N. Goudreau et al., DNS-GLY-(P-NO2)PHE-BETA-ALA, A SPECIFIC FLUOROGENIC SUBSTRATE FOR NEUTRAL ENDOPEPTIDASE-24.11, Analytical biochemistry, 219(1), 1994, pp. 87-95
A novel fluorogenic peptide, dansyl-Gly-(p-NO2) Phe-beta Ala (DGNPA),
was synthesized as a selective substrate for neutral endopeptidase 24.
11, an enzyme involved in enkephalin and atrial natriuretic peptide de
gradation and a marker of differentiation (CD10) on the surface of lym
phohematopoietic cells. Cleavage of the substrate Gly-(p-NO2)Phe amide
bond leads to an increase in fluorescence related to the disappearanc
e of the intramolecular quenching of the dansyl fluorescence by the ni
trophenyl residue. This new fluorogenic substrate is an improvement ov
er the commercially available dansyl-D-Ala-Gly-(p-NO2)Phe-Gly, as the
Gly(4) residue of the latter has been replaced by a beta-alanine, ther
efore eliminating a residual sensitivity of the peptide toward angiote
nsin converting enzyme. Moreover, deletion of the D-Ala(2) residue was
shown to increase the quenching efficiency, thus raising the sensitiv
ity of the assay, which was further improved by stopping the reaction
with dioxane. The present substrate has improved affinity (K-m = 37 mu
M, V = 0.72 mu mol min(-1) mg protein(-1)), selectivity, and sensitiv
ity over its precursor and was used in automated assays using 96-well
microplates and a fluorescence plate reader. (C) 1994 Academic Press,
Inc.