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 conditions of temperature and pH hold great promise for use in protein structural studies. However, the extreme stability of the peptide amide bond has placed limits on the number of reagents available. In addition, the development of new cleavage strategies has been hindered by the fact that no facile procedure exists for the detection and characterization of metal-assisted peptide hydrolysis. Here we describe a rapid assay in which a microplate reader 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 have extended our approach to a successful analysis of reaction kinetics. Aliquots from multiple time points are treated with fluorescamine in a single 96-well plate. Because the plate is scanned in a microplate reader in only 58 s, the assay is very convenient compared to conventional approaches which rely on NMR and HPLC to monitor individual reactions. Using our assay, rate constants and half-lives are easily derived from the kinetic data by means of linear regression curve fits of triplicate runs.
Grant
K B
KB
Department of Chemistry, Center for Biotechnology and Drug Design, Georgia State University, University Plaza, Atlanta, GA 30303-3083, USA. kbgrant@gsu.edu
Pattabhi
S
S
D001331:Automation; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D005450:Fluorescamine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D016014:Linear Models; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013696:Temperature
D010455:Peptides; D005450:Fluorescamine