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Abstract
The use of enzymes for protein labeling promises many advantages over the traditional chemical methods. Labels may be attached by addition via condensation reactions or by substitution through transpeptidations. These reactions compete with removal of the label by hydrolysis. Carboxypeptidase Y, a serine protease from yeast was examined as a possible catalyst for attachment of labels to proteins.
During the course of the reaction in the presence of an amino acid nucleophile, both the hydrolysis and transpeptidation reactions are observed. Initially, the transpeptidation product is formed in good yields, but after the substrate peptide is depleted, the transpeptidation product is hydrolyzed. The initial rate of formation of the transpeptidation product is proportional to the nucleophile concentration while the rate of the hydrolysis reaction is independent of the nucleophile concentration. A kinetic model and a computer program for simulation of the reaction are presented.
The structural requirements of the nucleophile were examined. Sulfonic and phosphonic acids groups were not effective in place of the carboxylic acid group. Separation of the amino and carboxylic acid groups by more than one carbon atom or removal of the acid group results in lower incorporation efficiency. The enzyme was found to lose activity on incubation at high temperature or pH and in the presence of organic solvents. Sufficient activity was retained in detergents and chaotropic agents to allow their inclusion in enzymatic labeling systems. No hydrolysis or transamidation of the side chain amide of asparagine was detected.
Application of these reactions to the labeling of antibodies produced variable amounts of label incorporation, especially at neutral pH. No incorporation was observed when the reactions were applied to a synthetic peptide corresponding to the carboxyl terminus of the antibody heavy chain. These results are explained in terms of the predicted structure of the peptide and the potential for variable degrees of hydrolysis or denaturation of the antibodies during isolation and storage.
