Identification of the second (buried) cysteine residue and of the C-terminal disulfide bridge of bovine spleen cathepsin B.
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.[1]References
- Identification of the second (buried) cysteine residue and of the C-terminal disulfide bridge of bovine spleen cathepsin B. Baudys, M., Meloun, B., Pohl, J., Kostka, V. Biol. Chem. Hoppe-Seyler (1988) [Pubmed]
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