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

Chemical reactions of proteins, determination of terminal and functional groups in proteins
Chemical reactions of proteins
Reactions for free amino groups

Alkylation. For the alkylation of Proteins, Reagents such as dimethyl sulfate, methyl iodide, methyl bromide, and diazomethane (CH₂N₂) are used. The reaction proceeds in a mildly alkaline medium at a Temperature of 20–25°, and its mechanism is analogous to that of the amino groups in free Amino Acids. In addition to amino groups, sulfhydryl groups and the hydroxyl groups of Tyrosine can also participate in this reaction.

Methylation reactions have been widely used to investigate the Introduction/13.html">Structure of Fibrous proteins, as well as in studies of Insulin. Upon interaction of the latter with methyl iodide, complete inactivation of the hormone was observed, indicating the crucial functional role of the amino groups.

Arylation. Among these reactions, we shall focus exclusively on the Methods for determining N-terminal amino acids using dinitrofluorobenzene and pipsyl chloride. The dinitrophenylation method was first proposed by Sanger in 1945; it consists in reacting dinitrofluorobenzene with the N-Terminal Groups of a protein or peptide in a mildly alkaline medium to form the corresponding dinitrophenyl derivatives. The bond between dinitrobenzene and the amino group is resistant to the action of strong acids, which allows the dinitrophenyl derivatives of proteins to be subjected to acid Hydrolysis.

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This releases stable dinitrophenyl Amino Acid Derivatives, which can be separated chromatographically, identified using reference compounds ("standards"), and quantified colorimetrically. As a result, we reveal The Nature of the N-terminal amino acid (or several N-terminal acids, if the protein consists of multiple polypeptide chains).

However, the method has limited Specificity, since dinitrofluorobenzene reacts not only with the α-amino groups of N-terminal amino acids, but also with the ε-amino groups of Lysine, the SH groups of cystine, the OH groups of tyrosine, and the imidazole groups of Histidine. Nevertheless, ε-DNP-lysine is easily distinguished using an appropriate standard, while all other derivatives are colorless and do not interfere with the determination.

A similar determination can be carried out using pipsyl chloride (p-iodophenylsulfonyl chloride). The method is akin to Sanger's method, and the chemical mechanism of the reaction has been outlined above (see Chapter II). A distinctive feature of this method is that pipsyl chloride is labeled with a radioactive iodine isotope, and the pipsyl derivatives of Amino acids are determined radiometrically after chromatographic Separation. The analysis results generally agree well with data obtained by the dinitrofluorobenzene method. A disadvantage of this method compared to the dinitrofluorobenzene one is that certain N-terminal groups, if deeply embedded within the protein molecule, may prove inaccessible to pipsyl chloride yet accessible to dinitrofluorobenzene. Therefore, this approach for analyzing N-terminal amino acids is used less frequently.

Acylation. A wide variety of compounds are used as acylating agents. Most of them react not only with amino groups, but sometimes (and even more actively) with phenolic OH groups, SH groups, and certain others. Here, we will consider only those reagents that preferentially react with amino groups. These include acetic anhydride, phenyl isocyanate and phenyl isothiocyanate, benzoyl chloride, and carbobenzoxy chloride.

Acetic anhydride is the most widely used reagent for protein acylation. It is more specific than ketene, does not cause Protein Denaturation, and requires no specialized equipment. To carry out the reaction, the protein is dissolved or suspended in a cooled sodium acetate solution, and acetic anhydride is slowly added to the suspension:

The acetylated derivatives of Hemoglobin and Trypsin obtained by this Procedure retained their biological activity unchanged. Serum albumin also underwent no denaturation, which was proven by determining the number of masked SH groups.

The reaction of the amino group with phenyl isothiocyanate has found widespread application in determining the N-terminal amino acids of proteins or Peptides. This method was first proposed by Edman in 1950. The chemical mechanism of the reaction has already been described above (see Chapter II), and here we shall only outline its mechanism. Upon the interaction of phenyl isothiocyanate with the α-amino groups of the N-terminal amino acids of a protein, a phenylthiocarbamyl protein derivative is formed. The reaction proceeds in an aqueous-pyridine or aqueous-dioxane medium at pH 8–9 and a temperature of 25–37°. When the phenylthiocarbamyl protein derivative is treated with an acid, cyclization and Cleavage of the N-terminal residues occur in the form of phenylthiohydantoins. The latter can be chromatographically separated and identified; they can also be cleaved down to free Amino Acids and the latter quantified. The protein remaining after the cleavage of the phenylthiohydantoin contains one less amino acid than the original one. It can be isolated and treated with phenyl isothiocyanate once again. In this manner, by sequentially cleaving one amino acid after another, their sequence in the N-terminal region of the protein can be determined. This technique has successfully determined the N-terminal sequence for ten or more amino acid residues in the molecules of insulin, β-corticotropin, the pituitary melanophore-stimulating hormone, and other proteins.

However, the method also has A number of drawbacks. The conditions for the cyclization and cleavage of phenylthiohydantoins vary among different proteins and must be optimized for each specific case. The chromatographic conditions for phenylthiohydantoins are insufficiently well-developed; their alkaline cleavage to the free acid is sometimes accompanied by the decomposition of the acid itself. These shortcomings can be circumvented by carrying out hydrolysis and establishing the Amino Acid Composition of both the original protein and the protein remaining after the cleavage of the phenylthiohydantoin. Nevertheless, such a procedure significantly complicates the Determination of the N-terminal amino acid.

Finally, the last acylation reaction under consideration is the reaction with carbobenzoxy chloride. Its mechanism is analogous to that for free amino acids. The reaction proceeds at a temperature of 0–25° in a mildly alkaline medium (pH 8), with amino groups reacting primarily and other basic groups reacting to a negligible extent. This reagent has been widely used to mask amino groups in the preparation of modified proteins, as well as in studying The Role of amino groups in the biological activity of tobacco mosaic virus (TMV), insulin, and other proteins.

Reaction with formaldehyde. It is well known that at room temperature and in a neutral medium, formaldehyde rapidly and reversibly combines with the amino and imino groups of proteins. In this process, an amino group can combine with one or two molecules of the aldehyde, whereas an imino group can combine with only one. The reaction likely proceeds as follows:

R—NH₂ + НСНО ⇄ R-NH—CH₂OH

R—NH—CH₂ОH + НCHО ⇄ R—N(CH₂ОH)₂

Proof of the reversibility of this process is the fact that formaldehyde can be removed from the protein by dialysis or dilution, provided the interaction was short-term and took place at room temperature.

Upon prolonged exposure of proteins to a dilute formaldehyde solution at pH 8–9 and 20–37°, slow and irreversible reactions take place, during which firm binding of formaldehyde occurs. This binding is due to The conversion of methylol groups (CH2OH) into intermolecular methylene bridges that link amino groups, on the one hand, and imidazole, guanidino, and indole groups, on the other:

R'—NH—CH₂OH + HN = R" → R'—NH—СН₂—N = R"

The presence of intermolecular cross-links was proven by an increase in molecular weight and a decrease in the solubility and Swelling capacity of formaldehyde-treated proteins. Processes of this nature occur during leather tanning and the preparation of Vaccines and toxoids. Incubation of toxins and vaccines with formaldehyde leads to The formation of stable derivatives with significantly attenuated activity and unaltered antigenicity.

Reaction with nitrous acid. Deamination of proteins with nitrous acid (the Van Slyke method) is one of the most convenient techniques for determining the number of free amino groups in proteins or the degree of substitution of these groups in their modified derivatives. Protein deamination is carried out in 0.5 M acetate buffer (pH 4) at 0° with a 1 M sodium nitrite solution for various time intervals. Under these conditions, the terminal α-amino groups of the protein react most readily, the ε-amino groups significantly slower, and the guanidino groups very slowly indeed.

A significant drawback of this reaction is the fact that a number of proteins cannot withstand such Treatment, becoming denatured and precipitating even before complete deamination is achieved. In this case, the Quantitative determination of amino groups, as well as The Study of The Effect of protein deamination on its biological activity, becomes impossible. Serum albumin is an example of such a protein. At the same time, during protein deamination, not only amino groups but also indole, imidazole, guanidino, and disulfide groups may enter into the reaction. Some authors have also noted The oxidation of sulfhydryl groups. Therefore, it is highly probable that protein denaturation is associated with secondary reactions of nitrous acid with the aforementioned functional groups.