Aerobic purification of hydrogenase from Rhizobium japonicum by affinity chromatography.

Abstract:

:We purified active hydrogenase from free-living Rhizobium japonicum by affinity chromatography. The uptake hydrogenase of R. japonicum has been treated previously as an oxygen-sensitive protein. In this purification, however, reducing agents were not added nor was there any attempt to exclude oxygen. In fact, the addition of sodium dithionite to aerobically purified protein resulted in the rapid loss of activity. Purified hydrogenase was more stable when stored under O2 than when stored under Ar. Sodium-chloride-washed hydrogen-oxidizing membranes were solubilized in Triton X-100 and deoxycholate and loaded onto a reactive red 120-agarose column. Purified hydrogenase elutes at 0.36 M NaCl, contains a nickel, and has a pH optimum of 6.0. There was 452-fold purification resulting in a specific activity of 76.9 mumol of H2 oxidized per min per mg of protein and a yield of 17%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed subunits with estimated molecular weights of 65,000 and 33,000. Hydrogenase prepared in this manner was used to raise and affinity purify antibodies against both subunits.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Stults LW,Moshiri F,Maier RJ

doi

10.1128/jb.166.3.795-800.1986

subject

Has Abstract

pub_date

1986-06-01 00:00:00

pages

795-800

issue

3

eissn

0021-9193

issn

1098-5530

journal_volume

166

pub_type

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