Evaluation of some fluorogenic substrates for continuous assay of aminopeptidase P

Three potential fluorogenic substrates for assay of aminopeptidase P (AP-P) have been prepared and evaluated, using enzyme purified from porcine kidney. They are based on internal quenching of the synthetic, fluorescent amino acid (R,S)-2-amino-3-(7-methoxy-4-coumaryl)propanoic acid ((R,S)-Amp) by a 2,4-dinitrophenyl (DNP) group. The compounds are X-Pro-Pro-(R,S)-Amp-NH2, where X is H-Lys(E-DNP), H-Orn(S-DNP), or L-2-amino-3-(DNP)aminopropionic acid. The first two were found to be excellent substrates for AP-P, with respective K-m values of 4.8 and 5.2 mu M. An advantageous feature is that under the conditions of assay, using 4-mm(2) cells, the substrates are without noticeable quenching effect on the fluorescence of Pro-Pro-(R,S)-Amp-NH2 (the product liberated by the action of AP-P). At concentrations greater than about 30-50 mu M, both substrates appear to inhibit the enzyme, but this has little practical consequence since assays can be carried out at substrate concentrations, giving up to approximately 80% of V-max without this inhibitory effect being noticeable. The Lys derivative was found to be a very useful substrate for a continuous assay for AP-P and equally good in a discontinuous assay of multiple samples using microtiter plates. The racemic center at the Amp residue did not prevent total hydrolysis of the Lys derivative, suggesting that subsite specificity in AP-P does not extend as far as the P3' position. (C) 1997 Academic Press.