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. 1996 May 1;315(Pt 3):1001–1005. doi: 10.1042/bj3151001

pH-dependence of the dithiol-oxidizing activity of DsbA (a periplasmic protein thiol:disulphide oxidoreductase) and protein disulphide-isomerase: studies with a novel simple peptide substrate.

L W Ruddock 1, T R Hirst 1, R B Freedman 1
PMCID: PMC1217253  PMID: 8645136

Abstract

A decapeptide containing two cysteine residues at positions 3 and 8 has been designed for use in monitoring the disulphide bond-forming activity of thiol:disulphide oxidoreductases. The peptide contains a tryptophan residue adjacent to one of the cysteine residues and an arginine residue adjacent to the other. Oxidation of this dithiol peptide to the disulphide state is accompanied by a significant change in tryptophan fluorescence emission intensity. This fluorescence quenching was used as the basis for monitoring the disulphide bond-forming activity of the enzymes protein disulphide-isomerase (PDI) and DsbA (a periplasmic protein thiol:disulphide oxidoreductase) in the pH range 4.0-7.5, where the rates of spontaneous or chemical oxidation are low. Reaction rates were found to be directly proportional to enzyme concentration, and more detailed analysis indicated that the rate-determining step in the overall process was the reoxidation of the reduced form of the enzyme by GSSG. The pH-dependence of the enzyme-catalysed reaction reflected primarily the pKa of the reactive cysteine residue at the active site of each enzyme. The data indicate a pKapp of 5.6 for bovine PDI and of 5.1 for Vibrio cholerae DsbA.

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Selected References

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