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-methoxy4-coumaryl)propanoic acid ((R,S)-Amp) by a 2, 4dinitrophenyl (DNP) group. The compounds are X-Pro-Pro-(R, S)-Amp-NH2, where X is H-Lys(epsilon-DNP), H-Orn(delta-DNP), or L-2-amino-3-(DNP)aminopropionic acid. The first two were found to be excellent substrates for AP-P, with respective Km values of 4.8 and 5.2 microM. An advantageous feature is that under the conditions of assay, using 4-mm2 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 microM, 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 Vmax 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.
Hawthorne
S J
SJ
Medical Biology Centre, The Queen's University of Belfast, Belfast, BT9 7BL, United Kingdom.
Harriott
P
P
Lim
J
J
Turner
A J
AJ
Walker
B
B
Williams
C H
CH
D000626:Aminopeptidases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001322:Autoanalysis / Q000379:methods; D005456:Fluorescent Dyes; D007668:Kidney / Q000201:enzymology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D013552:Swine
D005456:Fluorescent Dyes; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase