Chemistry and Biology of Proteins - F. Haurowitz 1953

Internal structure of globular proteins
Terminal d-carboxyl groups

Free protein carboxyl groups readily form esters with ethyl alcohol in the presence of small amounts of mineral acids [34]. Similar esters are produced by the action of alkylating agents on Proteins:

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However, the corresponding Methods are unsuitable for determining C-terminal carboxyl groups for two reasons. First, the resulting ester bonds are easily hydrolyzed; second, In addition to C-terminal carboxyl groups, the free carboxyl groups of glutamic and aspartic acids are also alkylated. Terminal C-terminal carboxyl groups can be distinguished from the carboxyl groups of dicarboxylic Amino Acids because the former react with thiocyanate to form thiohydantoin derivatives [35, 36].

It is clear from the formula given above that this reaction requires the presence of an N-terminal amino group, either free or incorporated into a peptide bond. Therefore, the free carboxyl groups of aspartic and glutamic acids cannot form a thiohydantoin ring. It was established in the author's laboratory that this reaction proceeds not only with Amino Acids and Peptides, but also with proteins. Unfortunately, it has not yet been possible to isolate amino acid hydantoin derivatives in these experiments [37, 38].