New fluorogenic substrates for N-arginine dibasic convertase.
N-Arginine dibasic (NRD) convertase is a recently described peptidase capable of selectively cleaving peptides between paired basic residues. The characterization of this unique peptidase has been hindered by the fact that no facile assay procedure has been available. Here we report the development of a rapid and sensitive assay for NRD convertase, based on the utilization of two new internally quenched fluorogenic peptides: Abz-GGFLRRVGQ-EDDnp and Abz-GGFLRRIQ-EDDnp. These peptides contain the fluorescent 2-aminobenzoyl moiety that is quenched in the intact peptide by a 2, 4-dinitrophenyl moiety. Cleavage by NRD convertase at the Arg-Arg sequence results in an increase of fluorescence. NRD convertase cleaves these peptides efficiently and with high specificity as observed by both HPLC and fluorescence spectroscopy. The rate of hydrolysis of the fluorogenic substrates is proportional to enzyme concentration, and obeys Michaelis-Menten kinetics. The kinetic parameters for the fluorescent peptides (Km values of approximately 1.0 microM, and Vmax values of approximately 1 microM/(min. mg) are similar to those obtained with peptide hormones as substrates.
Csuhai
E
E
Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0084, USA.
Juliano
M A
MA
Pyrek
J S
JS
Harms
A C
AC
Juliano
L
L
Hersh
L B
LB
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D008297:Male; D008666:Metalloendopeptidases / Q000032:analysis*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology
D005456:Fluorescent Dyes; D010455:Peptides; D008666:Metalloendopeptidases; C087976:nardilysin