Isolation and partial characterization of homogeneous nitrite reductase from etiolated bean shoots (Phaseolus angularis W.F. Wight)

Abstract

Methyl viologen-dependent nitrite reductase (EC 1.7.7.1) (NiR) was purified about 1780-fold with a yield of 3.5% from etiolated bean shoots with a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Butyl-Toyopearl chromatography, ferredoxin-Sepharose affinity chromatography and Ultrogel AcA44 gel filtration. The purified enzyme was apparently homogeneous as shown by polyacrylamide disc gel electrophoresis with a specific activity of 53.4 units/mg protein. The molecular weight of the enzyme was estimated to be 100 kilodaltons by gel filtration. Subunit analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded two protein bands with a large subunit molecular weight of 64 kilodaltons and a small subunit molecular weight of 35 kilodaltons. The purified enzyme could be stored at −20°C for several weeks without any loss of activity in the presence of 10% glycerol and 10 mM ß-mercaptoethanol.