New fluorogenic substrates for N-arginine dibasic convertase

Eva Csuhai, Maria Aparecida Juliano, Jan St Pyrek, Amy C. Harms, Luiz Juliano, Louis B. Hersh

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


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-GGFLR-RIQ-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 (K(m) values of ~1.0 μM, and V(max) values of ~ 1 μM/(min·mg) are similar to those obtained with peptide hormones as substrates.

Original languageEnglish
Pages (from-to)149-154
Number of pages6
JournalAnalytical Biochemistry
Issue number1
StatePublished - Apr 10 1999

Bibliographical note

Funding Information:
This work was supported in Brazil by Fundac¸ão de Amparo Pes-quisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´ico e Tecnológico (CNPq). This work was also supported in part by NIDA/NIH Grant DA02243. E.Cs. is a recipient of a NIDA/NIH Postdoctoral Fellowship DA05671.

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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