Assay for enzyme inhibition: detection of natural inhibitors of trypsin and chymotrypsin

A technique for quickly detecting nanogram quantities of low- and high-molecular-weight inhibitors of some serine proteases is described. The inhibitor solutions are spotted onto agar films which contain either L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK)-trypsin or tosyl lysine chloromethyl ketone (TLCK)-chymotrypsin. Enzyme inhibition is visualized as colorless zones on a pink background after the films were stained with the chromogenic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester. The method is used for rapidly testing both high-performance liquid chromatography fractions and thin-layer chromatograms to identify the inhibitors of trypsin and chymotrypsin in complex microbial extracts. The assay is quantitative so that it is possible to compare the specificity of the inhibitory fractions for trypsin and chymotrypsin. Results with standard inhibitors demonstrate the high sensitivity of the method, e.g., inhibition is detected with 1 ng of soybean trypsin inhibitor and 0.3 ng of antipain or chymostatin.