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 involving carboxyl groups

Esterification. Methyl alcohol is the most convenient agent for the esterification of carboxyl groups. The conditions for selective and complete esterification were developed by Fraenkel-Conrat and are as follows. The protein is suspended in a 100-fold excess of alcohol, and Hydrochloric acid is added portion-wise to the suspension as a catalyst to a final concentration of 0.1 N. The reaction proceeds for several days at 0—20°. The resulting ester is dialyzed and concentrated by lyophilization. Analysis of the products obtained showed that the number of methoxyl groups (OCH₃) in them is equal to the number of carboxyl groups in the original protein.

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A disadvantage of the method is the Denaturation of certain Proteins (e.g., serum albumin) under the action of even weaker acids. For those proteins that withstand the reaction conditions, this method can be used to determine The Role of carboxyl groups in biological activity. The esterification of these groups, for instance, proved detrimental to The activity of the lactogenic hormone and Insulin.

Reaction with anhydrous hydrazine. The reaction with anhydrous hydrazine is the only chemical METHOD FOR DETERMINING C-terminal Amino Acids that yields reliable results in many cases. The other two Methods—reduction with metal hydrides and the thiohydantoin method—generally yield irreproducible and unreliable results. The method was proposed by Akabori in 1852. The protein is heated with anhydrous hydrazine in a sealed ampoule at 100—120° for 8–10 hours. This causes Protein Cleavage, or Hydrazinolysis, As a result of which all amino acids except the C-terminal one form hydrazides. By Treatment with benzaldehyde, The amino acid hydrazides are converted into insoluble derivatives, which are removed by filtration. The free C-terminal Amino acids are then determined chromatographically.

The hydrazinolysis method has A number of specific drawbacks. For example, treatment with hydrazine causes partial or complete degradation of such Amino acids as Cysteine and cystine, as well as asparagine, glutamine, and dicarboxylic amino acids. The hydrazides of Certain amino acids prove insufficiently stable and may revert to free amino acids during subsequent Processing. This is a major flaw of the method and often renders it unsuitable for the Analysis of certain proteins. Nevertheless, this method is still widely used in many laboratories today. The reason for this is its relative simplicity and applicability to the Analysis of proteins in which the hydrazides of C-terminal amino acids are sufficiently stable.