Product analysis of bisulfite reductase activity isolated from Desulfovibrio vulgaris

Abstract

Bisulfite reductase was purified from extracts of Desulfovibrio vulgaris. By colorimetric analyses trithionate was found to be the major product, being formed in quantities 5 to 10 times more than two other detectable products, thiosulfate and sulfide. When [35 ]bisulfite was used as the substrate, all three products were radioactively labeled. Degradation of [35Sltrithionate showed that all of its sulfur atoms were equally labeled. In contrast, [35S]thiosulfate contained virtually all of the radioactivity in the sulfonate atom while the sulfane atom was unlabeled. These results, in conjunction with the finding that the sulfide was radioactive, led to the conclusion that bisulfite reductase reduced bisulfite to trithionate as the major product and sulfide as the minor product; the reason for the unusual labeling pattern found in the thiosulfate molecule was not apparent at this time. When bisulfite reductase was incubated with [35S]bisulfite in the presence of another protein fraction, FII, the thiosulfate formed from this reaction contained both sulfur atoms having equal radioactivity. This discovery, plus the fact that trithionate was not reduced to thiosulfate under identical conditions, led to the speculation that bisulfite could be reduced to thiosulfate by another pathway not involving trithionate. 733 on August 6, 2020 by guest

Characterization of a novel thiosulfate-forming enzyme isolated from Desulfovibrio vulgaris

“…When bisulfite reductase and TF were incubated with bisulfite, both trithionate and thiosulfate were detected, with thiosulfate being more predominant. This system constituted the thiosulfate-forming system as previously reported (8,31). Although it appeared that TF functioned as a trithionate reductase, incubation of TF with either bisulfite or trithionate alone resulted in no activity.…”

“…Portions (1 ml) of the enzyme were stored at -20°C and thawed once for each experiment. The major and minor bands, characteristic for pure bisulfite reductase from D. vulgaris (8,16,19,22), were observed by discontinuous gel electrophoresis.…”

“…Purification of bisulfite reductase. Bisulfite reductase was purified by a modification of the previously described procedure (8). The final bisulfite reductase preparation was passed through a Sephadex G-200 column (2.5 by 30 cm) equilibrated with 0.02 M potassium phosphate buffer, pH 7.5.…”

“…The KOH filter paper used to trap the 35SO2 was placed in 10 ml of counting solution and incubated in the dark for 4 h prior to liquid scintillation counting. The isolation of [35S]thiosulfate was accomplished by column chromatography on Dowex-1 and was degraded as previously described (8). Degradation of authentic sulfane and sulfonate-labeled thiosulfate, purchased.…”

“…Trithionate was synthesized by a modification of the method of Roy and Trudinger (26), as described earlier (4 Na235SO4 was periodically monitored by methods previously described (8). Na235SO3 was purchased from New England Nuclear Corp.…”