The action of proteolytic enzymes on N,N-dimethyl proteins. Basis for a microassay for proteolytic enzymes.

Abstract

N,N-Dimethyl proteins were prepared by reductive methylation of their amino groups with formaldehyde and NaBH4. Proteolysis of these alkylated proteins by trypsin, α-chymotrypsin, subtilisin, pepsin, and fungal protease was determined by direct measurement of the bonds split, with the use of trinitrobenzenesulfonic acid to determine directly the appearance of new terminal amino groups. The low blank values obtained with N,N-dimethyl proteins has resulted in a greatly increased sensitivity and accuracy not possible with unmodified proteins. On the basis of these studies, an assay of proteolytic activity is described, with N,N-dimethylcasein or N,N-dimethylhemoglobin as substrate, which is from 10 to several hundred times more sensitive than the standard caseinolytic assay of Kunitz.