1. α-Amino acids as structure components of proteins. Chromatographic analysis of amino acids

1. α-Amino acids as structure components of proteins. Chromatographic analysis of amino acids

2. Hydrolysis of simple proteins. Precipitation reactions of proteins.

3. Determination of the total protein contents in blood serum.

Next to water, proteins are the most abundant substances in most cells – from 10% to 20% of the cell’s mass. All proteins contain the elements carbon, hydrogen, oxygen, and nitrogen; most also contain sulfur. The presence of nitrogen in proteins sets them apart from carbohydrates and lipids, which generally do not contaiitrogen. The average nitrogen content of proteins is 15.4% by mass. Other elements, such as phosphorus and iron, are essential constituents of certain specialized proteins.

Casein, the main protein of milk, contains phosphorus, an element very important in the diet of infants and children. Hemoglobin, the oxygen-transporting protein of blood, contains iron.

Amino acids – the building blocks for proteins. The word protein comes from the Greek proteios, which means “of first importance.” This derivation alludes to the key role that proteins play in life processes.

А protein is in polymer in which the monomer units are amino acids. Thus the starting point for а discussion of proteins is an understanding of the structures and chemical properties of amino acids.

An amino acid is an organic compound that contains both an amino (–NН₃) group and a carboxyl (-СООН) group.

The amino acids found in proteins are always α-amino acids – that is, amino acids in which the amino group is attached to the α-carbon atom of the carboxylic acid carbon chain. The general structural formula for an α-amino acid is:

The R group present in an α-amino acid is called the amino acid side chain. The nature of this side chain distinguishes а-amino acids from each other. Side chains vary in size, shape, charge, acidity, functional groups present, hydrogen-bonding ability, and chemical reactivity.

Over 700 different naturally occurring amino acids are known, but only 20 of them, called standard amino acids, are normally present in proteins.

А standard amino acid is one of the 20 α-amino acids normally found in proteins. Amino acids are grouped according to side-chain polarity. In this system there are four categories: (1) nonpolar amino acids, (2) polar neutral amino acids, (3) polar acidic amino acids, and (4) polar basic amino acids. This classification system gives insights into how various types of amino acid side chains help determine the properties of proteins.

CLASSIFICATION OF AMINO ACIDS

Nonpolar amino acids contain one amino group, one carboxyl group, and a nonpolar side chain. When incorporated into а protein, such amino acids are hydrophobic (“water fearing”); that is, they are not attracted to water molecules. They are generally found in the interior of proteins, where there is limited contact with water. There are eight nonpolar amino acids.

The three types of polar amino acids have varying degrees of affinity for water. Within а protein, such amino acids are said to be hydrophilic (“water-loving”). Hydrophilic amino acids are often found on the surfaces of proteins.

Polar neutral amino acids contain one amino group, one carboxyl group, and а side chain that is polar but neutral. The side chain is neutral in that it is neither acidic nor basic in solution at physiological pH. There are seven polar neutral amino acids.

Polar acidic amino acids contain one amino group and two carboxyl groups, the second carboxyl group being part of the side chain. In solution at physiological рН, the side chain of а polar acidic amino acid bears а negative charge; the side-chain carboxyl group has lost its acidic hydrogen atom. There are two polar acidic amino acids: aspartic acid and glutamic acid.

Polar basic amino acids contain one amino groups and one carboxyl group, the second amino group being part of the side chain. In solution at physiological рН, the side chain of а polar basic amino acid bears а positive charge; the nitrogen atom of the amino group has accepted а proton. There are three polar basic amino acids: lysine, arginine, and histidine.

The names of the standard amino acids are often abbreviated using three-letter codes. Except in four cases, these abbreviations are the first three letters of the amino acid’s name. In addition, а new one-letter code for amino acid names is currently gaining popularity (particularly in computer applications). These abbreviations are used extensively when describing peptides and proteins, which contain tens and hundreds of amino acid units.

The essential amino acids. All of the 20 amino acids are necessary constituents of human protein. Adequate amounts of 11 of the 20 amino acids can be synthesized from carbohydrates and lipids in the body if а source of nitrogen is also available. Because the human body is incapable of producing 9 of these 20 acids fast enough or in sufficient quantities to sustaiormal growth, these 9 amino acids, called essential amino acids, must be obtained from food. Essential amino acids are amino acids that must be obtained from food. An adequate human diet must include foods that contain these essential amino acids.

The human body can synthesize small amounts of some of the essential amino acids, but not enough to meet its needs, especially in the case of growing children.

The 9 essential amino acids for adults are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. (In addition, arginine is essential for children).

А complete dietary protein contains all the essential amino acids in the same relative amounts in which human being require them. А complete dietary protein may or may not contain all the nonessential amino acids. Most animal proteins, including casein from milk and proteins found in meat, fish, and eggs, are complete proteins, although gelatin is an exception (it lacks tryptophan). Proteins from plants (vegetables, grains, and legumes) have quite diverse amino acid patterns, and some tend to be limiting in one or more essential amino acids. Some plant proteins (for example, corn protein) are far from complete. Others (for example, soy protein) are complete. Thus vegetarians must eat а variety of plant foods to obtain all of the essential amino acids in appropriate quantities.

Chirality and amino acids. Four different groups are attached to the α-carbon atom in all of the standard amino acids except glycine, where the R group is а hydrogen atom.

This means that the structures of 19 of the 20 standard amino acids possess а chiral center at this location, so enantiomeric forms (left- and right-handed forms) exist for each of these amino acids.

With few exceptions (in some bacteria), the amino acids found iature and in proteins are isomers. Thus, as is the case with monosaccharides, nature favors one mirror-image form over the other. Interestingly, for amino acids the L isomer is the preferred form, whereas for monosaccharides theisomer is preferred.

The rules for drawing Fischer projections for amino acid structures follow.

1. The – СООН group is put at the top of the projection, the R group at the bottom. This positions the carbon chain vertically.

2. The – NН₂ group is in а horizontal position. Positioning it on the left denotes the L isomer, and positioning it on the right denotes the D isomer.

Acid – base properties of amino acids. In pure form, amino acids are white crystalline solids with relatively high decomposition points. (Most amino acids decompose before they melt.) Also most amino acids are not very soluble in water because of strong intermolecular forces within their crystal structures. Such properties are those often exhibited by compounds in which charged species are present. Studies of amino acids confirm that they are charged species both in the solid state and in solution.

Both an acidic group (-СООН) and а basic group (-NН₂) are present on the same carbon in an α-amino acid.

We learned that ieutral solution, carboxyl groups have а tendency to lose protons (Н⁺), producing а negatively charged species:

–СООН = – СОО^– + Н⁺

We learned that ieutral solution, amino groups have а tendency to accept protons (Н⁺), producing а positively charged species:

–NH₂ + H⁺ == –NH₃⁺

As is consistent with the behavior of these groups, ieutral solution, the –СООН group of an amino acid donates а proton to the –NH₂ of the same amino acid. We can characterize this behavior as an internal acid — base reaction. The net result is that ieutral solution, amino acid molecules have the structure

Such а molecule is known as а zwitterion, from the German term meaning “double ion”. А zwitterion is а molecule that has а positive charge on one atom and а negative charge on another atom. Note that the net charge on а zwitterion is zero even though parts of the molecule carry charges. In solution and also in the solid state, α-amino acids are zwitterions.

Zwitterion structure changes when the pH of а solution containing an amino acid is changed from neutral either to acidic (low pH) by adding an acid such as НС1 or to basic (high pH) by adding а base such as NaOH. In an acidic solution, the zwitterion accepts а proton (Н⁺) to form а positively charged ion.

In basic solution, the –NH₃⁺ of the zwitterion loses а proton, and а negatively charged species is formed.

Thus, in solution, three different amino acid forms can exist (zwitterion, negative ion, and positive ion). The three species are actually in equilibrium with each other, and the equilibrium shifts with pH change. The overall equilibrium process can be represented as follows:

In acidic solution, the positively charged species on the left predominates; nearly neutral solutions have the middle species (the zwitterion) as the dominant species; in basic solution, the negatively charged species on the right predominates.

The previous discussion assumed that the side chain (R group) of an amino acid remains unchanged in solution as the pH is varied. This is the case for neutral amino acids but not for acidic or basic ones. For these latter compounds, the side chain can also acquire а charge, because it contains an amino or а carboxyl group that can, respectively, gain or lose а proton.

Because of the extra site that can be protonated or deprotonated, acidic and basic amino acids have four charged forms in solution.

The existence of two low-pH forms for aspartic acid results from the two carboxyl groups being deprotonated at different pH values. For basic amino acids, two high-pH forms exist because deprotonation of the amino groups does not occur simultaneously. The side-chain amino group deprotonates before the α-amino group.

The isoelectric point for an amino acid is the pH at which the total charge on the amino acid is zero. Every amino acid has а different isoelectric point. Fifteen of the 20 amino acids, those with nonpolar or polar neutral side chains, have isoelectric points in the range of 4.8 – 6.3. The three basic amino acids have higher isoelectric points (His = 7.59, Lys = 9.74, Arg = 10.76), and the two acidic amino acids have lower ones (Asp = 2.77, Glu = 3.22).

А рН below the isoelectric point favors the positively charged form of the amino acid. Conversely, а рН above the isoelectric point favors the negatively charged form of the amino acid.

When two electrodes (one positively charged and one negatively charged) are immersed in а solution containing an amino acid, molecules with а net positive charge are attracted to the negatively charged electrode, and negatively charged amino acid molecules migrate toward the positively charged electrode. The zwitterion form exhibits no net migration toward either electrode. This behavior is the basis for the measurement of isoelectric points. The pH of the solution is adjusted until no net migration occurs.

Mixtures of amino acids in solution can be separated by using their different migration patterns at various pH values. This type of analytical separation is called electrophoresis. Electrophoresis is the process of separating charged molecules on the basis of their migration toward charged electrodes.

REACTION OF AMINO ACIDS.

i) Reaction with alcohols – esters formation:

ii)                Reaction with ammonia – amides formation. The amides of aspartic and glutamic acid acids, asparagine and glutamine, play important role in the transport of ammonia in the body.

iii)  Decarboxylation. Amino acids may be decarboxylated by heat, acids, bases or specific enzymes to the primary amines:

Some of the decarboxylation reaction are of great importance in the body, decarboxylation of histidine to histamine:

In the presence of foreign protein introduced into the body, very large quantities of histamine are produced in the body and allergic reactions become evident. In extreme cases shock may result. The physiological effects of histamine may be neutralized or minimized by the use of chemical compounds known as antihistamines.

iv)   Salts are formed. All amino acids can react with some inorganic acids and bases and form two kind sold:

v)  Deamination:

1.     oxidation deamination – important pathway for the biodegradation of α-amino acids:

2.     hydrolitic deamination – reaction with nitrous acid. Amino acids react with nitrous acid to give hydroxy acid along with the evolution of nitrogen.

The nitrogen can be collected and measured. Thus this reaction constitutes one of the methods for the estimation of amino acids.

3.     intramolecular deamination – unsaturated acids are formed

4.     redaction deamination – saturated carboxylic acid formation:

vi)   Transamination:

vii)           Peptide formation. Two amino acids can react in а similar way – the carboxyl group of one amino acid reacts with the amino group of the other amino acid. The products are а molecule of water and а molecule containing the two amino acids linked by an amide bond.

Removal of the elements of water from the reacting carboxyl and amino groups and the ensuing formation of the amide bond are better visualized when expanded structural formulas for the reacting groups are used.

In amino acid chemistry, amide bonds that link amino acids together are given the specific name of peptide bond. А peptide bond is а bond between the carboxyl group of one amino acid and the amino group of another amino acid.

Under proper conditions, many amino acids can bond together to give chains of amino acids containing numerous peptide bonds. For example, four peptide bonds are present in а chain of five amino acids.

Short to medium-sized chains of amino acids are known as peptides. А peptide is а sequence of amino acids, of up to 50 units, in which the amino acids are joined together through amide (peptide) bonds. А compound containing two amino acids joined by а peptide bond is specifically called а dipeptide; three amino acids in а chain constitute а tripeptide; and so on. The name oligopeptide is loosely used to refer to peptides with 10 to 20 amino acid residues and polypeptide to larger peptides.

In all peptides, the amino acid at one end of the amino acid sequence has а free H₃N⁺ group, and the amino acid at the other end of the sequence has а free СОО^– group. The end with the free H₃N⁺ group is called the N-terminal end, and the end with the free СОО^– group is called the С-terminal end. By convention, the sequence of amino acids in а peptide is written with the N-trminal end amino acid at the left. The individual amino acids within а peptide chain are called amino acid residues.

The structural formula for а polypeptide may be written out in full, or the sequence of amino acids present may be indicated by using the standard three-letter amino acid abbreviations. The abbreviated formula for the tripeptide:

which contains the amino acids glycine, alanine, and serine, is Gly – Ala – Ser. When we use this abbreviated notation, by convention, the amino acid at the N-terminal end of the peptide is always written on the left.

The repeating chain of peptide bonds and α-carbon atoms in а peptide is referred to as the backbone of the peptide. The R group side chains are substituents on the backbone.

Peptides that contain the same amino acids but in different order are different molecules (structural isomers) with different properties. For example, two different dipeptides can be formed from one molecule of alanine and one molecule of glycine.

In the first dipeptide, the alanine is the N-terminal residue, and in the second molecule, it is the С-terminal residue. These two compounds are isomers with different chemical and physical properties.

The number of isomeric peptides possible increases rapidly as the length of the peptide chain increases. Let us consider the tripeptide Ala – Ser – Cys as another example. In addition to this sequence, five other arrangements of these three components are possible, each representing another isomeric tripeptide: Ala – Cys – Ser, Ser –Ala – Cys, Ser – Cys – Ala, Cys – Ala – Ser, and Cys – Ser – Ala. For а pentapeptide containing 5 different amino acids, 120 isomers are possible.

More than two hundred peptides have been isolated and identified as essential to the proper functioning of the human body. In general, these substances serve as hormones or neurotransmitters. Their functions range from controlling pain to controlling muscle contraction or kidney fluid excretion.

Two important hormones produced by the pituitary gland are oxytocin and vasopressin, Each hormone is а nonapeptide (nine amino acid residues) with six of the residues hells in the form of а loop by а disulfide bond formed from the interaction of two cysteine residues.

Oxytocin regulates uterine contractions and lactation. Vasopressin regulates the excretion of water by the kidneys; it also affects blood pressure. The structure of vasopressin differs from that of oxytocin at only two amino acid positions: the third and eighth amino acid residues. The result of these variations is а significant difference in physiological action.

Endorphins are peptides that bind at receptor sites in the brain to reduce pain. These compounds are synthesized by the brain itself. А subclass of such molecules, the enkephalins, are simple pentapeptides. Two important enkephalins are methionitlen enkephalin (Tyr – Gly – Gly – Phe – Met ) and leucine enkephalin (Tyr – Gly – Gly – Phe – Leu).

The action of the prescription painkillers morphine and codeine is based on their binding at the same receptor sites in the brain as naturally occurring enkephalins.

QUALITATIVE REACTIONS ON THE PROTEINS AND AMINO ACIDS

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

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

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

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

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

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

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

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

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

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

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

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

The primary structure of а protein is the sequence of amino acids present in its peptide chain or chains. Knowledge of primary structure tells us which amino acids are present, the number of each, their sequence, and the length and number of polypeptide chains.

The first protein whose primary structure was determined was insulin, the hormone that regulates blood-glucose level; а deficiency of insulin leads to diabetes. The sequencing of insulin, which took over 8 years, was completed in 1953. Today, thousands of proteins have been sequenced; that is, researchers have determined the order of amino acids within the polypeptide chain or chains.

The primary structure of а specific protein is always the same, regardless of where the protein is found within an organism. The structures of certain proteins are even similar among different species of animals. For example, the primary structures of insulin in cows, pigs, sheep, and horses are very similar both to each other and to human insulin. Until recently, this similarity was particularly important for diabetics who required supplemental injections of insulin.

An analogy is often drawn between the primary structure of proteins and words. Words, which convey information, are formed when the 26 letters of the English alphabet are properly sequenced. Proteins, which function biologically, are formed from the proper sequence of 20 amino acids. The proper sequence of letters in а word is necessary for it to make sense, just as the proper sequence of amino acids is necessary to make biologically active protein. Furthermore, the letters that form а word are written from left to right, as are amino acids in protein formulas. As any dictionary of the English language will document, a tremendous variety of words can be formed by different letter sequences. Imagine the number of amino acid sequences possible for а large protein. There are 1.55×10⁶⁶ sequences possible for the 51 amino acids found in insulin! From these possibilities, the body reliably produces only one, illustrating the remarkable precision of life processes. From the simplest bacterium to the human brain cell, only those amino acid sequences needed by the cell are produced.

The secondary structure of а protein is the arrangement in space of the atoms in the backbone of the protein. Three major types of protein secondary structure are known; the alpha helix, the beta pleated sheet, and the triple helix. The major force responsible for all three types of secondary structure is hydrogen bonding between а carbonyl oxygen atom of а peptide linkage and the hydrogen atom of an amino group (-NH) of another peptide linkage farther along the backbone. This hydrogen-bonding interaction may be diagrammed as follows:

The Alpha Helix The alpha helix (α-helix) structure resembles а coiled helical spring, with the coil configuration maintained by hydrogen bonds between N – Н and С= О groups of every fourth amino acid, as is shown diagrammatically in Figure.2.

Figure. Three representations of (а) the а helix protein structure. Hydrogen bonds between amide groups (peptide linkages) are shown in (b) and (с). (d) The top view of an а helix shows that amino acid side chains (R groups) point away from the long axis of the helix.

Figure. Two representations of the p pleated sheet protein structure. (а) А representation emphasizing the hydrogen bonds between protein chains. (b) А representation emphasizing the pleats and the location of the R groups.

Proteins have varying amounts of α-helical secondary structure, ranging from а few percent to nearly 100 %. In an α-helix, all of the amino acid side chains (R groups) lie outside the helix; there is not enough room for them in the interior. Figure.3d illustrates this situation. This structural feature of the α-helix is the basis for protein tertiary structure.

The beta pleated sheet (β-pleated sheet) secondary structure involves amino acid chains that are almost completely extended. Hydrogen bonds form between two different side-by-side protein chains (interchain bonds) as shown in Figure.3, or between different parts of а single chain that folds back on itself (intrachain bonds).

The term pleated sheet arises from the repeated zigzag pattern in the structure (Figure.3b). Amino acid side chains are located above and below the plane of the sheet.

Very few proteins have entirelyhelix or p pleated sheet structures. Instead, most proteins have only certain portions of their molecules in these conformations. The rest of the molecule assumes а “random structure.” It is possible to have both а helix and p pleated sheet structures within the same protein.

Collagen, the structural protein of connective tissue (cartilage, tendon, and skin), has а triple-helix structure. Collagen molecules are very long, thin, and rigid. Many such molecules, lined up alongside each other, combine to make collagen fibers. Cross-linking gives the fibers extra strength.

The tertiary structure of а protein is the overall three-dimensional shape that results from the attractive forces between amino acid side chains (R groups) that are widely separated from each other within the chain.

А good analogy for the relationships among the primary, secondary, and tertiary structures of а protein is that of а telephone cord. The primary structure is the long, straight cord. The coiling of the cord into а helical arrangement gives the secondary structure. The supercoiling arrangement the cord adopts after you hang up the receiver is the tertiary structure.

Interactions responsible for tertiary structure. Four types of attractive interactions contribute to the tertiary structure of а protein:

(1) covalent disulfide bonds,

(2) electrostatic attractions (salt bridges),

(3) hydrogen bonds,

(4) hydrophobic attractions.

All four of these interactions are interactions between amino acid R groups. This is а major distinction between tertiary-structure interactions and secondary-structure interactions. Tertiary-structure interactions involve the R groups of amino acids; secondary-structure interactions involve the peptide linkages between amino acid units.

Disulfide bonds, the strongest of the tertiary-structure interactions, result from the –SH groups of two cysteine molecules reacting with each other to form а covalent disulfide. This type of interaction is the only one of the four tertiary-structure interactions that involves а covalent bond. That – SH groups are readily oxidized to give а disulfide bond, – S – S –. Disulfide bonds may involve two cysteine units in the same chain or in different chains.

Figure. Four types of interactions betweenamino acid R groups produce thetertiary structure of а protein. (а) Disulfide bonds. (b) Electrostatic interactions (salt bridges). (с) Hydrogen bonds. (d) Hydrophobic interactions.

Electrostatic interactions, also called salt bridges, always involve amino acids with charged side chains. These amino acids are the acidic and basic amino acids. The two R groups, one acidic and one basic, interact through ion — ion attractions. Figure.b shows an electrostatic interaction.

Hydrogen bonds can occur between amino acids with polar R groups. А variety of polar side chains can be involved, especially those that possess the following functional groups:

Hydrogen bonds are relatively weak and are easily disrupted by changes in pH and temperature. Hydrophobic interactions result when two nonpolar side chains are close to each other, In aqueous solution, many proteins have their polar R groups outward, toward the aqueous solvent (which is also polar), and their nonpolar R groups inward (away from the polar water molecules). The nonpolar R groups then interact with each other. Hydrophobic interactions are common between phenyl rings and alkyl side chains. Although hydrophobic interactions are weaker than hydrogen bonds or electrostatic interactions, they are a significant force in some proteins because there are so many of them; their cumulative effect can be greater in magnitude than the effects of hydrogen bonding.

In 1959, а protein tertiary structure was determined for the first time. The determination involved myoglobin, а protein whose function is oxygen storage in muscle tissue. It involves а single chain of 153 amino acids with numerous а helix segments within the chain. The structure also contains а heme group, an iron-containing group with the ability to bind molecular oxygen.

Quaternary structure is the highest level of protein organization. It is found only in proteins that have structures involving two or more polypeptide chains that are independent of each other — that is, are not covalently bonded to each other. These multichain proteins are often called oligomeric proteins. The quaternary structure of а protein involves the associations among the separate chains in an oligomeric protein.

Figure.  А schematic diagram showing the tertiary structure of the single-chain protein myoglobin.

Most oligomeric proteins contain an eveumber of subunits (two subunits = а dimer four subunits = а tetramer, and so on). The subunits are held together mainly hydrophobic interactions between amino acid R groups.

The hydrophobic interactions maintaining quaternary structure are more easily interrupted than those for tertiary structure. For example, only small changes in cellular conditions can cause а tetrameric protein to fall apart, dissociating into dimers or perhaps four separate subunits, with а resulting temporary loss of protein activity. As conditions change back, the oligomer automatically re-forms, and normal protein function is restored.

An example of а protein with quaternary structure is hemoglobin, the oxygen-carrying protein in blood. It is а tetramer in which there are two identical α chains and two identical β chains. Each chain enfolds а heme group, the site where oxygen binds to the protein.

Figure. А schematic diagram showing the quaternary structure of the oxygen-carrying protein hemoglobin.

Globular and fibrous proteins

On the basis of structural shape, proteins can be classified into two major types: fibrous proteins and globular proteins. А fibrous protein is а protein that has а long, thin, fibrous shape. Such proteins are made up of long rod-shaped or string-like molecules that can intertwine with one another and form strong fibers. They are water-insoluble and generally have structural functions within the human body. А globular protein is а protein whose overall shape is roughly spherical or globular. Globular proteins either dissolve in water or form stable suspensions in water, which allows them to travel through the blood and other body fluids to sites where their activity is needed. Table. 2 gives examples of selected common fibrous and globular proteins.

Table. 2 Some common fibrous and globular proteins

The fibrous protein alpha keratin is particularly abundant iature, where it is found in protective coatings for organisms. It is the major protein constituent of hair, feathers, wool, fingernails and toenails, claws, scales, horns, turtle shells, quills, and hooves.

The structure of а typical alpha keratin that of hair is depicted in Figure. The individual molecules are almost wholly alpha-helical. Pairs of these helices twine about one another to produce а coiled coil. In hair, two of the coiled coils then further twist together to form а four-molecule protofilament.

Figure. The coiled-coil structure of the fibrous protein alpha keratin.

Finally, eight protofilaments combine in either а circular or а square arrangement to make а microfilament, which is the basic structural unit. Introduction of disulfide links (between cysteine residues) within the several levels of structure determine the “hardness” of the alpha keratin. “Hard” keratins, such as those found in horns and nails, have considerably more disulfide bridges than their softer counterparts found in hair, wool, and feathers.

Simple and Conjugated Proteins

Proteins are classified as either simple proteins or conjugated proteins. А simple protein is made up entirely of amino acid residues. More than one polypeptide chain may be present, but all chains contain only amino acids. А conjugated protein has other chemical components in addition to amino acids. These additional components, which may be organic or inorganic, are called prosthetic groups. А prosthetic group is а non-amino acid unit permanently associated with а protein.

Conjugated proteins may be further classified according to the nature of the prosthetic group. For example, proteins containing lipids, those containing carbohydrates, and those containing metal ions are called lipoproteins, glycoproteins, and metalloproteins, respectively. Table.3 gives further examples of the types of conjugated proteins.

1. Simple protein

Simple protein consists of only amino acids or their derivatives. When hydrolysed by acids, alkalies or enzymes, simple proteins yield only amino acids or their derivatives. They include the following groups.

(a) Albumins:

These are water soluble-proteins found in all body cells and also in the blood stream. Examples are lacto albumin found in milk and serum albumin found in blood.

(b) Globulins:

These are insoluble in water but are soluble in dilute salt solutions of strong acids and bases. Examples of globulins are lactoglobulin found in milk and ologlobulin.

(c) Glutelins:

These are soluble in dilute acids and alkalies. The protein glutenin from wheat is an example. They occur only in plant material.

(d) Prolamines:

These are soluble in 70-80% alcohol. They include fliadin from wheat and zein from corn. They are found only in plant material.

(e) Albumunoids:

Albuminoids or seleroproteins are insoluble in all neutral solvents and in dilute alkalies and acids. They are found in connective tissues and in hair and nails. Examples are keratin, found in the cornified layers of the skin, cortex of hair and nails, and collagen which is found in the white fibres of areolar tissue.

(f) Histones:

These are water soluble proteins in which basis amino acids predominate. They are rich in arginine or lysine. In eukaryotes the DNA of the chromosomes is associated with histones to form nucleoproteins.

(g) Protamines:

These are water soluble basic polypeptides with a low molecular weight (about 4,000 daltons). They are very rich in the amino acid arginine. The polypeptide chain consists of 28 amino acid residues, which include 19 arginines and 8-9 non basic amino acids. Protamines are found bound to DNA in spermatozoa of some fishes. Examples of protamines are salmine (in salmon) and sturine (in sturgeons)

(2) Conjugated proteins:

These consist of simple proteins in combination with some non-protein component. The non-protein groups are called prosthetic groups. Conjugated protein includes the following group.

Types of Conjugated Proteins

 (a) Nucleoproteins:

(Protein + nucleic acid). Nucleoproteins are proteins in combination with nucleic acids. In trout spermatozoa nucleoproteins constitute 90% of the solid material, and in certain erythrocyte nuclei almost 100% nucleoproteins are combinations of nucleic acids with the basic simple protein protamine. Nuclehistones are combinations of nucleic acids with the simple basic protein histone. In addition there are several acidic proteins, the non histone proteins.

(b) Glycoproteins:

(Protein+Carbohydrate): Glycoproteins are proteins link to carbohydrate. In most glycoproteins the linkage is between asparagines (Ans) and N-acetyl-D-glocosamine (GICNAC). Salivary glands and mucous glands of the digestive tract, however, secrete mucoproreins in which the linkage is between N-acetylglycosamine and serinel threonine of the protein. Glcoproteins are of two main catagories, intracellular and secretory. Intracellular glycoproteins are present in cell membranes and have an important role in membrane interaction and recognition. Examples of secretory glycoproteins are: Plasma glycoproteins, secreted by the liver, thyroglobulin, secreted by the thyroid gland, immunoglobins, secreted by plasma cells, ovoalbumins, secreted by the oviduct in the hen, ribonuclease, the enzyme which breaks down RNA, and deoxyribonuclease, the enzyme which breaks down DNA.

(c) Phosphoproteins (Protein+phosphate):

Phosphoproteins are proteins in combination with a phosphate-containing radical other than a nucleic acid or a phospholipid. Examples of phosphoproteins are casein of milk and ovovitelline in eggs.

(d) Chromoproteins:

These are proteins in combination with a proshetic group that is a pigmen. Examples are the respiratory pigments haemoglobin and haemocyanin, visual purple or rhodopsin found in the rods of the eye, flavoproteins and cytochromes.

(e) Lipoproteins:

These are proteins conjugated with lipids. There are four types of lipoproteins, high density lipoproteins (HDL) or a-lipoproteins, low density lipoproteins (VLDL) or pre-β lipoproteins and chylomicrons.

(f) Metalloproteins:

These are proteins conjugated to metal ion (s) which are not part of the prosthetic group. They include caeruloplasmin, an enzyme with oxidase activity that may transport copper in plasma, and siderophilin that is found to iron.

Protein hydrolysis. When а protein or polypeptide in а solution of strong acid or strong base is heated, the peptide bonds of the amino acid chain are hydrolyzed and free amino acids are produced. The hydrolysis reaction is the reverse of the formation reaction for а peptide bond. Amino and carboxylic acid functional groups are regenerated.

Let us consider the hydrolysis of the tripeptide Ala – Gly – Cys under acidic conditions. Complete hydrolysis produces one unit each of the amino acids alanine, glycine, and cysteine. The equation for the Note that the product amino acids in this reaction are written in positive ion form because of the acidic reaction conditions.

Protein digestion is simply enzyme-catalyzed hydrolysis of ingested protein. The free amino acids produced from this process are absorbed through the intestinal wall into the bloodstream and transported to the liver. Here they become the raw materials for the synthesis of new protein tissue. Also, the hydrolysis of cellular proteins to amino acids is an ongoing process, as the body resynthesizes needed molecules and tissue.

Protein denaturation is the partial or complete disorganization of а protein’s characteristic three-dimensional shape as a result of disruption of its secondary, ternary, and quaternary structural interactions. Because the biological function of а protein depends on its three-dimensional shape, the result of denaturation is loss of biological activity. Protein denaturation does not affect the primary structure of а protein.

Although some proteins lose all of their three-dimensional structural characteristics upon denaturation, most proteins maintain some three-dimensional structure. For а few small proteins, it is possible to find conditions under which the effects of denaturation can be reversed; this restoration process in which the protein is “refolded” is called renaturation. Denaturation is irreversible, however, for most proteins.

Loss of water solubility is а frequent physical consequence of protein denaturation. The precipitation out of bkological solution of denatured protein is called coagulation.

А most dramatic example of protein denaturation occurs when egg white (а concentrated solution of the protein albumin) is poured onto а hot surface. The clear albumin solution immediately changes into а white solid with а jelly-like consistency. А similar process occurs when hamburger juices encounter а hot surface. А brown, jelly-like solid forms.

One of the reasons why many foods are cooked is to denature the protein present so that it is more easily digested. It is easier for digestive enzymes to “work on” denatured (unraveled) protein. Cooking foods also kills microorganisms through protein denaturation. For example, ham and bacon can harbor parasites that cause trichinosis. Cooking the ham or bacon denatures parasite protein.

In surgery, heat is often used to seal small blood vessels. This process is called cauterization. Small wounds can also be sealed by cauterization. Heat-induced denaturation is used in sterilizing surgical instruments and in canning foods; bacteria are destroyed when the heat denatures their protein.

The body temperature of а patient with fever may rise to 102 ⁰F, 103 ⁰F, or even 104 ⁰F without serious consequences. А temperature above 106 ⁰F (41 ⁰С) is extremely dangerous, for at this level, the enzymes of the body begin to be inactivated. Enzymes, which function as catalysts for almost all body reactions, are protein. Inactivation of enzymes, through denaturation, can have lethal effects on body chemistry.

The effect of ultraviolet radiation 6om the sun, а nonionizing radiation, is similar to that of heat. Denatured skin proteins cause most of the problems associated with sunburn.

А curdy precipitate of casein, the principal protein in milk, is formed in the stomach when the hydrochloric acid of gastric juice denatures milk. The curdling of milk that takes place when milk sours or cheese is made results from the presence of lactic acid, а byproduct of bacterial growth. Yogurt is prepared by growing lactic acid – producing bacteria in skim milk. The coagulated denatured protein gives yogurt its semi-solid consistency.

Serious eye damage can result from eye tissue contact with acids or bases, when irreversibly denatured and coagulated protein causes а clouded cornea. This reaction is part of the basis for the rule that students wear protective eyewear in the chemistry laboratory.

Alcohols are an important type of denaturing agent. Denaturation of bacterial protein takes place when isopropyl or ethyl alcohol is used as а disinfectant. This accounts for the common practice of swabbing the skin with alcohol before giving an injection. Interestingly, pure isopropyl or ethyl alcohol is less effective than the commonly used 70% alcohol solution. Pure alcohol quickly denatures and coagulates the bacterial surface, thereby forming an effective barrier to further penetration by the alcohol. The 70% solution denatures more slowly and allows complete penetration to be achieved before coagulation of the surface proteins takes place.

Glucoproteins are conjugated protein that contain carbohydrates or carbohydrate derivatives in addition to amino acids. The carbohydrate content of glycoproteins is variable (from а few percent up to 85%), but it is fixed for any specific glycoprotein.

Glycoproteins include а number of very important substances; two of these, collagen and immunoglobulins, are described in this section. Many of the proteins in plasma (cell) membranes are actually glycoproteins. The blood group markers of the АВО system are also glycoproteins in which the carbohydrate content can reach 85%.

Collagen, the most abundant of all proteins in humans (30% of total body protein), is а major structural material in tendons, ligaments, blood vessels, and skin; it is also the organic component of bones and teeth. The predominant structural feature within collagen molecules, three chains of amino acids wrapped (wound) into а triple helix, has already been considered.

The rich content of the amino acid proline (up to 20%) in collagen is one reason why it has а triple-helix conformation rather than the simpler а helix structure. Proline amino acid residues do not fit into regular а helices because of the cyclic’ nature of the side chain present and its accompanying different “geometry.”

An additional structural feature of collagen is the presence of the nonstandard amino acids 4-hydroxyproline (5%) and 5-hydroxylysine (1%) – derivatives of the standard amino acids proline and lysine.

The presence of carbohydrate units (mostly glucose, galactose, and their disaccharides) attached by glycosidic linkages to collagen at its 5-hydroxylysine residues causes collagen to be classified as а glycoprotein. The function of the carbohydrate groups in collagen is related to cross-linking; they direct the assembly of collagen triple helices into more complex aggregations called collagen fibrils.

Collagen molecules (triple helices) are very long, thin, and rigid. Many such molecules, lined up alongside each other, combine to make collagen fibrils. Cross-linking between helices gives the fibrils extra strength. The greater the number of cross links, the more rigid the fibril is. The stiffening of skin and other tissues associated with aging is thought to result, at least in part, from an increasing amount of cross-linking between collagen molecules. The process of tanning, which converts animal hides to leather, involves increasing the degree of cross-linking.

When collagen is boiled in water, under basic conditions, it is converted to the water-soluble protein gelatin. This process involves both denaturation and hydrolysis. Heat acts as а denaturant, causing rupture of the hydrogen bonds supporting collagen’s triple-helix structure. Regions in the amino acid chains where prolinе and hydroxyproline concentrations are high are particularly susceptible to hydrolysis, which breaks up the polypeptide chains. Meats become more tender when cooked because of the conversion of some collagen to gelatin. Tougher cuts of meat (more cross-linking), such as stew meat, need longer cooking times.

Immunoglobulins are among the most important and interesting of the soluble proteins in the human body. Immunoglobulins are glycoprotein molecules produced by an organism as а protective response to the invasion of microorganisms or foreign molecules. Different classes of immunoglobulins, identified by differing carbohydrate content and molecular mass, exist.

Immunoglobulins serve as antibodies to combat invasion of the body by antigens. Antigens are foreign substances, such as bacteria and viruses, that invade the body. Antibodies are molecules that counteract specific antigens. The immune system of the human body has the capability to produce immunoglobulins that respond to several thousand different antigens.

All types of immunoglobulin molecules have а similar basic structure, which includes the following features:

1. Four polypeptide chains are present: two identical heavy (Н) chains and two identical light (L) chains.

2. The Н chains, which usually contain 400 — 500 amino acid residues, are approximately twice as long as the Ь chains.

3. Both the Н and Ь chains have constant and variable regions. The constant regions have the same amino acid sequence from immunoglobulin to immunoglobulin, and the variable regions have а different amino acid sequence in each immunoglobulin.

4. The carbohydrate content of various immunoglobulins varies from 1% to 12% by mass.

5. The secondary and tertiary structures are similar for all immunoglobulins. They involve а Y-shaped conformation, with disulfide linkages between Н and L chains stabilizing the structure.

The interaction of an immunoglobulin molecule with an antigen occurs at the “tips” (upper-most part) of the Y structure. These tips are the variable-composition region of the immunoglobulin structure. It is here that the antigen binds specifically, and it is here that the amino acid sequence differs from one immunoglobulin to another.

Each immunoglobulin has two identical active sites and can thus bind to two molecules of the antigen it is “designed for.” The action of many such immunoglobulins of given type in concert with each other creates an antigen – antibody complex that precipitates from solution. Eventually, an invading antigen can be eliminated from the body through such precipitation. The bonding of an antigen to the variable region of an immunoglobulin occurs through dipole — dipole interactions and hydrogen bonds rather than covalent bonds.

The importance of immunoglobulins is amply and tragically demonstrated by the effects of AIDS (acquired immune deficiency syndrome). The AIDS virus upsets the body’s normal production of immunoglobulins and leaves the body susceptible to what would otherwise not be debilitating and deadly infections.

Individuals who receive organ transplants must be given drugs to suppress the production of immunoglobulins against foreign proteins in the new organ, thus preventing rejection of the organ. The major reason for the increasing importance of organ transplants is the successful development of drugs that can properly manipulate the body’s immune system.

Many reasons exist for а mother to breast- еес1 а newborn infant. One of the most important is immunoglobulins. During the first two or three days of lactation, the breasts produce colostrum, а premilk substance containing immunoglobulins from the mother’s blood. Colostrum helps protect the newborn infant from those infections to which the mother has developed immunity. These diseases are the ones in her environment – precisely those the infant needs protection from. Breast milk, once it is produced, is а source of immunoglobulins for the infant for а short time. (After the first week of nursing, immunoglobulin concentrations in the milk decrease rapidly.) Infant formula used as a substitute for breast milk is almost always nutritionally equivalent, but it does not contain immunoglobulins.

Lipoproteins are conjugated proteins composed of both lipids and amino acids. The major function of such proteins is to help suspend lipids and transport them through the blood-stream. Lipids, in general; are insoluble in blood (an aqueous medium) because of their nonpolar nature.

The presence or absence of various types of lipoproteins in the blood appears to have implications for the health of the heart and blood vessels. Lipoprotein levels in the blood are now used as an indicator of heart attack risk.

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 100 cm long. Starch or silica gel granules are hydrophilic and are surrounded by a layer of a tightly bound water, which serve as a stationary phase, past which flows a moving phase of an immiscible solvent containing the mixture to be separated. The mixture of solutes undergoes a microscopic partition process between the fixed water layer and the flowing solvent.

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

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

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

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

Molecular-Exclusion Chromatography. One of the most useful and powerful tools for separating pro­teins from each other on the basis of size is molecular-exclusion chromatography, also known as gel-filtration or molecular-sieve chromatography. It differs from ion-exchange chromatography, which separates solutes on the basis of their electric charge and acid-base properties. In molecular-exclusion chromatography the mixture of pro­teins is allowed to flow by gravity down a column packed with beads of an inert, highly hydrated polymeric material that has previously been washed and equilibrated with the buffer alone. Common column materials are Sephadex, the commercial name of a polysaccharide derivative; Bio-Gel, a commercial polyacryl-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 particular ligand 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 iormal conditions. Hyperaminoacidemia – the increasing of amino acid level in blood plasma. The possible causes of such state are liver diseases, diabetus mellitus, acute and chronical kidney failure, congenital enzymopathy.

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

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

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

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

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

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

The mo­lecular weight of proteins and its determination.

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

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

Determination of the Molecular Weight from Osmotic-Pressure Measurements

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

Determination of Molecular Weight by Sedimentation Analysis

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

Determining Molecular Weight by Light Scattering

When a beam of light is passed through a protein solution in a darkened room, the path of the beam can be seen because the light is scattered by the protein molecules. This is called the Tyndal 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 without denaturation. 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 a semipermeable 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 the isoelectric 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 precipitated isoelectric protein remains in its native conformation and can be redissolved in a medium having an appropriate pH and salt concentration.

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

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

Effect of Temperature on Solubility of Proteins.

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

Separation Procedures Based on Electric Charge.

Electrophoretic Methods. This method can separate a protein mix­ture on the basis of both electric charge and molecular size. For this purpose, special paper, gels of potato starch or polyacrylamide 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 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.

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.

Denaturation.

Most protein molecules retain their biological activity only within a very limited range of temperature and pH. Exposing soluble or globular proteins to extremes of pH or to high tem­peratures for only short periods causes most of them to un­dergo a physical change known as denaturation, in which the most visible effect is a decrease in solubility. Since no covalent bonds in the backbone of the polypeptide chain are broken during this relatively mild treatment, the primary structure remains intact. Most globular proteins undergo denaturation when heated above 60 to 70°C. Formation of an insoluble white coagulum when egg white is boiled is a common example of protein denaturation. But the most sig­nificant consequence of denaturation is that the protein usually loses its characteristic biological activity; e.g., heating usually destroys the catalytic ability of enzymes.

Denaturation is the unfolding of the characteristic na­tive folded structure of the polypeptide chain of globular protein molecules. When thermal agitation causes the native folded structure to uncoil or unwind into a randomly looped chain, the protein loses its biological activity. In certain cases denaturation of native proteins into biologically inactive forms is not irreversible. An unfolded protein molecule simetime may spontaneously returns to its native biologically ac­tive form in the test tube, a process called renaturation. If the denatured protein was an enzyme, its catalytic activity returns on renaturation, without change in the specificity of the reaction catalyzed.

Using of protein capacity to salting-out and denaturation in medical practice.

The salting-out of proteins is used for separation and purification of proteins without loss of their native properties. For example, this method can be used for receiving of pure native enzymes.

Salting-out of proteins is also used in laboratory medicine for diagnostics.

Principle of denaturation is used during the aseptic processing of operational rooms, surgical instruments et c.

In poisoning by salts of Pb, Hg and some other metals patient is administrated the solution of protein (milk, egg white). In stomach the complex compounds between protein and metals are formed. As result protein is denaturated. This prevent the further absorption of metals in the intestine.

The total contents of proteins in blood plasma is 65-85 g/l.

There are a lot of proteins in blood plasma distinguished by the physical, chemical and functional properties: transport proteins, enzymes, proenzymes, inhibitors of enzymes, hormones, antibodies, antitoxins, factors of coagulation and anticoagulators and others. Hypoproteinemia – decrease of the total contents of proteins in blood plasma. This state occurs in old people as well as in pathological states accompanying with the oppressing of protein synthesis and activation of decomposition of tissue proteins (starvation, hard infectious diseases, state after hard trauma and operations, cancer).

The quantity of separate protein fractions depend on the method of separation. In paper electrophoresis blood plasma proteins can be separated on 5 fractions: albumins (40-50 g/l), α₁-globulins (3-6 g/l), α₂-globulins (4-9 g/l), b-globulins (6-11 g/l) and γ‑globulins (7-15 g/l).

Albumins – multidispersed fraction of blood plasma which are characterized by the high electrophoretic mobility and mild dissolubility in water and saline solutions. Due to high hydrophilic properties albumins bind a significant amount of water, and the volume of their molecule under hydratation is doubled. Hydrative layer formed around the serum albumins provides to 70-80 % of oncotic pressure of blood plasma proteins, that can be applied in clinical practice at albumins transfusion to patients with tissue edemas. The decreasing of albumins concentration in blood plasma, for example under disturbance of their synthesis in hepatocytes at liver failure, can cause the water transition from vessels into the tissues and development of oncotic edemas.

Albumins execute also important physiological function as transporters of a lot of metabolites and diverse low molecular weight structures. The molecules of albumins have several sites with centers of linkage for molecules of organic ligands, which are affixed by the electrostatic and hydrophobic bonds. Serum albumins can affix and convey fatty acids, cholesterol, cholic pigments (bilirubin and that similar), vitamins, hormones, some amino acids, toxins and medicines.

Globulins – heterogenous fraction of blood proteins which execute transport
(α₁-globulins – transport of lipids, thyroxine, corticosteroid hormones; α₂-globulins – transport of lipids, copper ions; b-globulins – transport of lipids, iron) and protective (participation  of b-globulins in immune reactions as antitoxins; γ-globulins as immunoglobulins) functions.

A ratio between concentrations of albumines and globulins (so called “protein coefficient”) in blood plasma is often determined in clinical practice. In helthy people this coefficient is 1,5-2,0.

Immunoglobulins (Ig A, Ig G, Ig E, Ig M) – proteins of γ-globulin fraction of blood plasma executing the functions of antibodies which are the main effectors of humoral immunity.

C-reactive protein. This protein received the title owing to its capacity to react with C-polysaccharide of a pneumococcus forming precipitates. According to its chemical nature C-reactive protein is glycoprotein.

In blood plasma of healthy people the C-reactive protein is absent but it occurs at pathological states accompanied by an inflammation and necrosis of tissues. The availability of C-reactive protein is characteristic for the acute period of diseases – “protein of an acute phase”. The determination of C-reactive protein has diagnostic value in an acute phase of rheumatic disease, at a myocardial infarction, pneumococcal, streptococcal, staphylococcal infections.

Crioglobulin – the protein of the γ-globulin fraction. Like to the C-reactive protein crioglobulin absent in blood plasma of the healthy people and occurs at leukoses, rheumatic disease, liver cirrhosis, nephroses. The characteristic physico-chemical feature of crioglobulin is its dissolubility at standard body temperature (37 ^oC) and capacity to form the sediment at cooling of a blood plasma up to 4 ^oC.

α₂-macroglobulin – protein of α₂-globulin fraction, universal serum proteinase inhibitor. Its contents (2,5 g/l) in blood plasma is highest comparing to another proteinase inhibitors.

The biological role of α₂-macroglobulin consists in regulation of the tissue proteolysis systems which are very important in such physiological and pathological processes as blood clotting, fibrinolysis, processes of immunodefence, functionality of a complement system, inflammation, regulation of vascular tone (kinine and renin-angiothensine system).

α₁-antitrypsin – glycoprotein with a molecular weight 55 kDa. Its concentration in blood plasma is 2-3 г/л. The main biological property of this inhibitor is its capacity to form complexes with proteinases oppressing proteolitic activity of such enzymes as trypsin, chemotrypsin, plasmin, trombin.

Fibronectin – glycoprotein of blood plasma that is synthesized and secreted in intercellular space by different cells. Fibronectin present on a surface of cells, on the basal membranes, in connective tissue and in blood. Fibronectin has properties of a «sticking» protein and contacts with the carbohydrate groups of gangliosides on a surface of plasma membranes executing the integrative function in intercellular interplay. Fibronectin also plays important role in the formation of the pericellular matrix.

Haptoglobin – protein of α₂-globulin fraction of blood plasma. Haptoglobin has capacity to bind a free haemoglobin forming a complex that refer to γ-globulins electrophoretic fraction. Normal concentration in blood plasma – 0,10-0,35 g/l.

Haptoglobin-hemoglobin complexes are absorbed by the cells of reticulo-endothelial system, in particular in a liver, and oxidized to cholic pigments. Such haptoglobin function promotes the preservation of iron ions in an organism under conditions of a physiological and pathological erythrocytolysis.

Transferrin – glycoprotein belonging to the b-globulin fraction. It binds in a blood plasma iron ions (Fe³⁺). The protein has on the surface two centers of linkage of iron. Transferrin is a transport form of iron delivering its to places of accumulation and usage.

Ceruloplasmin – glycoprotein of the α₂-globulin fraction. It can bind the copper ions in blood plasma. Up to 3 % of all copper contents in an organism and more than 90 % copper contents in plasma is included in ceruloplasmin. Ceruloplasmin has properties of ferroxidase oxidizing the iron ions. The decrease of ceruloplasmin in organism (Wilson disease) results in exit of copper ions from vessels and its accumulation in the connective tissue that shows by pathological changes in a liver, main brain, cornea.

BLOOD PROTEINS

Blood proteins, also termed serum proteins or plasma proteins, are proteins present in blood serum. They serve many different functions, including transport of lipids, hormones, vitamins and metals in the circulatory system and the regulation of acellular activity and functioning and in the immune system. Other blood proteins act as enzymes, complement components, protease inhibitors or kinin precursors. Contrary to popular belief, hemoglobin is not a blood protein, as it is carried within red blood cells, rather than in the blood serum.

Serum albumin accounts for 55 % of blood proteins, and is a major contributor to maintaining the osmotic pressure of plasma to assist in the transport of lipids and steroid hormones. Globulins make up 38% of blood proteins and transport ions, hormones and lipids assisting in immune function. Fibrinogen comprise 7% of blood proteins; conversion of fibrinogen to insoluble fibrin is essential for blood clotting. The remainder of plasma proteins (1%) is made up of regulatory proteins such as enzymes, proenzymes and hormones. All blood proteins are synthesized in liver except for the gamma globulins.

Separating serum proteins by electrophoresis is a valuable diagnostic tool as well as a way to monitor clinical progress. Current research regarding blood plasma proteins is centered on performing proteomics analyses of serum/plasma in the search for biomarkers. These efforts started with two-dimensional gel electrophoresis efforts in the 1970s and in more recent times this research has been performed using LC-tandem MS based proteomics. The normal laboratory value of serum total protein is around 7 g/dL.

FAMILIES OF BLOOD PROTEINS

Blood protein	Normal level	%	Function
Albumins	3.5-5.0 g/dl	55%	create oncotic pressure and transport insoluble molecules
Globulins	1.0-1.5 g/dl	38%	participate in immune system
Fibrinogen	0.2-0.45 g/dl	7%	Blood coagulation
Regulatory proteins		<1%	Regulation of gene expression
Clotting factors		<1%	Conversion of fibrinogen into Fibrin

 

SERUM ALBUMIN is the most abundant plasma protein in mammals. Albumin is essential for maintaining the oncotic pressure needed for proper distribution of body fluids between intravascular compartments and body tissues. It also acts as a plasma carrier by non-specifically binding several hydrophobic steroid hormones and as a transport protein for hemin and fatty acids. Too much serum albumin in the body can be harmful.

Function.  Major contributors to oncotic pressure (known also as colloid osmotic pressure) of plasma; carriers for various substances.

Albumin is a soluble, monomeric protein which comprises about one-half of the blood serum protein. Albumin functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones and plays a role in stabilizing extracellular fluid volume. Albumin is a globular un-glycosylated

 serum protein of molecular weight 65,000. Albumin is synthesized in the liver as preproalbumin which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce the secreted albumin.

Types Serum albumin is widely distributed in mammals. Examples include:

·         The human version is human serum albumin.

·         Bovine serum albumin, or BSA, is commonly used in immunodiagnostic procedures, clinical chemistry reagents, cell culture media, protein chemistry research and molecular biology laboratories (usually to leverage its non-specific protein binding properties).

Physical properties. Albumin (when ionized in water at pH 7.4, as found in the body) is negatively charged. The glomerular basement membrane is also negatively charged in the body; some studies suggest that this prevents the filtration of albumin in the urine. According to this theory, that charge plays a major role in the selective exclusion of albumin from the glomerular filtrate. A defect in this property results in nephrotic syndrome leading to albumin loss in the urine. Nephrotic syndrome patients are sometimes given albumin to replace the lost albumin.

Because smaller animals (for example rats) function at a lower blood pressure, they need less oncotic pressure to balance this, and thus need less albumin to maintain proper fluid distribution.

Structure. The general structure of albumin is characterized by several long α (alpha) helices, this allows it to maintain a relatively static shape, something essential for regulating blood pressure.

Serum albumin contains eleven distinct binding domains for hydrophobic compounds. One hemin and six long-chain fatty acids can bind to serum albumin at the same time.

The globulins are a family of globular proteins that have higher molecular weights and water solubility values than the albumins. Some globulins are produced in the liver, while others are made by the immune system. Globulins, albumin, and fibrinogen are the major blood proteins. The normal concentration of globulins in the blood is about 2.6-4.6 g/dL.

The term “globulin” is sometimes used synonymously with “globular protein”. However, albumins are also globular proteins, but are not globulins. All other serum globular proteins are globulins.

TYPES OF GLOBULINS

All globulins fall into one of the following four categories:

·       Alpha 1 globulins

·       Alpha 2 globulins

·       Beta globulins

·       Gamma globulins (one group of gamma globulins are the immunoglobulins, which are also known as “antibodies”)

Globulins can be distinguished from one another using serum protein electrophoresis.

Reference ranges for blood tests, comparing blood content of globulins (shown in purple at right) with other constituents.

Sizes

Globulins exists in various sizes. The lightest globulins are the alpha globulins, which typically have molecular weights of around 92 kDa, while the heaviest class of globulins are the gamma globulins, which typically weight about 120 kDa. Being the heaviest, the gamma globulin are among the slowest to segregate in gel electrophoresis. Since they are immunologically active, they are also called “immunoglobulins

“.

PLANT GLOBULINS

Non-human globulin proteins exist as well, such as cucurbitin from squashes and vicilin and legumin from legumes and peas, functioning as protein storage within seeds. These proteins can cause allergic reactions if they bind with human IgE antibodies.

Pseudoglobulins and euglobulins

Pseudoglobulins are a class of globulins that are more soluble in ammonium sulfate than euglobulins. Pseudoglobulins are also soluble in pure water, while euglobulins are not.

FIBRINOGEN (factor I) is a soluble, 340 kDa plasma glycoprotein, that is converted by thrombin into fibrin during blood clot formation. Fibrinogen is synthesized in the liver by the hepatocytes. The concentration of fibrin in the blood plasma is 200-400 mg/dL (normally measured using the Clauss method.

During normal blood coagulation, a coagulation cascade activates the zymogen prothrombin by converting it into the serine protease thrombin. Thrombin then converts the soluble fibrinogen into insoluble fibrin strands. These strands are then cross-linked by factor XIII to form a blood clot. FXIIIa stabilizes fibrin further by incorporation of the fibrinolysis inhibitors alpha-2-antiplasmin and TAFI (thrombin activatable fibrinolysis inhibitor, procarboxypeptidase B), and binding to several adhesive proteins of various cells. Both the activation of Factor XIII by thrombin and plasminogen activator (t-PA) are catalyzed by fibrin. Fibrin specifically binds the activated coagulation factors factor Xa and thrombin and entraps them in the network of fibers, thus functioning as a temporary inhibitor of these enzymes, which stay active and can be released during fibrinolysis. Recent research has shown that fibrin plays a key role in the inflammatory response and development of rheumatoid arthritis.

Fibrinogen deficiency. Congenital fibrinogen deficiency (afibrinogenemia) or disturbed function of fibrinogen has been described in a few cases.

It can lead to either bleeding or thromboembolic complications, or is clinically without pathological findings. More common are acquired deficiency stages that can be detected by laboratory tests in blood plasma or in whole blood by means of thrombelastometry. Acquired deficiency is found after hemodilution, blood losses and/or consumption such as in trauma patients, during some phases of disseminated intravascular coagulation (DIC), and also in sepsis. In patients with fibrinogen deficiency, the correction of bleeding is possible by infusion of fresh frozen plasma (FFP), cryoprecipitate (a fibrinogen-rich plasma fraction) or by fibrinogen concentrates. There is increasing evidence that correction of fibrinogen deficiency or fibrinogen polymerization disorders is very important in patients with bleeding.

Diagnostic use. Fibrinogen levels can be measured in venous blood. Normal levels are about 1.5-3 g/L, depending on the method used. In typical circumstances, fibrinogen is measured in citrated plasma samples in the laboratory, however the analysis of whole-blood samples by use of thrombelastometry (platelet function is inhibited with cytochalasin D) is also possible. Higher levels are, amongst others, associated with cardiovascular disease (>3.43 g/L). It may be elevated in any form of inflammation, as it is an acute-phase protein; for example, it is especially apparent in human gingival tissue during the initial phase of periodontal disease.  Fibrinogen levels increase in pregnancy to an average of 4.5 g/l, compared to an average of 3 g/l ion-pregnant people.

It is used in veterinary medicine as an inflammatory marker: In horses, a level above the normal range of 1.0-4.0 g/L suggests some degree of systemic inflammatory response.

Low levels of fibrinogen can indicate a systemic activation of the clotting system, with consumption of clotting factors faster than synthesis. This excessive clotting factor consumption condition is known as disseminated intravascular coagulation or “DIC.” DIC can be difficult to diagnose, but a strong clue is low fibrinogen levels in the setting of prolonged clotting times (PT or aPTT), in the context of acute critical illness such as sepsis or trauma. Besides low fibrinogen level, fibrin polymerization disorders that can be induced by several factors, including plasma expanders, can also lead to severe bleeding problems. Fibrin polymerization disorders can be detected by viscoelastic methods such as thrombelastometry.

Physiology. In its natural form, fibrinogen can form bridges between platelets, by binding to their GpIIb/IIIa surface membrane proteins; however, its major function is as the precursor to fibrin.

Fibrinogen, the principal protein of vertebrate blood clotting, is a hexamer, containing two sets of three different chains (α, β, and γ), linked to each other by disulfide bonds. The N-terminal sections of these three chains contain the cysteines that participate in the cross-linking of the chains. The C-terminal parts of the α, β and γ chains contain a domain of about 225 amino-acid residues, which can function as a molecular recognition unit. In fibrinogen as well as in angiopoietin, this domain is implicated in protein-protein interactions. In lectins, such as mammalian ficolins and invertebrate tachylectin 5A, the fibrinogen C-terminal domain binds carbohydrates. On the fibrinogen α and β chains, there is a small peptide sequence (called a fibrinopeptide). These small peptides are what prevent fibrinogen from spontaneously forming polymers with itself.

The conversion of fibrinogen to fibrin occurs in several steps. First, thrombin cleaves the N-terminus of the fibrinogen alpha and beta chains to fibrinopeptide A and B respectively. The resulting fibrin monomers polymerize end to end to from protofibrils, which in turn associate laterally to form fibrin fibers. In a final step, the fibrin fibers associate to form the fibrin gel.