Jack Bean Urease VI. Determination of thiol and disulfide content: Reversible inactivation of the enzyme by the blocking of the unique cysteine residue

Abstract

The total thiol content of highly pure jack bean urease (urea amidohydrolase, EC 3.5.1.5) has been determined by titration with 5,5′-dithiobis(2-nitrobenzoic acid) in the presence of 6 M guanidinium chloride. Urease contains 15 thioi groups per 96.6-kDa subunit. Coupled with amino acid analysis data, this result establishes that urease contains one cystine disulfide bond per 96.6-kDa subunit. Slow loss of enzymatic activity in the presence of β-mercaptoethanol and oxygen is due to the formation of a mixed disulfide which involves the unique active-site cysteine residue. Enzymatic activity can be fully restored by treatment of inactive urease with 0.1 M dithiothreitol at pH 7.3. Urease is quite stable when stored in 0.05 M sulfite/0.02 M phosphate buffer (pH 7.2, 1 mM in EDTA).