Rapid micro methods for the determination of exo- and endopeptidase activities in plant extracts

Abstract

Rapid and sensitive photometric assays for aminopeptidase, carboxypeptidase, and endopeptidase activities were developed. The methods of aminopeptidase and carboxypeptidase determinations were based on the hydrolysis of artificial substrates ( l-leucine- p-nitroanilide and N-carbobenzoxy- l-phenylalanine- l-alanine, respectively). Endopeptidase activities were assayed either with acetylated casein or with azocasein as substrate. The utilization of microtitration plates and of a multichannel photometer allowed rapid, sensitive, and accurate measurements. The amounts of enzyme extract and of substrates were reduced considerably compared with other methods for the detection of peptide hydrolase activities. The methods described are suitable for routine measurements in large series (e.g., elution profiles from columns, screening procedures).