Fluorogenic substrates for bacterial aminopeptidase P and its analogs detected in human serum and calf lung.
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 (rho-nitro-L-phenylalanyl) quenches the fluorescence of the ABz group (omicron-aminobenzoyl). Both substrates were readily cleaved by aminopeptidase P from Escherichia coli, releasing rho-nitro-L-phenylalanine and causing a proportional increase in fluorescence. Complete hydrolysis of the two substrates resulted in a 7.5-fold and 3.4-fold fluorescence increase, respectively. Applying this fluorogenic assay, we were able to detect and measure quantitatively amino-peptidase P-like activity in the human serum and calf-lung extracts. Substrate II was shown to be 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.
D000626:Aminopeptidases / Q000097:blood / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002863:Chromogenic Compounds / Q000138:chemical synthesis / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005470:Fluorometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D009842:Oligopeptides / Q000138:chemical synthesis; D013379:Substrate Specificity
D002863:Chromogenic Compounds; D009842:Oligopeptides; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase