Initial Purification of Uptake Hydrogenase from Frankia sp. strain ArI-5
Uicker, William C.
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The actinomycete symbiont Frankia contains a membrane bound uptake hydrogenase that serves to improve the relative efficiency of nitrogen-fixation in the microbe. The enzyme catalyzes the oxidation of molecular hydrogen (H2 --> 2H+ + 2e-). In this work in progress, a purification of the hydrogenase of Frankia strain ArI-5 is attempted under aerobic conditions. To de-repress the hydrogenase, cultures were grown in BFAD media without reduced nitrogen. Cells were lysed in a French pressure cell at ca. 20,000 psi, in a buffer containing 0.01 M Tris-Cl (pH 7.0), 0.3 mg/mL PMSF, 0.001 M EDTA, 0.1 M KCI, and 10% glycerol. Cellular debris was removed by centrifugation, and membranes were pelleted at 130,OOOxg for 2 hours. Membrane-bound proteins were solubilized using 0.5% Triton X-100 and 0.1% deoxycholate. Aliquots were quick-frozen and stored at -70°C. Enzyme activity was assayed by amperometric determination of hydrogen concentration, using methylene blue as the terminal electron acceptor. Detergent-solubilized protein extracts exhibited hydrogenase activity which was inhibited in a concentration dependent manner by free Reactive Red 120. Final purification was accomplished via affinity chromatography on Reactive Red 120- agarose. Hydrogenase activity was monitored throughout the purification process, indicating that this method may be used in future purification. To our knowledge, this is the first attempted purification of hydrogenase from this organism.