Spinach nitrate reductase: Further purification and removal of ‘nicked’ sub-units by affinity chromatography

Abstract

NADH-nitrate reductase (EC 1.6.6.1) from spinach (Spinacea oleracea L. v. Noorman) has been purified 3400-fold by a multi-stage procedure involving streptomycin sulphate, (NH₄)₂SO₄, hydroxylapatite, molecular seiving and two stages of affinity chromatography using blue-Sepharose and 5′ AMP-Sepharose. The enzyme has a specific activity of 51 μmol NO₂⁻ produced min⁻¹ mg⁻¹ and a functional haem with extinction coefficients (mM) of 127 at 412 nm (oxidized) and 172 at 423 (NADH-reduced). Gel electrophoresis indicates two sub-units of approx. 110 000 and 120 000.