A Simple Procedure for Purification of NADH-Specific Dihydropteridine Reductase from Mammalian Liver1

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Abstract
A simple and convenient purification method which can yield a homogeneous preparation from even a small amount of starting material was devised for NADH-specific dihydropteridine reductase from rat liver. The procedure is essentially composed of two steps, i.e., affinity chromatography on Matrex gel blue A and hydrophobic chromatography on Phenyl-Sepharose. Prior to the Matrex gel blue A chromatography, the crude extract of rat liver was oxidized to dissociate NADH, bound in the enzyme-NADH complex, from the enzyme. Low molecular weight substances in the extract were removed by Sephadex G-25 gel filtration; then the enzyme was purified by successive chromatographies on Matrex gel blue A and Phenyl-Sepharose columns. Thus about 0.1 mg of purified enzyme was obtained from 3 g of rat liver with 40%. recovery. The preparation showed a single protein band on polyacrylamide and SDS-gel electrophoresis. Using NADH and tetra-hydro-6-methylpterin, the maximal velocity of the enzyme was determined to be 64.2 μmol quinonoid-dihydro-6-methylpterin reduced/min/mg. Km values of the enzyme were 0.85, μM and 3.4 μM for NADH and quinonoid-dihydro-6-methylpterin, respectively. This simple purification method was also applicable to livers from other mammalian sources such as human and bovine.