Abstract
Recently we developed the new synthetic substrates for various proteases, α-naphthylester derivatives such as acetyl-Tyr-α-naphthylester (ATNE), tosyl-L-Lys-α-naphthylester (TLNE) and L-Pro-L-Phe-L-Arg-a-naphthylester (PPANE). We found that these substrates were highly sensitive to the various trypsin-like enzymes and chymotrypsin, and also that they could be used in preparing zymograms of various proteases.
In this work, in order to clear the possibility for clinical application, we studied on the development of micro-assay of human urinary kallikrein and Clesterase, a first component of human complement, using PPANE and ATNE as substrates, respectively. With PPANE as substrate, the minimum detectable concentration of human urinary kallikrein, was about 0.001KU. Thus use of PPANE provided a highly sensitive method for detection of human urinary kallikrein. Kallikrein could be determined with a 25μl samples of human urine. Also, with ATNE as substrate, Cl esterase could be determined with a 100μl samples of human serum.
Further, we examined the proteases in normal and metaplastic stomach mucosas which has been considered to be a possible precancerous stage by preparing zymograms of these tissue extracts with TLNE and PPANE as substrates. With both substrates, the zymogram of metaplastic mucosa showed an additional band besides those of normal mucosa.
These results suggest that the α-naphthylester derivatives are very available for the investigation of proteases in various pathological conditions.
