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

Research output: Contribution to journalArticle

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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
LanguageEnglish
Pages13-17
Number of pages5
JournalAnalytical Biochemistry
Volume253
Issue number1
DOIs
Publication statusPublished - 1997

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Fluorescent Dyes
Assays
Substrates
Quenching
Derivatives
Enzymes
Hydrolysis
Fluorescence
X-Pro aminopeptidase
Amino Acids
Acids

Cite this

Hawthorne, S.J. ; Harriott, P. ; Lim, J. ; Turner, A.J. ; Walker, B. ; Williams, C.H. / Evaluation of some fluorogenic substrates for continuous assay of aminopeptidase P. In: Analytical Biochemistry. 1997 ; Vol. 253, No. 1. pp. 13-17.
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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",
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Evaluation of some fluorogenic substrates for continuous assay of aminopeptidase P. / Hawthorne, S.J.; Harriott, P.; Lim, J.; Turner, A.J.; Walker, B.; Williams, C.H.

In: Analytical Biochemistry, Vol. 253, No. 1, 1997, p. 13-17.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Evaluation of some fluorogenic substrates for continuous assay of aminopeptidase P

AU - Hawthorne, S.J.

AU - Harriott, P.

AU - Lim, J.

AU - Turner, A.J.

AU - Walker, B.

AU - Williams, C.H.

N1 - cited By 9

PY - 1997

Y1 - 1997

N2 - 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

AB - 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

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DO - 10.1006/abio.1997.2320

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T2 - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

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