Solubilization and Reconstitution of a Particulate Hydrogenase from Vibrio succinogenes

Abstract

Abstract Hydrogenase activity was found to be associated with the particulate matter of cells of Vibrio succinogenes after lysis with EDTA at pH 9.0. Solubilization of the enzyme was effected by treatment of the particulate fraction at pH 11.0. While the particulate hydrogenase was unaffected by sulfhydryl compounds, the solubilized enzyme had an absolute sulfhydryl requirement. Proteolytic enzymes such as pronase and trypsin, as well as ethylenediaminetetraacetate, could substitute for β-mercaptoethanol in stimulating soluble hydrogenase activity. Incubation at pH 7.0 of the pH 11.0-soluble hydrogenase with the pH 11.0-extracted particulate fraction resulted in the incorporation of most or all of the hydrogenase into the particulate fraction. The reconstituted, particulate hydrogenase, which no longer required sulfhydryl compounds for activity, could be separated into a soluble, hydrogenase-containing fraction and an inactive, insoluble fraction by re-exposure to pH 11.0. These results suggest that solubilization and reconstitution of the particulate system is a reversible, pH-dependent process.