamin deficiency. Recently, malaria patients in Thailand were found to be severely thiamin deficient more frequently than non-infected individuals. Malarial infection leads to a large increase in the metabolic demand for glucose. Because thiamin is required for enzymes involved in glucose metabolism, the stresses induced by malarial infection could exacerbate thiamin deficiency in predisposed individuals. HIV-infected individuals, whether or not they had developed AIDS, were also found to be at increased risk for thiamin deficiency. The lack of association between thiamin intake and evidence of deficiency in these HIV-infected individuals suggests that they had an increased requirement for thiamin. Further, chronic alcohol abuse impairs intestinal absorption and utilization of thiamin; thus, alcoholics have increased requirements for thiamin.


Excessive loss


Excessive loss of thiamin may precipitate thiamin deficiency. By increasing urinary flow,  diuretics may prevent reabsorption of thiamin by the kidneys and increase its excretion in the urine, although this remains quite controversial. Individuals with kidney failure requiring  hemodialysis lose thiamin at an increased rate and are at risk for thiamin deficiency. Alcoholics who maintain a high fluid intake and urine flow rate may also experience increased loss of thiamin, exacerbating the effects of low thiamin intake.


Anti-thiamin factors (ATF)


The presence of anti-thiamin factors (ATF) in foods also contributes to the risk of thiamin deficiency. Certain plants contain ATF, which react with thiamin to form an oxidized, inactive product. Consuming large amounts of tea and coffee (including decaffeinated), as well as chewing tea leaves and betel nuts, have been associated with thiamin depletion in humans due to the presence of ATF. Thiaminases are enzymes that break down thiamin in food. Individuals who habitually eat certain raw freshwater fish, raw shellfish, and ferns are at higher risk of thiamin deficiency because these foods contain thiaminase that normally is inactivated by heat in cooking. In Nigeria, an acute neurologic syndrome (seasonal ataxia) has been associated with thiamin deficiency precipitated by a thiaminase in African silkworms, a traditional high-protein food for some Nigerians.



 Food sources


A varied diet should provide most individuals with adequate thiamin to prevent deficiency. In the U.S. the average dietary thiamin intake for young adult men is about 2 mg/day and 1.2 mg/day for young adult women. A survey of people over the age of 60 found an average dietary thiamin intake of 1.4 mg/day for men and 1.1 mg/day for women. However, institutionalization and poverty both increase the likelihood of inadequate thiamin intake in the elderly.Whole grain cereals, legumes (e.g., beans and lentils), nuts, lean pork, and yeast are rich sources of thiamin. Because most of the thiamin is lost during the production of white flour and polished (milled) rice, white rice and foods made from white flour (e.g., bread and pasta) are fortified with thiamin in many Western countries. A number of thiamin-rich foods are listed in the table below along with their thiamin content in milligrams (mg). For more information on the nutrient content of foods, search the  USDA food composition database.




Thiamin (mg)

Lentils (cooked)

1/2 cup


Peas (cooked)

1/2 cup


Long grain brown rice (cooked)

1 cup


Long grain white rice, enriched (cooked)

1 cup


Long grain white rice, unenriched (cooked)

1 cup


Whole wheat bread

1 slice


White bread, enriched

1 slice


Fortified breakfast cereal

1 cup


Wheat germ breakfast cereal

1 cup


Pork, lean (cooked)

3 ounces*


Brazil nuts

1 ounce



1 ounce


Spinach (cooked)

1/2 cup



1 fruit



1/2 fruit



1 cup


Egg (cooked)

1 large


Riboflavin (Vitamin B2)



Riboflavin is a water-soluble B vitamin, also known as vitamin B2. In the body, riboflavin is primarily found as an integral component of the coenzymes, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). Coenzymes derived from riboflavin are termed flavocoenzymes, and  enzymes that use a flavocoenzyme are called flavoproteins.


 Oxidation-reduction (redox) reactions


Living organisms derive most of their energy from oxidation-reduction (redox) reactions, which are processes that involve the transfer of electrons. Flavocoenzymes participate in redox reactions in numerous metabolic pathways. Flavocoenzymes are critical for the  metabolism of carbohydrates, fats, and proteins. FAD is part of the  electron transport (respiratory) chain, which is central to energy production. In conjunction with  cytochrome P-450, flavocoenzymes also participate in the metabolism of drugs and toxins.


Antioxidant functions


Glutathione reductase is a FAD-dependent enzyme that participates in the  redox cycle of glutathione. The glutathione redox cycle plays a major role in protecting organisms from  reactive oxygen species, such as hydroperoxides. Glutathione reductase requires FAD to regenerate two molecules of reduced glutathione from oxidized glutathione. Riboflavin deficiency has been associated with increased oxidative stress. Measurement of glutathione reductase activity in red blood cells is commonly used to assess riboflavin nutritional status.


Glutathione peroxidase, a selenium-containing enzyme, requires two molecules of reduced glutathione to break down hydroperoxides (see diagram).


Xanthine oxidase, another FAD-dependent enzyme,  catalyzes the oxidation of hypoxanthine and xanthine to uric acid. Uric acid is one of the most effective water-soluble  antioxidants in the blood. Riboflavin deficiency can result in decreased xanthine oxidase activity, reducing blood uric acid levels.


Nutrient Interactions


B-complex vitamins


Because flavoproteins are involved in the metabolism of several other vitamins (vitamin B6, niacin, and folic acid), severe riboflavin deficiency may affect many enzyme systems. Conversion of most naturally available vitamin B6 to its coenzyme form, pyridoxal 5'-phosphate (PLP), requires the FMN-dependent enzyme, pyridoxine 5'-phosphate oxidase (PPO). At least two studies in the elderly have documented significant interactions between indicators of vitamin B6 and riboflavin nutritional status. The  synthesis of the niacin-containing coenzymes, NAD and NADP, from the  amino acid, tryptophan, requires the FAD-dependent enzyme, kynurenine mono-oxygenase. Severe riboflavin deficiency can decrease the conversion of tryptophan to NAD and NADP, increasing the risk of niacin deficiency. Methylene tetrahydrofolate reductase (MTHFR) is a FAD-dependent enzyme that plays an important role in maintaining the specific folate coenzyme required to form  methionine from  homocysteine (see  diagram). Along with other B vitamins, increased riboflavin intake has been associated with decreased plasma homocysteine levels. Recently, increased plasma riboflavin levels were associated with decreased plasma homocysteine levels, mainly in individuals  homozygous for the C677T  polymorphism of the MTHFR gene and in individuals with low folate intake. Such results illustrate that chronic disease risk may be influenced by complex interactions between genetic and dietary factors.



Riboflavin deficiency alters iron metabolism. Although the mechanism is not clear, research in animals suggests that riboflavin deficiency may impair iron absorption, increase intestinal loss of iron, and/or impair iron utilization for the synthesis of hemoglobin. In humans, improving riboflavin nutritional status has been found to increase circulating hemoglobin levels. Correction of riboflavin deficiency in individuals who are both riboflavin and iron deficient improves the response of iron-deficiency anemia to iron therapy.




Ariboflavinosis is the medical name for clinical riboflavin deficiency. Riboflavin deficiency is rarely found in isolation; it occurs frequently in combination with deficiencies of other water-soluble vitamins. Symptoms of riboflavin deficiency include sore throat, redness and swelling of the lining of the mouth and throat, cracks or sores on the outsides of the lips (cheliosis) and at the corners of the mouth (angular stomatitis), inflammation and redness of the tongue (magenta tongue), and a moist, scaly skin inflammation (seborrheic dermatitis). Other symptoms may involve the formation of blood vessels in the clear covering of the eye (vascularization of the cornea) and decreased red blood cell count in which the existing red blood cells contain normal levels of hemoglobin and are of normal size (normochromic normocytic anemia). Severe riboflavin deficiency may result in decreased conversion of vitamin B6 to its  coenzyme form (PLP) and decreased conversion of tryptophan to niacin (see Nutrient Interactions).**http%3A/**http%3A/


Preeclampsia is defined as the presence of elevated blood pressure, protein in the urine, and  edema (significant swelling) during pregnancy. About 5% of women with preeclampsia may progress to eclampsia, a significant cause of maternal death. Eclampsia is characterized by seizures, in addition to high blood pressure and increased risk of hemorrhage (severe bleeding). A study in 154 pregnant women at increased risk of preeclampsia found that those who were riboflavin deficient were 4.7 times more likely to develop preeclampsia than those who had adequate riboflavin nutritional status. The cause of preeclampsia-eclampsia is not known. Decreased intracellular levels of flavocoenzymes could cause  mitochondrial dysfunction, increase  oxidative stress, and interfere with nitric oxide release and thus blood vessel dilation—all of these changes have been associated with preeclampsia. However, a small  randomized, placebo-controlled, double-blind trial in 450 pregnant women at high risk for preeclampsia found that supplementation with 15 mg of riboflavin daily did not prevent the condition.


Risk factors for riboflavin deficiency


Alcoholics are at increased risk for riboflavin deficiency due to decreased intake, decreased absorption, and impaired utilization of riboflavin. Additionally, anorexic individuals rarely consume adequate riboflavin, and lactose intolerant individuals may not consume milk or other dairy products which are good sources of riboflavin. The conversion of riboflavin into FAD and FMN is impaired in  hypothyroidism and  adrenal insufficiency. Further, people who are very active physically (athletes, laborers) may have a slightly increased riboflavin requirement. However, riboflavin supplementation has not generally been found to increase exercise tolerance or performance.


Disease Prevention


 Age-related  cataracts are the leading cause of visual disability in the U.S. and other developed countries. Research has focused on the role of nutritional  antioxidants because of evidence that light-induced oxidative damage of  lens proteins may lead to the development of age-related cataracts. A  case-control study found significantly decreased risk of age-related cataract (33% to 51%) in men and women in the highest  quintile of dietary riboflavin intake (median of 1.6 to 2.2 mg/day) compared to those in the lowest quintile (median of 0.08 mg/day in both men and women) (17). Another case-control study reported that individuals in the highest quintile of riboflavin nutritional status, as measured by red blood cell glutathione reductase activity, had approximately one half the occurrence of age-related cataract as those in the lowest quintile of riboflavin status, though the results were not statistically significant (18). A cross-sectional study of 2,900 Australian men and women, 49 years of age and older, found that those in the highest quintile of riboflavin intake were 50% less likely to have cataracts than those in the lowest quintile. A  prospective study of more than 50,000 women did not observe a difference between rates of cataract extraction between women in the highest quintile of riboflavin intake (median of 1.5 mg/day) and women in the lowest quintile (median of 1.2 mg/day). However, the range between the highest and lowest quintiles was small, and median intake levels for both quintiles were above the current RDA for riboflavin. A recent study in 408 women found that higher dietary intakes of riboflavin were inversely associated with five-year change in lens opacification. Although these observational studies provide support for the role of riboflavin in the prevention of cataracts, placebo-controlled intervention trials are needed to confirm the relationship.


Disease Treatment


Migraine headaches

Some evidence indicates that impaired  mitochondrial oxygen metabolism in the brain may play a role in the pathology of  migraine headaches. Because riboflavin is the precursor of the two flavocoenzymes (FAD and FMN) required by the flavoproteins of the mitochondrial  electron transport chain, supplemental riboflavin has been investigated as a treatment for migraine. A randomized placebo-controlled trial examined the effect of 400 mg of riboflavin/day for three months on migraine prevention in 54 men and women with a history of recurrent migraine headaches. Riboflavin was significantly better than placebo in reducing attack frequency and the number of headache days, though the beneficial effect was most pronounced during the third month of treatment. A more recent study by the same investigators found that treatment with either a medication called a beta-blocker or high-dose riboflavin resulted in clinical improvement, but each therapy appeared to act on a distinct pathological mechanism: beta-blockers on abnormal cortical information processing and riboflavin on decreased brain mitochondrial energy reserve. A small study in 23 patients reported a reduction in median migraine attack frequency after supplementation with 400 mg of riboflavin daily for three months. Additionally, a 3-month randomized, double-blind, placebo-controlled study that administered a combination of riboflavin (400 mg/day), magnesium, and feverfew to migraine sufferers reported no therapeutic benefit beyond that associated with taking a placebo containing 25 mg/day of riboflavin). Compared to baseline measurements in this trial, both the placebo and treatment groups experienced some benefits with respect to the mean number of migraines, migraine days, or migraine index. Although these findings are preliminary, data from most studies to date suggest that riboflavin supplementation might be a useful adjunct to pharmacologic therapy in migraine prevention.



 Food sources

 Most plant and animal derived foods contain at least small quantities of riboflavin. In the U.S., wheat flour and bread have been enriched with riboflavin (as well as thiamin, niacin, and iron) since 1943. Data from large dietary surveys indicate that the average intake of riboflavin for men is about 2 mg/day and for women is about 1.5 mg/day; both intakes are well above the RDA. Intake levels were similar for a population of elderly men and women (1). Riboflavin is easily destroyed by exposure to light. For instance, up to 50% of the riboflavin in milk contained in a clear glass bottle can be destroyed after two hours of exposure to bright sunlight. Some foods with substantial amounts of riboflavin are listed in the table below along with their riboflavin content in milligrams (mg). For more information on the nutrient content of foods, search the  USDA food composition database.




Riboflavin (mg)

Fortified cereal

1 cup

0.59 to 2.27

Milk (nonfat)

1 cup (8 ounces)


Cheddar cheese

1 ounce


Egg (cooked)

1 large



1 ounce


Salmon (cooked)

3 ounces*


Halibut (broiled)

3 ounces


Chicken, light meat (roasted)

3 ounces


Chicken, dark meat (roasted)

3 ounces


Beef (cooked)

3 ounces


Broccoli (boiled)

1/2 cup chopped


Asparagus (boiled)

6 spears


Spinach (boiled)

1/2 cup


Bread, whole wheat

1 slice


Bread, white (enriched)

1 slice





The most common forms of riboflavin available in supplements are riboflavin and riboflavin 5'-monophosphate. Riboflavin is most commonly found in multivitamin and vitamin B-complex preparations (26).




 No toxic or adverse effects of high riboflavin intake in humans are known. Studies in cell culture indicate that excess riboflavin may increase the risk of  DNA strand breaks in the presence of chromium (VI), a known  carcinogen (27). This may be of concern to workers exposed to chrome, but no data in humans are available. High-dose riboflavin therapy has been found to intensify urine color to a bright yellow (flavinuria), but this is a harmless side effect. The Food and Nutrition Board did not establish a tolerable upper level of intake (UL) when the RDA was revised in 1998 (1).


Drug interactions


Several early reports indicated that women taking high-dose oral contraceptives (OC) had diminished riboflavin nutritional status. However, when investigators controlled for dietary riboflavin intake, no differences between OC users and non-users were found (1). Phenothiazine derivatives like the anti-psychotic medication chlorpromazine and tricyclic antidepressants inhibit the incorporation of riboflavin into FAD and FMN, as do the anti-malarial medication, quinacrine, and the cancer chemotherapy agent, adriamycin (4). Long-term use of the anti-convulsant, phenobarbitol may increase destruction of riboflavin, by liver enzymes, increasing the risk of deficiency (3).


Niacin (Vitamin B5)



Nicotinic Acid


Niacin exists in two forms, nicotinic acid and nicotinamide. Both forms are readily absorbed from the stomach and the small intestine. Niacin is stored in small amounts in the liver and transported to tissues, where it is converted to coenzyme forms. Any excess is excreted in urine. Niacin is one of the most stable of the B vitamins. It is resistant to heat and light, and to both acid and alkali environments. The human body is capable of converting the amino acid tryptophan to niacin when needed. However, when both tryptophan and niacin are deficient, tryptophan is used for protein synthesis.

Structure of NAD+


There are two coenzyme forms of niacin: nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phophate (NADP+). They both help break down and utilize proteins, fats, and carbohydrates for energy. Niacin is essential for growth and is involved in hormone synthesis.

Pellagra results from a combined deficiency of niacin and tryptophan. Long-term deficiency leads to central nervous system dysfunction manifested as confusion, apathy, disorientation, and eventually coma and death. Pellagra is rarely seen in industrialized countries, where it may be observed in people with rare disorder of tryptophan metabolism (Hartnup's disease), alcoholics, and those with diseases that affect food intake.


 The liver can synthesize niacin from the essential aminoacid  tryptophan, but the synthesis is extremely slow; 60 mg of tryptophan are required to make one milligram of niacin. Dietary niacin deficiency tends to occur only in areas where people eat corn, the only grain low in niacin, as a staple food, and that don't use lime during maize (corn) meal/flour production. Alkali lime releases the tryptophan from the corn so that it can be absorbed in the gut, and converted to niacin.;_ylu=X3oDMTA4NDgyNWN0BHNlYwNwcm9m/SIG=11sgnhnkl/EXP=1175707612/**http%3A/

Niacin plays an important role in the production of several sex and stress-related hormones, particularly those made by the adrenal gland. Niacin, when taken in large doses, increases the level of high density lipoprotein (HDL) or "good" cholesterol in blood, and is sometimes prescribed for patients with low HDL, and at high risk of heart attack. Niacin (but not niacinamide) is also used in the treatment of hyperlipidemia because it reduces very low density lipoprotein (VLDL), a precursor of low density lipoprotein (LDL) or "bad" cholesterol, secretion from the liver, and inhibits cholesterol synthesis.

The main problem with the clinical use of niacin for dyslipidemia is the occurrence of skin flushing, even with moderate doses.

Recommended intake is expressed as milligrams of niacin equivalents (NE) to account for niacin synthesized from tryptophan. High doses taken orally as nicotinic acid at 1.5 to 2 grams per day can decrease cholesterol and triglyceride levels, and along with diet and exercise can slow or reverse the progression of heart disease. 


" No Flush vitamin b3, niacin.(Strenght  not  exactly  as  Shown  on  bottle.) "


The nicotinamide form of niacin in multivitamin and B-complex tablets do not work for this purpose. Supplementation should be under a physician's guidance.


Food sources

 Good sources of niacin include yeast, meat, poultry, red fishes (e.g., tuna, salmon), cereals (especially fortified cereals), legumes, and seeds. Milk, green leafy vegetables, coffee, and tea also provide some niacin. In plants, especially mature cereal grains like corn and wheat, niacin may be bound to sugar molecules in the form of glycosides, which significantly decrease niacin bioavailability.


In the United States, the average dietary intake of niacin is about 30 mg/day for young adult men and 20 mg/day for young adult women. In a sample of adults over the age of 60, men and women were found to have an average dietary intake of 21 mg/day and 17 mg/day, respectively. Some foods with substantial amounts of niacin are listed in the table below along with their niacin content in milligrams (mg). Food composition tables generally list niacin content without including niacin equivalents (NE) from tryptophan, or any adjustment for niacin bioavailability. For more information on the nutrient content of specific foods, search the  USDA food composition database; data included in the table below are from this database.




Niacin (mg)

Chicken (light meat)

3 ounces* (cooked without skin)


Turkey (light meat)

3 ounces (cooked without skin)


Beef (lean)

3 ounces (cooked)


Salmon (chinook)

3 ounces (cooked)


Tuna (light, packed in water)

3 ounces


Bread (whole wheat)

1 slice


Cereal (unfortified)

1 cup


Cereal (fortified)

1 cup


Pasta (enriched)

1 cup (cooked)



1 ounce (dry roasted)



1 cup (cooked)


Lima beans

1 cup (cooked)


Coffee (brewed)

1 cup



Common side effects of nicotinic acid include flushing, itching, and gastrointestinal disturbances such as nausea and vomiting. Hepatotoxicity (liver cell damage), including elevated liver enzymes and jaundice, has been observed at intakes as low as 750 mg of nicotinic acid/day for less than three months (34, 35). Hepatitis has been observed with timed-release nicotinic acid at dosages as little as 500 mg/day for two months, although almost all reports of severe hepatitis have been associated with the timed-release form of nicotinic acid at doses of 3 to 9 grams per day used to treat high cholesterol for months or years (8). Immediate-release (crystalline) nicotinic acid appears to be less toxic to the liver than extended release forms. Immediate-release nicotinic acid is often used at higher doses than timed-release forms, and severe liver toxicity has occurred in individuals who substituted timed-release niacin for immediate-release niacin at equivalent doses (33). Skin rashes and dry skin have been noted with nicotinic acid supplementation. Transient episodes of low blood pressure (hypotension) and headache have also been reported. Large doses of nicotinic acid have been observed to impair glucose tolerance, likely due to decreased insulin sensitivity. Impaired glucose-tolerance in susceptible (pre-diabetic) individuals could result in elevated blood glucose levels and clinical diabetes. Elevated blood levels of uric acid, occasionally resulting in attacks of gout in susceptible individuals, have also been observed with high-dose nicotinic acid therapy (34). Nicotinic acid at doses of 1.5 to 5 grams/day has resulted in a few case reports of blurred vision and other eye problems, which have generally been reversible upon discontinuation. People with abnormal liver function or a history of liver disease, diabetes, active peptic ulcer disease, gout, cardiac arrhythmias, inflammatory bowel disease, migraine headaches, and alcoholism may be more susceptible to the adverse effects of excess nicotinic acid intake than the general population (8).



 Nicotinamide is generally better tolerated than nicotinic acid. It does not generally cause flushing. However, nausea, vomiting, and signs of liver toxicity (elevated liver enzymes, jaundice) have been observed at doses of 3 grams/day (33). Nicotinamide has resulted in decreased insulin sensitivity at doses of 2 grams/day in adults at high risk for insulin-dependent diabetes


Pantothenic Acid (Vitamin B3)

Pantothenic Acid

Pantothenic Acid


Pantothenic acid, also called vitamin B3, is a water-soluble vitamin required to sustain life. Pantothenic acid is needed to form coenzyme-A (CoA), and is critical in the metabolism and synthesis of carbohydrates, proteins, and fats. Its name is derived from the Greek pantothen meaning "from everywhere" and small quantities of pantothenic acid are found in nearly every food, with high amounts in whole grain cereals, legumes, eggs, meat, and royal jelly.

Pantothenic acid is stable in moist heat. It is destroyed by vinegar (acid), baking soda (alkali), and dry heat. Significant losses occur during the processing and refining of foods. Pantothenic acid is released from coenzyme A in food in the small intestine. After absorption, it is transported to tissues, where coenzyme A is resynthesized. Coenzyme A is essential for the formation of energy as adenosine triphosphate (ATP) from carbohydrate, protein, alcohol, and fat.


 Coenzyme A is also important in the synthesis of fatty acids, cholesterol, steroids, and the neurotransmitter acetylcholine, which is essential for transmission of nerve impulses to muscles.

Coenzyme A


Dietary deficiency occurs in conjunction with other B-vitamin deficiencies. Pantothenic acid is used in the synthesis of coenzyme A (abbreviated as CoA). Coenzyme A may act as an acyl group carrier to form acetyl-CoA and other related compounds; this is a way to transport carbon atoms within the cell. The transfer of carbon atoms by coenzyme A is important in cellular respiration, as well as the biosynthesis of many important compounds such as fatty acids, cholesterol, and acetylcholine. Dietary deficiency occurs in conjunction with other B-vitamin deficiencies. In studies, experimentally induced deficiency in humans has resulted in headache, fatigue, impaired muscle coordination, abdominal cramps, and vomiting.

 In studies, experimentally induced deficiency in humans has resulted in headache, fatigue, impaired muscle coordination, abdominal cramps, and vomiting.


Biotin (Vitamin B8)


Biotin is a water soluble vitamin and a member of Vitamin B complex.  Also known as Vitamin H, Bios II, Co-enzyme R.  Its natural form is D-biotin.  It was isolated from liver in 1941 by Dr. Paul Gyorgy.



        co-enzyme in wide variety of body metabolic reactions

        needed for production of energy from carbohydrates, fats and proteins

        needed for interconversions

        essential for maintenance of healthy skin, hair, sweat glands, nerves, bone marrow and glands producing sex hormones


        Brewer's Yeast




        fish, fatty, white

        meats, especially pig liver and kidney



        wheat bran

        wheat germ

        wholemeal grains

        unpolished brown rice





        by newborn children being fed on dried milk

        during stress situations

        when on antibiotic therapy


        seborrheic dermatitis

        Leiner's Disease

        alopecia (hair falling out in handfuls)

        scalp disease

        skin complaints

        preventing cot death (given to babies)


        leaching into cooking

        drying of milk for baby foods


In babies:

        dry scaling of the scalp and face

        persistent diarrhea

In adults:


        diminished reflexes


        hair loss

        increase in blood cholesterol levels

        loss of appetite

        muscular pains


        pale, smooth tongue



        specific anemia

        deficiency may be induced by excessive intake of raw egg whites, which contain the protein Avidin which immobilizes Biotin


        toxicity unknown

High quality Vitamin B (Biotin) can be purchased from Global Herbal Supplies




Biotin is the most stable of B vitamins. It is commonly found in two forms: the free vitamin and the protein-bound coenzyme form called biocytin. Biotin is absorbed in the small intestine, and it requires digestion by enzyme biotinidase, which is present in the small intestine. Biotin is synthesized by bacteria in the large intestine, but its absorption is questionable. Biotincontaining coenzymes participate in key reactions that produce energy from carbohydrate and synthesize fatty acids and protein.

Avidin is a protein in raw egg white, which can bind to the biotin in the stomach and decrease its absorption. Therefore, consumption of raw whites is of concern due to the risk of becoming biotin deficient. Cooking the egg white, however, destroys avidin. Deficiency may develop in infants born with a genetic defect that results in reduced levels of biotinidase. In the past, biotin deficiency was observed in infants fed biotin-deficient formula, so it is now added to infant formulas and other baby foods.


Vitamin B6



Pyridoxal, pyridoxamine and pyridoxine are collectively known as vitamin B6. All three compounds are efficiently converted to the biologically active form of vitamin B6, pyridoxal phosphate. This conversion is catalyzed by the ATP requiring enzyme, pyridoxal kinase.

Pyridoxal Phosphate


Vitamin B6 is present in three forms: pyridoxal, pyridoxine, and pyridoxamine. All forms can be converted to the active vitamin-B6 coenzyme in the body. Pyridoxal phosphate (PLP) is the predominant biologically active form. Vitamin B6 is not stable in heat or in alkaline conditions, so cooking and food processing reduce its content in food. Both coenzyme and free forms are absorbed in the small intestine and transported to the liver, where they are phosphorylated and released into circulation, bound to albumin for transport to tissues. Vitamin B6 is stored in the muscle and only excreted in urine when intake is excessive.


Vitamin B6 Benefit



PLP participates in amino acid synthesis and the interconversion of some amino acids. It catalyzes a step in the synthesis of hemoglobin, which is needed to transport oxygen in blood. PLP helps maintain blood glucose levels by facilitating the release of glucose from liver and muscle glycogen. It also plays a role in the synthesis of many neurotransmitters important for brain function. This has led some physicians to prescribe megadoses of B6 to patients with psychological problems such as depression and mood swings, and to some women for premenstrual syndrome (PMS). It is unclear, however, whether this therapy is effective. PLP participates in the conversion of the amino acid tryptophan to niacin and helps avoid niacin deficiency. Pyridoxine affects immune function, as it is essential for the formation of a type of white blood cell.

Populations at risk of vitamin-B6 deficiency include alcoholics and elderly persons who consume an inadequate diet. Individuals taking medication to treat Parkinson's disease or tuberculosis may take extra vitamin B6 with physician supervision. Carpal tunnel syndrome, a nerve disorder of the wrist, has also been treated with large daily doses of B6. However, data on its effectiveness are conflicting.


Folic Acid, Folate, Folacin (Vitamin B9)




Folic Acid


Folacin or folate, as it is usually called, is the form of vitamin B9 naturally present in foods, whereas folic acid is the synthetic form added to fortified foods and supplements. Both forms are absorbed in the small intestine and stored in the liver. The folic acid form, however, is more efficiently absorbed and available to the body. When consumed in excess of needs, both forms are excreted in urine and easily destroyed by heat, oxidation, and light.


Vitamin B9 Benefits


Folic acid is a water soluble vitamin and is a member of the Vitamin B complex. Also known as Folacin, pteroyl-L-glutamic acid (PGA), vitamin Bc or vitamin M. Folic acid and its derivatives (mostly the tri and heptaglutamyl peptides) are widespread in nature. It is a specific growth factor for certain micro-organisms.  Found in yeast and liver in 1935.

All forms of this vitamin are readily converted to the coenzyme form called tetrahydrofolate (THFA), which plays a key role in transferring single-carbon methyl units during the synthesis of DNA and RNA, and in interconversions of amino acids. Folate also plays an important role in the synthesis of neurotransmitters. Meeting folate needs can improve mood and mental functions.



involved in the formation of new cells

involved in the metabolism of ribonucleic acids (RNA) and deoxyribonucleic acids (DNA), essential for protein synthesis, formation of blood and transmission of genetic code

essential during pregnancy to reduce the risk of neural tube defects (birth defects affecting the brain and/or spinal cord)essential for the normal growth and development of the fetus

involved in the biosynthesis of purines, serines and glycine

involved in some functions associated with Vitamin B12

necessary for building resistance to diseases in the thymus gland of new born babies and infants

may reduce the risk of cervical dysplasia

necessary for red blood cell production

Food Source


Brewers's Yeast

citrus fruits, peeled


fatty fish

fresh nuts

green leafy vegetables

meats, especially pig liver and kidney



pulses, such as lentils

roasted nuts

soy products, such as tofu

unpolished brown rice

wheat germ

wheat bran

wheat grains

Effective With




Pantothenic Acid

Vitamin C

Increased Intakes Needed

by alcohol drinkers

by the elderly

during pregnancy and breastfeeding

if taking contraceptive pill

if taking the drugs, Aspirin, Cholestyramine,  Isethionate, Isoniazid, Methotrexate,  Pentamidime, Phenytoin (may be neutralized), Primidone, Pyrimethamine, Triamterene,Trimethoprim

Used For

malabsorption in geriatric patients

megaloblastic anemia

mental deterioration



Destroyed By

leached into cooking water

processing and cooking of vegetables, fruits and dairy products

unstable to oxygen at high temperatures but protected by Vitamin C

Symptoms of Deficiency






Deficiency Leads To

Various conditions relating to childbirth:


birth defects, such as neural tube defect which causes spina bifida

hemorrhage following birth

premature birth

premature separation of the placenta from the uterus


As well as:

megaloblastic anemia (red blood cells are large and uneven with a shortened life span)

mild mental symptoms, such as forgetfulness and confusion

Symptoms of Toxicity

Folic Acid has a low toxicity but occasionally the following symptoms occur:

abdominal distension

flatulence (gas/wind)


loss of appetite



sleep disturbance

symptoms of fever

temperature rise

Long term high doses may cause Vitamin B12 losses from the body


Folate deficiency is one of the most common vitamin deficiencies. Early symptoms are nonspecific and include tiredness, irritability, and loss of appetite. Severe folate deficiency leads to macrocytic anemia, a condition in which cells in the bone marrow cannot divide normally and red blood cells remain in a large immature form called macrocytes. Large immature cells also appear along the length of the gastrointestinal tract, resulting in abdominal pain and diarrhea.


Vitamin B9 Source


Pregnancy is a time of rapid cell multiplication and DNA synthesis, which increases the need for folate. Folate deficiency may lead to neural tube defects such as spina bifida (failure of the spine to close properly during the first month of pregnancy) and anencephaly (closure of the neural tube during fetal development, resulting in part of the cranium not being formed). Seventy percent of these defects could be avoided by adequate folate status before conception, and it is recommended that all women of childbearing age consume at least 400 micrograms (μg) of folic acid each day from fortified foods and supplements. Other groups at risk of deficiency include elderly persons and persons suffering from alcohol abuse or taking certain prescription drugs.


Vitamin B12

Vitamin B12 is found in its free-vitamin form, called cyanocobalamin, and in two active coenzyme forms. Absorption of vitamin B12 requires the presence of intrinsic factor, a protein synthesized by acid-producing cells of the stomach. The vitamin is absorbed in the terminal portion of the small intestine called the ileum. Most of body's supply of vitamin B12 is stored in the liver.


Vitamin B12 Benefits


Vitamin B12



Vitamin B12 is defficiently conserved in the body, since most of it is secreted into bile and reabsorbed. This explains the slow development (about two years) of deficiency in people with reduced intake or absorption. Vitamin B12 is stable when heated and slowly loses its activity when exposed to light, oxygen, and acid or alkaline environments.


Vitamin B12 Eczema


Vitamin B12 coenzymes help recycle folate coenzymes involved in the synthesis of DNA and RNA, and in the normal formation of red blood cells. Vitamin B12 prevents degeneration of the myelin sheaths that cover nerves and help maintain normal electrical conductivity through the nerves.

Active center of tetrahydrofolate (THF). Note that the N5 position is the site of attachment of methyl groups, the N10 the site for attachment of formyl and formimino groups and that both N5 and N10 bridge the methylene and methenyl groups


Vitamin-B12 deficiency results in pernicious anemia, which is caused by a genetic problem in the production of intrinsic factor. When this occurs, folate function is impaired, leading to macrocytic anemia due to interference in normal DNA synthesis. Unlike folate deficiency, the anemia caused by vitamin-B12 deficiency is accompanied by symptoms of nerve degeneration, which if left untreated can result in paralysis and death.


Since vitamin B12 is well conserved in the body, it is difficult to become deficient from dietary factors alone, unless a person is a strict vegan and consumes a


diet devoid of eggs and dairy for several years. Deficiency is usually observed when B12 absorption is hampered by disease or surgery to the stomach or ileum, damage to gastric mucosa by alcoholism, or prolonged use of anti-ulcer medications that affect secretion of intrinsic factor. Agerelated decrease in stomach-acid production also reduces absorption of B12 in elderly persons. These groups are advised to consume fortified foods or take a supplemental form of vitamin B12.









For many years, choline was not considered a vitamin because the body makes enough of it to meet its needs in most age groups. However, research now shows that choline production in the body is not enough to cover requirements. Choline is not considered a B vitamin because it does not have a coenzyme function and the amount in the body is much greater than other B vitamins. Choline not only helps maintain the structural integrity of membranes surrounding every cell in the body, but also can play a role in nerve signaling, cholesterol transport, and energy metabolism. An "adequate intake" is 550 milligrams per day for men and 425 milligrams per day for women. Choline is widely found in foods, so it is unlikely that a dietary deficiency will occur.


Vitamin C (Ascorbic Acid)

Ascorbic Acid

 In 1746, James Lind, a British physician, conducted the first nutrition experiment on human beings in an effort to find a cure for scurvy.

James Lind


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James Lind (1716 – 1794),a British Royal Navy surgeon who, in 1774, identified that a quality in fruit prevented the disease of scurvy in what was the first recorded controlled experiment

However, it was not until nearly 200 years later that ascorbic acid, or vitamin C, was discovered. Vitamin C participates in many reactions by donating electrons as hydrogen atoms. In a reducing reaction, the electron in the hydrogen atom donated by vitamin C combines with other participating molecules, making vitamin C a reducing agent, essential to the activity of many enzymes. By neutralizing free radicals, vitamin C may reduce the risk of heart disease, certain forms of cancer, and cataracts.

Vitamin C is needed to form and maintain collagen, a fibrous protein that gives strength to connective tissues in skin, cartilage, bones, teeth, and joints. Collagen is also needed for the healing of wounds.

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When added to meals, vitamin C increases intestinal absorption of iron from plant-based foods. High concentration of vitamin C in white blood cells enables the immune system to function properly by providing protection against oxidative damage from free radicals generated during their action against bacterial, viral, or fungal infections.


Vitamin C Deficit


Vitamin C also recycles oxidized vitamin E for reuse in cells, and it helps folic acid convert to its active form, (THF). Vitamin C helps synthesize carnitine, adrenaline, epinephrine, the neurotransmitter serotonin, the thyroid hormone thyroxine, bile acids, and steroid hormones.


A deficiency of vitamin C causes widespread connective tissue changes throughout the body. Deficiencies may occur in people who eat few fruits and vegetables, follow restrictive diets, or abuse alcohol and drugs. Smokers also have lower vitamin-C status. Supplementation may be prescribed by physicians to speed the healing of bedsores, skin ulcers, fractures, burns, and after surgery. Research has shown that doses up to 1 gram per day may have small effects on duration and severity of the common cold, but not on the prevention of its occurrence.


Ascorbic acid

Ascorbic acid is required in the diet of only a few vertebrates — man, monkeys, the guinea pig, and certain fishes. Some insects and other invertebrates also require ascorbic acid, but most other higher animals and plants can synthesize ascorbic acid from glucose or other simple precursors. Ascorbic acid is not present in microorganisms, nor does it seem to be required.

Ascorbic acid is a strong reducing agent, readily losing hydrogen atoms to become dehydroascorbic acid, which also has vitamin C activity. However, vitamin activity is lost when the lactone ring of dehydroascorbic acid is hydrolyzed to yield diketogulonic acid.