Highly sensitive method for determination of esterase activity of alpha-chymotrypsin and alpha-chymotrypsin-like...

A new substrate, Dns-L-phenylalanine ethyl ester, with high UV absorption has been developed for the determination of the esterase activity of alpha-chymotrypsin and alpha-chymotrypsin-like enzymes. The product, generated by the enzyme action, Dns-L-phenylalanine, was clearly separated from the ester substrate by micro reversed-phase high-performance liquid chromatography. The substrate was highly stable under the enzyme assay conditions used. As little as 0.15 ng of alpha-chymotrypsin and 1.49 ng of subtilisin BPN' could be detected when a long reaction time was employed. Hydrolyses of the substrate by alpha-chymotrypsin and alpha-chymotrypsin-like enzymes were blocked by specific inhibitors of the enzymes.