Weis M, Cotgreave I C, Moore G A, Norbeck K, Moldéus P
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Biochim Biophys Acta. 1993 Mar 10;1176(1-2):13-9. doi: 10.1016/0167-4889(93)90171-k.
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.
研究了单溴联苯胺（mBBr）的氨基酸残基特异性及其与细胞内蛋白质半胱氨酸残基的可及性。mBBr 与游离氨基酸半胱氨酸和牛血清白蛋白的巯基选择性反应。将分离的肝细胞与 mBBr 孵育，导致在细胞溶质、线粒体和微粒体部分中形成与蛋白质结合的 mBBr 荧光，且呈浓度依赖性，高达 16 mM 的 mBBr 并未使其完全饱和。蛋白质的 SDS-PAGE 分析表明，在高 mBBr 浓度下出现的与蛋白质结合的 mBBr 荧光增加的主要部分是由于与蛋白质的共价结合。通过 SDS-PAGE 可去除一小部分（微粒体部分为 10 - 16%）与蛋白质结合的 mBBr 荧光，因此推断这是由于荧光 mBBr 反应产物的物理截留所致。以总蛋白质半胱氨酸残基的消耗程度来测定，mBBr 在分离的微粒体中的可及性与 N - 乙基马来酰亚胺（NEM）相似。相比之下，在细胞溶质中，与 NEM 相比，被 mBBr 标记的蛋白质半胱氨酸残基数量明显较少。在两个细胞器部分中，对苯二醌（p - BQ）是最有效的巯基消耗试剂。结论是，当高浓度使用时，mBBr 是分析细胞蛋白质巯基状态及其外源物诱导变化的合适试剂；然而，应该考虑到：（i）mBBr 和特定外源物对细胞蛋白质巯基残基的相对可及性可能不同；（ii）在定量蛋白质巯基水平时，应去除 mBBr 的物理截留荧光反应产物。
