Fluorogenic Substrates for Bacterial Aminopeptidase P and Its Analogs Detected in Human Serum and Calf Lung

Publisher Website
Google Scholar
Abstract
A sensitive fluorimetric assay was developed for bacterial aminopeptidase P, based on intramolecularly quenched fluorogenic substrates. Two substrates were synthesized, Phe(NO2)-Pro-HN-CH2-CH2-NH-ABz (substrate I) and Phe(NO2)-Pro-Pro-HN-CH2-CH2-NH-ABz (substrate II), in which the Phe(NO2) group (p-nitro-L-phenylalanyl) quenches the fluorescence of the ABz group (o-aminobenzoyl). Both substrates were readily cleaved by aminopeptidase P from Escherichia coli, releasing p-nitro-L-phenylalanine and causing a proportional increase in fluorescence. Complete hydrolysis of the 2 substrates resulted in a 7.5-fold and 3.4-fold fluorescence increase, respectively. Applying this fluorogenic assay, aminopeptidase P-like activity could be detected and measured quantitatively in human serum and calf-lung extracts. Substrate II was specifically cleaved by aminopeptidase P in these preparations, while substrate I was apparently cleaved by other enzymes as well. In both preparations, the enzyme activity was independent of Co2+ ions, and Pro-HN-CH2-CH2-NH-ABz(Cbz) was inhibitory. The kinetic constant Km was determined as 0.35 mM and 0.28 mM for the human serum and the calf-lung enzymes, respectively. The enzyme activity was only slightly dependent on pH in the range 7.0-8.4.

This publication has 12 references indexed in Scilit: