Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Protein Chemistry
Amino Acid Composition of Proteins
General Properties of Amino Acids

Acid-base properties. These properties of Amino acids determine many of the physicochemical and biological characteristics of Proteins. Furthermore, almost all Methods for the isolation and identification of Amino acids are based on these properties. Amino acids are readily soluble in Water. They crystallize from neutral aqueous solutions in the form of dipolar (amphoteric) ions (zwitterions) rather than as undissociated molecules (the latter Structure is shown for convenience; however, all amino acids exist in the zwitterionic structure at physiological pH values).

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* These amino acids are formed after the completion of Protein Synthesis in The Cell ribosome As a result of post-synthetic chemical modification.

Upon dissolution in water, a crystalline amino acid, such as Alanine, can act either as an acid (a proton donor):

or as a base (a proton acceptor):

If The amino acid side chains (radicals) are neutral, they have virtually no effect on the dissociation of the α-carboxyl or α-amino groups, and the pK values (the negative logarithm of the dissociation constant) remain relatively constant. Consequently, the dissociation curves of almost all neutral amino acids overlap and can be examined using alanine as an example. If a strong acid (0.1 M HCl solution) or a strong base (0.1 M NaOH solution) is gradually added to an aqueous solution of alanine (e.g., 0.1 M), we obtain The titration curve of alanine, which is typical for all neutral amino acids (Fig. 1.6).

The apparent pK' values for the α-carboxyl and α-amino groups (i.e., the pH values at which these groups are, on average, half-dissociated) differ quite significantly, being pK₁ = 2.34 and pK₂ = 9.69. At low pH values (below pK₁'), almost all alanine molecules are fully protonated and carry a positive charge. In other words, at a high hydrogen ion concentration in the solution, the tendency for hydrogen to dissociate from the alanine structure is negligible. The titration curve shows that the transition point between the curve branches lies at pH 6.02. This means that at this pH value, the net (or average) electrical charge of the alanine molecule is zero, and it does not migrate in an electric field toward either the anode or the cathode (the isoelectric state). This pH value is termed the isoelectric point and is designated as pI. The isoelectric point of amino acids lacking additional NH₂ or COOH groups represents the arithmetic mean of the two pK' values:

respectively, for alanine

The isoelectric point of several Other Amino Acids containing additional acidic or basic groups (aspartic and glutamic acids, Lysine, Arginine, Tyrosine, etc.) depends, in addition, on the acidity or basicity of their side chains. For lysine, for example, pI must be calculated from the half-sum of the pK' values for the α- and ε-NH₂ groups. Thus, in the pH range from 4.0 to 9.0, Almost all amino acids exist predominantly in the form of zwitterions with a protonated amino group and a dissociated carboxyl group. It should be noted that at the physiological pH values of Tissues and Blood (7.1 and 7.4, respectively), amino acids (with the exception of Histidine) do not exhibit measurable buffer capacity. They acquire this capacity only at pH values close to their pK values (i.e., at pH 1.7–3.2 and 8.6–10.8).

Fig. 1.6. Curves obtained by titrating a 0.1 M alanine solution with 0.1 M HCl (a) and 0.1 M NaOH (b).

Stereochemistry of amino acids. A crucial property of amino acids liberated during the Hydrolysis of natural proteins under conditions that preclude racemization is their optical activity. When dissolved in water (or in HCl), they are capable of rotating the plane of polarized light (Glycine is the sole exception). This property is associated with the presence of an asymmetric carbon atom in the α-position of the molecule of all Natural Amino Acids (i.e., a carbon atom whose four valence bonds are occupied by different substituents). The specific rotation values to the right or left serve as a quantitative measure of optical activity, and for Most amino acids ranges from 10 to 30°. Approximately half of the protein amino acids are dextrorotatory, denoted by a "+" sign (Ala, Ile, Glu, Lys, etc.), while slightly fewer than half are levorotatory (Phe, Trp, Leu, etc.), denoted by a "-" sign. All these amino acids belong to the L-series, and the magnitude and sign of optical rotation depend on The Nature of the amino acid side chains and the pH of the solution in which the optical rotation is measured.

The stereochemistry of amino acids is conventionally evaluated not by optical rotation, but on The basis of the absolute configuration of all four substituent groups arranged around the asymmetric carbon atom at the vertices of a tetrahedral model. The absolute configuration of amino acids is traditionally correlated stereochemically with a compound arbitrarily chosen for comparison, namely glyceraldehyde, which also contains an asymmetric carbon atom. The L- and D-stereoisomers of glyceraldehyde are presented below, alongside the spatial configurations of L- and D-alanine:

All amino acids formed during the hydrolysis of natural proteins under conditions excluding racemization belong to the L-series. Thus, natural amino acids possess a spatial arrangement analogous to the configuration of L-glyceraldehyde. It should be emphasized once again that the symbols L and D denote the stereochemical configuration belonging to the L- or D-series, whereas the signs "+" and "-" indicate the direction of Rotation of the plane of polarization of light. Among protein amino acids, There are two (Threonine and isoleucine) that contain two asymmetric carbon atoms each. Consequently, if not in nature, at least in the laboratory, it is possible to obtain four stereoisomeric forms of these amino acids*. All four isomers are known for threonine. If threonine isolated from natural proteins is conventionally designated by the symbol L, its mirror image is referred to as D-threonine. The other two isomers, termed diastereoisomers or alloforms, can also exist in L- and D-forms. The structural configurations of all four stereoisomers of threonine can be represented by the following formulas:

As noted, D-Amino acids have not been detected in protein molecules**, but they are widespread in living nature.

For instance, D-isomers of glutamic acid, alanine, valine, phenylalanine, leucine, and several others have been discovered in bacterial cell walls; amino acids with D-configuration are also constituents of certain Antibiotics, notably actinomycins, bacitracin, and gramicidins A and S.

The Amino Acid Composition (Qualitative and quantitative) of many thousands of proteins derived from various sources has been elucidated (Table 1.4).

Analysis of the data in Table 1.4 reveals A number of regular patterns. Dicarboxylic acids and their amides account for up to 25–27% of all amino acids in most proteins. These same amino acids, together with leucine and lysine, comprise about 50% of all amino acids. At the same time, amino acids such as Cysteine, Methionine, Tryptophan, and histidine account for no more than 1.5–3.5%. Protamines and Histones exhibit a high content of the basic amino acids arginine and lysine, 26.4% and 85.2%, respectively (see "Chemistry of Simple Proteins").

Chemical Reactions for the detection and Determination of Amino acids in protein hydrolysates. The course of organic chemistry details numerous Chemical reactions characteristic of the α-amino and α-carboxyl groups of amino acids (acylation, alkylation, nitration, Esterification, etc.). Here, we will discuss general Color Reactions for detecting individual Amino Acids and amino acids incorporated into proteins, based on the Chemical Nature of the amino acid side chains (Table 1.5).

* During purely chemical (rather than enzymatic) synthesis of amino acids in the laboratory, an optically inactive mixture of L- and D-isomers is usually formed, designated as DL-amino acids, or racemates.

** D-Amino acids obviously do not play a major physiological role in animal and human organisms, although very active Enzymes catalyzing their degradation are present in Organs and tissues (see Chapter 12, "Intermediary METABOLISM of Amino Acids in Tissues"). Furthermore, L- and D-Amino acids differ in taste: the former are bitter, while the latter are sweet.

Table 1.4. Amino acid composition of certain natural proteins, in percentage

The reaction of amino acids with ninhydrin is successfully used for their detection in BIOLOGICAL OBJECTS AND quantitative determination. At Stage I of the reaction, reduced ninhydrin is formed due to the Oxidative Deamination of amino acids (accompanied by concurrent Decarboxylation of amino Acids):

At stage II, the resulting ammonia reacts with equimolar amounts of oxidized and reduced ninhydrin to form a blue-violet product, the color intensity of which (measured at 570 nm) is proportional to The amount of the amino acid:

Methods for the Quantitative determination of amino acids have been developed based on the ninhydrin reaction, notably paper partition Chromatography, first introduced in 1944 (A. Martin and R. Synge). Due to its high sensitivity, this same reaction is employed in automatic amino acid analyzers. Such an instrument was first designed by D. Spackman, S. Moore, and W. Stein (Fig. 1.7). After separating an amino acid mixture in columns packed with special ion-exchange resins (sulfonated polystyrene cation exchanger), the eluent stream from the Column enters a mixer along with a ninhydrin solution; the intensity of the resulting color is automatically measured using a photoelectric colorimeter and recorded with a chart recorder. This method has found widespread application in clinical practice for the analysis of blood, urine, and CEREBROSPINAL FLUID. It enables a complete qualitative amino acid profile of biological fluids to be obtained within 2-3 hours and reveals the presence of unusual nitrogen-containing substances, which is of great DIAGNOSTIC AND PROGNOSTIC significance.

Table 1.5. Reactions used for the identification and semiquantitative determination of Amino Acids and Proteins

Fig. 1.7. Operation of an automatic amino acid analyzer (schematic diagram by Spackman, Moore, and Stein).

1 - mixer; 2 - photoelectric colorimeter; 3 - chart recorder.

Automatic amino acid analyzers are continuously being improved, leading to higher sensitivity and faster analysis times. For instance, modern High-Performance Liquid Chromatography (HPLC) instruments make it possible to analyze a protein hydrolysate within 45 minutes while determining amino acid concentrations in the picomole range (Fig. 1.8).

Amino acid mixtures can also be successfully separated by paper Electrophoresis. At pH 6.0, acidic and basic Amino acids can be effectively separated from neutral ones. Under these conditions, negatively charged (acidic) amino acids migrate toward the anode, while positively charged ones move toward the cathode. Neutral amino acids remain at the origin line.

To separate neutral amino acids, electrophoresis is typically carried out at pH 1.8–2.0, where all of them migrate toward the anode with small but detectable differences in mobility. Following electrophoresis, the positions of amino acids on the electrophoretogram are identified using chemical reactions, and after elution of the colored products, they are quantified.

Fig. 1.8. HPLC of amino acids according to Zeux and Volter. Separation on a column (3 × 250 mm) packed with an ion-exchange resin (polystyrenedivinylbenzene). Amino acid concentration 500 pmol/L; detection reagent is fluorescamine, which forms a strongly fluorescent compound with the amino group.

1 - Asp; 2 - Thr; 3 - Ser; 4 - Glu; 5 - Gly; 6 - Ala; 7 - Cys; 8 - Val; 9 - Met; 10 - Ile; 11 - Leu; 12 - Tyr; 13 - Phe; 14 - Lys; 15 - His; 16 - Arg.