Evaluation of some fluorogenic substrates for continuous assay of aminopeptidase P

S.J. Hawthorne, P. Harriott, J. Lim, A.J. Turner, B. Walker, C.H. Williams

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Abstract

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(ϵ-DNP), H-Orn(δ-DNP), or L-2-amino-3-(DNP)aminopropionic acid. The first two were found to be excellent substrates for AP-P, with respectiveKmvalues of 4.8 and 5.2 μm. An advantageous feature is that under the conditions of assay, using 4-mm2cells, 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 μm, 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% ofVmaxwithout 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
Original languageEnglish
Pages (from-to)13-17
Number of pages5
JournalAnalytical Biochemistry
Volume253
Issue number1
DOIs
Publication statusPublished - 1997

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