Purification and Properties of Vitamin B12-dependent Ribonucleotide Reductase fromRhizobium meliloti

Abstract

Ribonucleotide reductase from Rhizobium meliloti grown on the cobalt-deficient medium containing methionine was purified about 37-fold by streptomycin treatment, ammonium sulfate fractionation and column chromatographies on 1st DEAE-cellulose, 2nd DEAE-cellulose and Sepharose 6B. In agar gel electrophoresis, the purified enzyme moved as a single protein band. In sedimentation velocity experiments and analytical gel filtration, the purified enzyme was proved to aggregate and disaggregate. The s20, w of the enzyme at high concentration was about 27.1. From the intercept of the extrapolated curve line, the s20, w of approximately 20 was obtained. In analytical gel filtration, the location of elution peak varied with the protein concentration placed on the column. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme exhibited two bands, the molecular weights of which were estimated to be approximately 130,000 (a major band) and 110,000 (a minor band). The proportion of the major band to the minor...