Immunological and enzymatic properties of the Desulfovibrio desulfuricans hydrogenase

Abstract

Affinity-purified antibodies specific for the purified, active periplasmic hydrogenase of Desulfovibrio desulfuricans were prepared. Immunodiffusion and enzyme-linked immunosorbent assay (ELISA) methods showed distinct differences between the native form of hydrogenase and the enzyme modified by heat, acid, active-site, and group-specific chemical treatments. The ELISA method indicated two zones of sensitivity, one for a native form of enzyme and another for irreversibly inactivated hydrogenase. The ELISA technique was also used to estimate the maximum specific activity of the enzyme in cell extracts. Immunoglobulins directed towards only the inactive form could not be obtained. A mixed immunoglobulin population directed towards both active and inactive forms of the enzyme was obtained by affinity chromatography on inactive hydrogenase – Sepharose. Immunological and enzymatic studies of the periplasmic hydrogenases in the Desulfovibrio genus showed strain dependence of the enzyme concentration in these microorganisms. The hydrogenases of two strains of Escherichia coli and several other bacteria were examined and showed strong cross-reaction with the hydrogenase of Desulfovibrio desulfuricans. It was found that the periplasmic and membrane-bound hydrogenases of Desulfovibrio desulfuricans ATCC 7757 differed in both enzymatic and immunological properties.