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.
Baudys
M
M
Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague.
Meloun
B
B
Pohl
J
J
Kostka
V
V
D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000032:analysis*; D002417:Cattle; D003545:Cysteine / Q000032:analysis*; D004220:Disulfides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D010434:Pepsin A; D010455:Peptides / Q000302:isolation & purification; D013154:Spleen / Q000032:analysis*; D013820:Thermolysin; D014357:Trypsin; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D004220:Disulfides; D010455:Peptides; C038560:4-dimethylaminoazobenzene-4'-iodoacetamide; D004124:p-Dimethylaminoazobenzene; D014357:Trypsin; D002401:Cathepsin B; D010434:Pepsin A; D013820:Thermolysin; D003545:Cysteine