A Sensitive Colorimetric Assay for Various Proteases Using Naphthyl Ester Derivatives as Substrates

Abstract

A convenient and highly sensitive colorimetric assay for various proteases, such as trypsin, chymotrypsin, plasmin, thrombin, and urokinase is described. The substrates used are α-naphthyl ester derivatives of N^α-tosyl-L-lysine, N^α-acetylglycyl-L-lysine, and N^α-acetyl-L-tyrosine, and activity is assayed by colorimetric determination of α-naphthol released from them. Use of these α-naphthyl ester derivatives made the method more sensitive than the use of the corresponding methyl or ethyl ester derivatives.
The minimum detectable concentrations of trypsin, chymotrypsin, plasmin, thrombin and urokinase in this method were about 0.002μg, 0.01μg, 0.002 CU, 0.01 IU, and 2 IU, respectively.
The Km values of trypsin and thrombin for TLNE were 0.11mM and 0.15mM, while those for TLME were 2.5mM and 6.7mM, respectively; the K_m values of chymotrypsin for ATNE and ATEE were 0.18mM and 0.7mM, respectively; and the K_m values of urokinase for AGLNE and AGLME were 0.17mM and 4mM, respectively.
Zymograms of various proteases were easy to prepare using these α-naphthyl ester substrates, and zymograms of trypsin and chymotrypsin were made with TLNE and ATNE, respectively, as substrates.