Baudys M, Meloun B, Pohl J, Kostka V
Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague.
Biol Chem Hoppe Seyler. 1988 May;369 Suppl:169-74.
Quantitative differences were found when bovine spleen cathepsin B was subjected to SH-group titration in the presence and in the absence of denaturing agents, as well as when the pH of the titration buffer was increased. The intra- and interchain thiol-disulfide exchange reactions accompanying the denaturation of cathepsin B were investigated by polyacrylamide gel electrophoresis in SDS and by gel filtration experiments. An identical behavior in these experiments showed also cathepsin B whose active site Cys29 only had been carboxymethylated; these findings suggested the presence of one additional SH-group. After conditions preventing thiol-disulfide exchange reactions, had been developed, the second SH-group (Cys240) was demonstrated independently in carboxymethylated cathepsin B by labeling with 4-(dimethylamino)azobenzene-4'-iodoacetamide and by selective isolation of the SH-peptide containing Cys240 on thiopropyl-Sepharose. As the second important result, a disulfide bridge formed by Cys148 and Cys252 in the C-terminal part of the chain was identified.
当在有和没有变性剂存在的情况下对牛脾组织蛋白酶B进行巯基滴定，以及当滴定缓冲液的pH值升高时，发现了定量差异。通过SDS聚丙烯酰胺凝胶电泳和凝胶过滤实验研究了组织蛋白酶B变性过程中伴随的链内和链间硫醇-二硫键交换反应。仅活性位点Cys29被羧甲基化的组织蛋白酶B在这些实验中也表现出相同的行为；这些发现表明存在另一个巯基。在开发出防止硫醇-二硫键交换反应的条件后，通过用4-（二甲基氨基）偶氮苯-4'-碘乙酰胺标记以及在硫丙基-琼脂糖上选择性分离含有Cys240的SH肽，在羧甲基化的组织蛋白酶B中独立证明了第二个巯基（Cys240）。作为第二个重要结果，鉴定出了由链C末端部分的Cys148和Cys252形成的二硫键。
