Use of a fluorescence microplate reader for the detection and characterization of metal-assisted peptide hydrolysis

Metal ions and complexes that hydrolyze peptides under nondenaturing condit ions of temperature and pH hold great promise for use in protein structural studies. However, the extreme stability of the peptide amide bond has plac ed limits on the number of reagents available. In addition, the development of new cleavage strategies has been hindered by the fact that no facile pr ocedure exists for the detection and characterization of metal-assisted pep tide hydrolysis. Here we describe a rapid assay in which a microplate reade r is used to detect fluorescence produced by the reaction of fluorescamine with hydrolyzed peptides. We have employed this assay to detect Zn(II) and Pd(II)-assisted peptide hydrolysis in multiple samples and in each case hav e extended our approach to a successful analysis of reaction kinetics. Aliq uots from multiple time points are treated with fluorescamine in a single 9 6-well plate. Because the plate is scanned in a microplate reader in only 5 8 s, the assay is very convenient compared to conventional approaches which rely on NMR and HPLC to monitor individual reactions. Using our assay, rat e constants and half-lives are easily derived from the kinetic data by mean s of linear regression curve fits of triplicate runs. (C) 2001 Academic Pre ss.