Protein Chemistry - Part 1 - General Protein Chemistry - Ashmarin I. P. 1968

Chemical composition of proteins
Determination of amino acids in protein hydrolysates and their fractionation

A wide variety of Methods are used for the Quantitative determination of Amino acids, which can be divided into the following groups: 1) chemical methods; 2) enzymatic methods; 3) isotope labeling methods; 4) microbiological methods; and 5) chromatographic methods, which also serve as Separation techniques.

Determination of Amino acids in a mixture. Among chemical methods, determining primary amino groups by converting their nitrogen into a gaseous state (the Van Slyke method) and the ninhydrin method are of particular importance. These yield the total amino acid content. Structure/129.html">Specific methods have been developed for individual groups of amino acids. For instance, The oxidation of hydroxyamino acids with periodic acid is carried out According to the following reaction:

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The amount of evolved ammonia is used to estimate the total content of these acids. Specific, frequently colorimetric reactions exist for determining Histidine, Cysteine, Arginine, and several Other Amino Acids; these will be discussed below in the section dedicated to individual amino acids (§ 4).

The group of basic amino acids (histidine, Lysine, arginine), acidic amino acids (glutamic and aspartic acids), as well as phenylalanine, can be determined via decarboxylation using specific Enzymes (typically of bacterial origin), followed by the quantitative measurement of the resulting carbon dioxide.

The isotope dilution method is also successfully employed to determine amino acids in hydrolysates. A known amount of a labeled amino acid preparation, such as ¹⁴C-labeled Glycine, is added to the protein hydrolysate. The glycine is then isolated, and its radioactivity is compared with that of the added preparation. The amount of glycine in the hydrolysate is calculated based on the degree of isotope dilution. The advantage of this method is that it does not require the complete Isolation of the amino acid.

Highly sensitive microbiological Methods for the quantitative determination of Amino acids have been developed. These are based on selecting microbial strains for which a specific amino acid acts as a growth-limiting factor. For example, Tryptophan serves as such a limiting factor for *Streptococcus plantarum*, with Microbial growth slowing down significantly when The amino acid concentration drops by 1 µg/ml. The dependence of growth on concentration is linear, making it possible to determine the concentration of the amino acid by measuring culture density (turbidimetrically). These methods were most thoroughly developed in the works of S. R. Mardashov and his school. They are particularly useful when high-sensitivity determinations are required without the need to separate the amino acid mixture.

Chromatographic methods. The most efficient separation of amino acids is achieved using chromatographic techniques. It is precisely through the application of these analytical approaches (paper Chromatography, ion-exchange resins, etc.) that significant breakthroughs in studying the Amino Acid Composition of Proteins and Polypeptides have been made.

The separation of protein hydrolysate components by chromatography is based on the fact that different amino acids have unequal partition coefficients between Water and a water-immiscible (yet partially water-soluble) solvent. Kieselguhr, Cellulose, silica gel, and filter paper can serve as the aqueous or so-called stationary phase, with their pores and capillaries holding a specific amount of water. The supporting medium can act either as an inert carrier or play an active role in adsorbing the substances to be separated.

Various water-saturated Solvents (butanol, phenol, collidine) are used as the Mobile phase. The amino acid mixture is applied to the adsorbent, after which the solvent is allowed to flow through it. Because the partition coefficients of different amino acids vary, they travel different distances from the origin point within the same time span. The ratio of the distance traveled by a given amino acid to the distance traveled by the solvent front is designated as R_f, a characteristic value for each amino acid under specific experimental conditions (adsorbent type, solvent system, etc.). The R_f value is always less than one. For a given solvent system, it is a reproducible value that AIDS in identifying an unknown amino acid.

Paper chromatography. As already noted, in paper chromatography, the stationary phase is the water contained within the paper capillaries. The movement of each component in the mixture results from a continuous exchange between the stationary aqueous phase and the mobile organic phase. The greater the solubility of a component in the organic liquid (compared to water), the faster it moves. Since the effective number of extraction and binding steps is very large, a small difference in solubility translates into a substantial difference in migration rates. The Procedure for separating an amino acid mixture by paper chromatography is as follows. A small amount of the sample solution is applied to the paper near the edge of a strip. After drying, the edge of the strip is immersed in a trough containing an organic solvent saturated with water. Driven by capillary forces, the solvent slowly creeps up the paper, washing the substances away from the application point. For each grade of paper ("slow" or "fast"), the moving liquid front advances at a characteristic rate. The direction of solvent flow can be upward ( ascending chromatography) or downward (descending chromatography).

In radial chromatography, separation is carried out on circular disks rather than paper strips; the amino acid mixture is applied to the center of the disk, where the solvent is also introduced (e.g., via a capillary). In radial chromatography, individual Amino acids are detected not as spots, but as distinct arcs.

In some cases, two-dimensional chromatography is employed to achieve a more complete separation of complex multicomponent systems. The test mixture is applied to the corner of a large sheet of paper and chromatographed in a descending stream of a solvent (e.g., phenol-water-ammonia). Once separation is complete, the sheet is dried, and the procedure is repeated using a different solvent in a direction perpendicular to the first. Two-dimensional chromatography achieves the separation of all amino acid hydrolysate components into distinct, independent spots (Fig. 8). This method is used exclusively for qualitative characterization due to significant amino acid losses caused by trace amounts being adsorbed by the paper.

Fig. 8. Example of a two-dimensional chromatogram of 18 amino acids (from Bailey, 1965).

To detect and quantitatively determine amino acids after separation, the dried chromatograms are treated with a 0.5% solution of ninhydrin in acetone. Amino acids appear as purple spots; the spots are cut out, the colored complex is extracted with an alcoholic copper sulfate solution, and the color intensity is measured at a wavelength of 575 mµ. The concentrations of the mixture components are determined using calibration curves constructed for each amino acid. Cystine is determined after converting it into cysteic acid via prior oxidation of the protein with performic acid (see Chapter IV, § 1).

Paper chromatography is a rather labor-intensive and time-consuming method. The analytical error in some cases may exceed ±5%.

Currently, a more accurate and rapid METHOD FOR DETERMINING the amino acid composition of proteins using ion-exchange resins has been developed.

Ion-exchange chromatography. The method of ion-exchange chromatography was developed by American scientists Moore and Stein. In 1958, this technique served as the basis for the automatic analyzer, which allows the amino acid composition of PROTEINS AND Peptides to be determined with high speed and precision. Ion-exchange resins consist of an organic polymer prepared in the form of beads of various sizes. Strong cation exchangers—polystyrene resins whose active ionogenic group is SO₃H—are used to separate amino acids. At any pH value, this group exists as the SO^-₃ anion with H⁺ counterions in solution. The polystyrene chains are periodically cross-linked by molecular bridges, creating a three-dimensional network structure within the resin. This structure allows water, electrolytes, and amino acids to penetrate its interior.

Sulfopolystyrene resins are typically used in the sodium form. To achieve this, the H⁺-form of the resin is pre-treated with alkali (rinsing with 1–2 N NaOH). During this process, its SO₃H functional groups are converted into SO₃Na groups.

Amino acid sorption is carried out from a buffer solution at pH 2.

Under these conditions, carboxyl groups are un-dissociated, while amino groups bind a proton and become positively charged, meaning the amino acids act as cations. When they are sorbed onto the resin, an exchange with Na⁺ ions takes place. Diamino acids, carrying two positive charges, are sorbed more strongly than monoamino acids. Furthermore, the strength of binding depends on the dissociation constants of the amino acid NH₃ groups. Slow elution of the amino acids is then performed using Buffer solutions at a pH that causes partial dissociation of the carboxyl and amino groups, thereby weakening the electrostatic interaction with the active groups of the resin. Neutral and acidic amino acids are eluted at pH 3.25–4.25, whereas basic amino acids elute at pH 5.28–5.35. Naturally, they emerge in an order corresponding to differences in their sorption strength on the resin. The eluate is collected in small fractions, achieving a sharp separation of individual amino acids.

All of the aforementioned operations are performed automatically in an instrument known as the amino acid analyzer. During the analysis, micro-dosing pumps pump the buffer through a sulfopolystyrene Column, with automated switching from one elution buffer to another at scheduled intervals. Upon exiting the column, the liquid containing the amino acids is automatically mixed with ninhydrin supplied by a second micro-pump, and the mixture flows through a thin plastic tubing placed in a thermostat heated to 100 °C. Under these conditions, a colored complex is formed between the Amino Acid and ninhydrin. Afterward, the colored solution passes through the cuvette of a photocolorimeter, and a self-recording galvanometer records the optical density curve on a paper strip (Fig. 9). The molar ratios of the amino acids in the given protein are determined from the areas of the characteristic peaks.

Fig. 9. Chromatographic fractionation of a synthetic amino acid mixture on Amberlite IR-120 columns (Moore et al., 1958):

a — 0.9 × 150 cm column; 1 μmol of each amino acid applied (0.5 μmol of cystine): 1 — cysteic acid, 2 — Methionine sulfoxide, 3 — aspartic acid, 4 — methionine sulfone, 5 — Threonine, 6 — Serine, 7 — glutamic acid, 8 — Proline, 9 — glycine, 10 — Alanine, 11 — cystine, 12 — valine, 13 — methionine, 14 — alloisoleucine, 15 — isoleucine, 16 — leucine, 17 — Tyrosine, 18 — phenylalanine; b — 0.9 × 15 cm column, identical amounts of amino acids applied:

1 — phenylalanine, 2 — tyrosine, 3 — lysine, 4 — histidine, 5 — ammonia, 6 — arginine.