Comparison of the membrane-bound and detergent-solubilised hydrogenase from paracoccus denitrificans. Isolation of the hydrogenase.
The hydrogenase from Paracoccus denitrificans is an integral membrane protein and has been solubilised by Triton X-100. The membrane-bound and detergent-solubilised forms of the enzyme have been compared. Both forms of the enzyme show a pH optimum for reduction of benzyl viologen at pH 8.5--9.0 and are both inhibited by concentrations of NaCl greater than 30 mM. An Arrhenius plot of the activity of hydrogenase in the membrane shows no 'break'. The form of the Arrhenius plot and the activation energy are not significantly changed on solubilisation of the enzyme. The Km and V values for benzyl viologen, methyl viologen and H2 are unaltered when the enzyme is extracted from the membrane. Therefore, solubilisation of hydrogenase from the membrane by Triton X-400 is unlikely to disrupt the native conformation of the enzyme. The detergent-solubilised hydrogenase has subsequently been purified using ammonium sulphate precipitation, sucrose density gradient centrifugation and chromatography on hydroxyapatite. The overall yield of activity is 23%, with a final purification of over 100-fold.
D002462:Cell Membrane / Q000201:enzymology*; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D012995:Solubility
D011092:Polyethylene Glycols; D006859:Hydrogen; D010088:Oxidoreductases