Accessibility of hepatocyte protein thiols to monobromobimane.
The amino-acid residue specificity of monobromobimane (mBBr) and its accessibility to cellular protein cysteine residues were investigated. mBBr reacted selectively with the sulfhydryl group of both the free amino acid cysteine and bovine serum albumin. Incubation of isolated hepatocytes with mBBr resulted in a concentration-dependent formation of protein-bound mBBr fluorescence in the cytosolic, mitochondrial and microsomal fractions, which was not fully saturated with up to 16 mM mBBr. SDS-PAGE resolution of the proteins revealed that the major portion of increased protein-bound mBBr fluorescence that occurred at high mBBr concentrations was due to covalent binding to proteins. A minor portion (10-16% in the microsomal fraction) of protein-bound mBBr fluorescence was removed by SDS-PAGE and is therefore concluded to be due to physical entrapment of fluorescent mBBr reaction products. The accessibility of mBBr, assayed as the degree of depletion of total protein cysteine residues, was similar to N-ethylmaleimide (NEM) in isolated microsomes. By contrast, in the cytosol a markedly lower amount of protein cysteine residues were labelled by mBBr as compared to NEM. In both organelle fractions p-BQ was the most efficient thiol-depleting reagent. It is concluded that mBBr is a suitable reagent for the analysis of the cellular protein thiol status and of its xenobiotic-induced alterations when used at high concentrations; however, it should be considered that, (i) the relative accessibility of mBBr and a particular xenobiotic to cellular protein thiol residues may be different, and (ii) physically entrapped fluorescent reaction products of mBBr should be removed when quantitating protein thiol levels.
Weis
M
M
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Cotgreave
I C
IC
Moore
G A
GA
Norbeck
K
K
Moldéus
P
P
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005456:Fluorescent Dyes; D008099:Liver / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013997:Time Factors
D000596:Amino Acids; D016227:Benzoquinones; D001643:Bridged Bicyclo Compounds; D005456:Fluorescent Dyes; D011506:Proteins; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; C004532:quinone; D003545:Cysteine; D005033:Ethylmaleimide; C028381:monobromobimane