An improved spectrophotometric assay for leucine aminopeptidase

Abstract

A sensitive assay to determine the activity of leucine aminopeptidase (EC 3.4.11.1), using L-leucine thiobenzyl ester as substrate, was developed. Hydrolysis of the ester by leucine aminopeptidase can be monitored in the presence of 5,5-dithiobis-(2-nitrobenzoic acid) by continuous spectrophotometric measurement at 412 nm. Comparison with some amide substrates showed that the thiol ester provides a much more sensitive assay, its specificity constant (Vmax./Km) being some 3000-fold higher than that of leucine p-nitroanilide.