17] Thiosulfate and trithionate reductases

Abstract

Publisher Summary This chapter describes purification and assay for trithionate and thiosulfate reductases from Desulfovibrio vulgaris (Hildenborough) (DvH) NCIMB 8303. Trithionate and thiosulfate are both implicated as intermediates during the respiratory sulfate reduction process occurring in dissimilatory sulfate-reducing bacteria (SRB). The most convenient method for assaying thiosulfate-forming enzyme (TF) under anaerobic conditions is by using manometry. Because the electrons for reducing bisulfite or trithionate to thiosulfate are derived from molecular hydrogen, the stoichiometry of the reaction may be determined. All purification steps are carried out at 0-4. The enzyme is stable to repeated lyophilization or freezing or thawing. The enzyme solution is colorless, and a single protein band is observed after polyacrylamide gel electrophoresis (PAGE). The assay for thiosulfate reduction can be accomplished by spectrophotometric methods or by manometry. The assay measures the production of sulfide from thiosulfate during the purification of thiosulfate reductase from D. vulgaris. The optimum pH for activity is 8.0–9.0 and the molecular weight, by sedimentation equilibrium studies, is 16,300, and by amino-acid analysis is 15,500.