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

Chemical reactions of proteins, determination of terminal and functional groups in proteins

A number of practical and theoretical problems can be solved through the Chemical Reactions of Proteins. For instance, one such task is studying The sequence of Amino Acids in polypeptide chains and identifying their terminal groups. Treating proteins with various selective Reagents is frequently employed to identify The Structure of the groups responsible for their biological activity—in other words, to elucidate the relationship between a protein's chemical structure and its function. Finally, this same approach makes it possible to obtain modified proteins that find widespread application in industry and medicine.

Since protein Chemical reactions are governed by the presence of functional groups in their molecules that are also characteristic of amino acids (such as carboxyl and amino groups, SH-groups, the phenolic OH-groups of Tyrosine, and hydroxy acids, among others), they are evidently analogous to the reactions of these same groups in free amino acids. However, the reactivity of polar groups located in the side chains of proteins is frequently reduced. This is due to the fact that the Functional groups of proteins are often shielded—concealed by the folds and loops of the macromolecule's polypeptide chains.

Denaturation disrupts the native conformation of a protein, exposing shielded groups and increasing its reactivity. This was first discovered for the SH-groups of egg albumin. It was found that denaturation induced by heat Treatment, the action of Hydrochloric acid, alcohol, or urea solutions, as well as ultraviolet radiation, leads to the exposure of SH-groups that yield a positive nitroprusside test. Slightly later, analogous Methods revealed the unmasking of Disulfide Bonds, the phenolic OH-group of tyrosine, and the indole group of Tryptophan. The increased reactivity of several proteins toward ketene and fluorodinitrobenzene following denaturation indicated the masking of a portion of their basic groups. Finally, an analysis of titration curves and studies on the Esterification of certain proteins pointed to the shielding of some acidic groups.

Because the shielding of individual functional groups varies among different proteins due to the peculiarities of their tertiary structure, the reactivity of identical groups can also differ from one protein to another. Naturally, this complicates the precise Determination of the number of functional groups in a given protein.

Protein chemical reactions also differ from those of amino acids in that the majority of protein reagents interact with more than one functional group. For example, ketene, which is used for the Acetylation of protein amino groups, reacts not only with them but also with phenolic and sulfhydryl groups. To prevent these Side Reactions, treatment should preferably be carried out either in a 1 M acid solution or at pH 10, which does not always favorably affect the biological activity of certain proteins. Reagents such as dinitrofluorobenzene, nitrous acid, and phenylisothiocyanate also react with amino groups with insufficient Specificity, and the specificity of each varies depending on the protein. Consequently, it is often necessary to select conditions for each individual protein that maximize reaction specificity, which requires testing several different reagents for every protein.

Admittedly, There are also strictly Specific Reagents for certain functional groups in proteins. For instance, most reagents for SH-groups are highly selective; acetic anhydride reacts selectively with amino groups, and esterifying agents (methyl and ethyl alcohols) with carboxyl groups; sulfuric acid and phosphoric anhydride are selective toward the OH-groups of hydroxy acids. However, for a number of proteins, esterification conditions or treatment with sulfuric acid and phosphoric anhydride prove destructive, leading to Protein Denaturation or breakdown. Here we encounter a third distinctive feature of protein chemical reactions: conducting reactions under conditions that are satisfactory for Amino acids can result in protein denaturation. Therefore, these reactions must be carried out in the mildest manner possible to avoid irreversible structural changes.

To summarize the foregoing, The behavior of one protein toward a specific reagent and reaction conditions does not necessarily coincide with that of another protein. While certain reagents may prove sufficiently specific toward specific functional groups of a protein under given conditions—and the protein itself does not undergo denaturation during the reaction—for another protein, these conditions may prove destructive, and the reagent itself may lose its group specificity. Therefore, preliminary experiments with each individual protein are required to determine the optimal reaction conditions for specific functional groups.

Some chemical reactions of proteins will be examined below; wherever possible, selective reagents, reaction conditions and duration, and the lability of the resulting bond are indicated. Particular attention is paid to the conditions under which maximum reaction specificity is manifested and denaturation is absent.