Intramolecularly‐Quenched Fluorescent Peptides as Fluorogenic Substrates of Leucine Aminopeptidase and Inhibitors of Clostridial Aminopeptidase

Amos CARMEL, Efrat KESSLER, Arieh YARON

Research output: Contribution to journalArticlepeer-review

Abstract

Fluorogenic oligopeptide derivatives of the type Lys(ABz)‐X‐ONBzl, where ABz is o‐amino‐benzoyl (anthraniloyl), X stands for Ala, Phe, or Ala‐Ala, and ONBzl is p‐nitrobenzyloxy, were synthesized and shown to be hydrolyzed by leucine aminopeptidase. The hydrolysis is accompanied by an increase in fluorescence due to disruption of the intramolecular quenching of the fluorescent anthraniloyl moiety by the nitrobenzyl ester group. The spectral characteristics of the compounds are not consistent with an energy transfer mechanism according to Förster, therefore the quenching is assumed to be caused by a direct encounter between the quenching and the fluorescent groups. The change in fluorescence that accompanies the enzymic hydrolysis of the first peptide bond was used for quantitative measurement of the activity of leucine aminopeptidase and for the determination of some of its kinetic parameters. A bacterial aminopeptidase from Clostridium histolyticum that is very similar to leucine aminopeptidase in its substrate specificity did not hydrolyze the above peptide derivatives. The hydrolysis of leucine p‐nitroanilide by this enzyme was found to be inhibited by the three peptides and the corresponding inhibition constants were determined.

Original languageEnglish
Pages (from-to)617-625
Number of pages9
JournalEuropean Journal of Biochemistry
Volume73
Issue number2
DOIs
StatePublished - Mar 1977
Externally publishedYes

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