Introduction into
biochemistry. Aminoacid
composition, structure, physical-chemical properties, classification and
functions of simple and complex proteins
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.
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Protein is crucial for tissue repair, building and preserving muscle,
and making important enzymes and hormones.
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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).
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Table 2.1 Classification of
Proteins According to biological function. |
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Type: |
Example: |
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Enzymes- Catalyze
biological reactions |
ß-galactosidase |
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Transport and
Storage |
Hemoglobin |
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Movement |
Actin |
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Immune Protection |
Immunoglobulins |
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Regulatory
Function within cells |
Transeription Factors |
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Hormones |
Insulin |
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Structural |
Collagen |
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.
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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.
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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.
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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.
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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)
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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:
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.
That means that the two
simplest amino acids, glycine and alanine, would be shown as:
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.
Either:
Or:
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:
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:
If you look carefully,
you will spot the abbreviations for glycine (Gly) and alanine (
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.
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.
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:
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.
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.
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.
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
The folded chains are again held together
by hydrogen bonds involving exactly the same groups as in the alpha-helix.
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.
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.
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
A 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 lipoproteins, glycoproteins, phosphoproteins,hemoproteins, flavoproteins, metalloproteins, phytochromes, cytochromes 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:
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.
Charge:
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.
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Hydrophobicity:
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.
Solubility:
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.
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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 |
|
|
Technique |
Protein Property Exploited |
|
Bulk
Methods |
|
|
Ammonium
sulfate precipitation |
Solubility |
|
Filtration |
Size |
|
Chromatography
Methods |
|
|
Ion-Exchange |
Charge |
|
Gel
Filtration (Gel Permeation) |
Size
or molecular wt. |
|
Hydrophobic
Interaction |
Hydrophobicity |
|
Affinity |
Biological
Activity |
|
Reversed
Phase |
Hydrophobicitiy |
|
Chromatofocusing |
pI
(Charge) |
|
Electrophoresis |
|
|
Native
Gel |
Mass/charge |
|
Denaturing
Gel (SDS-PAGE) |
Mass
(Molecular weight) |
|
IEF |
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.
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·
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 (
·
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.
Denaturation
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
|
|
Heat |
hydrogen bonds are broken by increased translational and
vibrational energy. |
|
Ultraviolet Radiation |
Similar to heat |
|
Strong Acids or Bases |
salt formation; disruption of hydrogen bonds. |
|
Urea Solution |
competition for hydrogen bonds. |
|
Some Organic Solvents |
change in dielectric constant and hydration of ionic
groups. |
|
Agitation |
shearing of hydrogen bonds. |
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.
Spectroscopy
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
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-
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: |
|
Globular Proteins |
|
Members
of this class serve regulatory, maintenance and catalytic roles in living
organisms. |
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.
Chromatography.
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
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.
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
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.
QUALITATIVE
REACTIONS ON THE PROTEINS AND AMINO ACIDS
Biuret test. The protein is warmed gently with
10 % solution of sodium hydroxide and then à drop of very
dilute copper sulphate solution is added, the formation of
reddish - violet colour indicates the presence of peptide
link, – ÑÎ – 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, Í2N –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 (à solution of mercuric
and mercurous nitrates in nitric acid containing à little nitrous
acid) followed by heating the solution give à 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 à protein solution and the mixture is heated to boil, blue to
violet colour appears on cooling. The colour is due to the
formation of à complex compound.
The test is
also given by ammonia, ammonium salts, and certain
amines. Ninhydrin is also used as à reagent for the quantitative
determination of free carboxyl groups in solutions of amino acids.
Nitroprusside test. Proteins containing
free -SH groups (of cysteine) give à reddish colour with sodium nitroprusside in ammonical solution.
Proteins are
polypeptides that contain more than 50 amino acid units. The dividing line
between à polypeptide and à protein is arbitrary. The
important point is that proteins are polymers containing à 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.