Fluorescence labelling of NADPH-cytochrome P-450 reductase with the monobromomethyl derivative of syn-9,10-dioxabimane.
The kinetics of thiol-group alkylation in NADPH-cytochrome P-450 reductase during its inactivation by monobromobimane has been studied using the fluorimetric determination of S-bimane-L-cysteine by high-performance liquid chromatography. Loss of activity during the reaction of NADPH-cytochrome P-450 reductase with monobromobimane is caused by the alkylation of one single critical cysteine residue, which can be protected against thiol-specific reagents by NADP(H). The chemical stability of the bimane group allows the digestion of bimane-labelled NADPH-cytochrome P-450 reductase by CNBr. The critical cysteine residue could be located in a CNBr-cleaved peptide purified to homogeneity with Mr 10 500 +/- 1 000 and valine as N-terminus.
D000596:Amino Acids / Q000032:analysis; D001643:Bridged Bicyclo Compounds / Q000032:analysis / Q000494:pharmacology*; D001952:Bridged-Ring Compounds / Q000494:pharmacology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors*; D010446:Peptide Fragments / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D001643:Bridged Bicyclo Compounds; D001952:Bridged-Ring Compounds; D005456:Fluorescent Dyes; D010446:Peptide Fragments; C039433:S-bimanylcysteine; D009251:NADPH-Ferrihemoprotein Reductase; D003545:Cysteine; C028381:monobromobimane