PREPARATION AND SOME PROPERTIES OF A SOLUBLE NITRATE REDUCTASE FROM RHIZOBIUM JAPONICUM.

Abstract

A method was developed for the preparation of a soluble nitrate reductase extract of Rhizobium japonicum cells derived from pure cultures of the organisms or from soy-bean nodules. In the procedure the bacterial suspension, glass beads and small quantities of sodium hydrosulfite and benzylviologen were sealed in a Tygon plastic tube. The cells were broken by a series of grinding and freezing and thawing operations. The cell-free extract was collected by centrifugation. The soluble enzyme preparation is extremely susceptible to oxidation. It was necessary to maintain completely reduced conditions throughout the purification procedures and the experimental operations in which properties of the enzyme were investigated. A simple assay procedure was developed in which benzylviologen (reduced by sodium hydrosulfite) was employed as the electron donor. Since excess reducing agent was present in assay tubes, assays could be conducted under aerobic conditions and still maintain reduced conditions in the reaction mixtures. The excess sodium hydrosulfite was oxidized at the end of the assay periods by vigorously shaking each tube in air. After this the nitrite in each reaction mixture was determined by a colorimetric procedure. The soluble enzyme was purified about 11-fold by (NH4)2SO4 fractionation and chromatography on a calcium phosphate column. Attempts to purify extracts containing sodium hydrosulfite but lacking benzylviologen were unsuccessful. NADH2 and succinate, which were effective electron donors for a particulate enzyme preparation from R. japonicum, were completely ineffective donors for nitrate reduction with the soluble preparation. A series of compounds were tested in an attempt to find a naturally occuring electron donor, but reduced benzyl-or methyl-viologen were the only compounds that functioned with the soluble enzyme. The effects of pH, substrate concentration, and various inhibitors on the activity of the enzyme, were investigated.