Stepwise Reduction of Disulfide Bonds in Taka-amylase A

Publisher Website
Google Scholar
Abstract
When Taka-amylase A (EG 3.2.1.1) containing one masked sulfhydryl and four disulfide groups was incubated with sodium borohydride at 29∼30°C in an aqueous solution, titrable sulfhydryl groups increased stepwise as a function of the reduction time. At the first and the second plateaus, two sulfhydryl groups and an additional one group were concluded to be derived from the most labile disulfide bond out of the four and from the masked sulfhydryl group, respectively. The above conclusion was confirmed by preparing radioautograms of the peptic digests of the S-carboxymethylated reduced-amylases, in which the sub-stituent was labeled with C14. Such a stepwise appearance of the sulfhydryl groups was quite sensitive to the protein confirmation, and could not be observed when the reduction was carried out at higher temperatures or in the presence of denaturants.