Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analyses.
Procedures which allow rapid, quantitative, and selective fluorescent labeling of protein cyst(e)ine residues prior to electrophoresis by reaction with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) under mild conditions are described. After labeling, the protein(s) of interest is easily monitored throughout electrophoresis and subsequent electroblotting or electroelution procedures. The stoichiometry of labeling and therefore the number of cysteine and/or half-cystine residues can be measured spectrophotometrically or fluorometrically and the derived cyst(e)ine adduct can also be quantitated by amino acid analysis and identified in protein sequencing. N-terminal blockage is not observed under the conditions utilized, nor are any other amino acid side chains modified. The procedures described allow complete, rapid, and facile reduction and alkylation of proteins with simultaneous incorporation of a fluorophore, permitting sensitive detection in subsequent manipulation of the proteins. Quantitative fluorescence prelabeling also allows the generation, purification, and sequencing of peptide fragments containing cyst(e)ine residues for determination of internal sequences and residues involved in disulfide bonds.
Kirley
T L
TL
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
D000478:Alkylation; D003545:Cysteine / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005456:Fluorescent Dyes; D015394:Molecular Structure; D010069:Oxadiazoles; D010084:Oxidation-Reduction; D010720:Phosphines; D011506:Proteins / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis
D005456:Fluorescent Dyes; D010069:Oxadiazoles; D010720:Phosphines; D011506:Proteins; C043255:tri-n-butylphosphine; D012710:Serum Albumin, Bovine; C046364:4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine