Purification and partial characterization of NADPH-cytochrome P450 reductase from Gentiana triflora flowers

Reduced form of nicotineamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase was solubilized from a microsomal fraction of Gentiana triflora flowers by 3-(3 Cholamidopropyl)-dimethylammonio]-1-propane sulfonate detergent and purified to electrophoretic homogeneity. The purification was achieved by adenosine 2', 5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. A Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. Western blot analysis showed that the purified protein cross-reacted with polyclonal antibody raised against rabbit anti-Gentiana triflora NADPH-cytochrome P450 reductase antibodies. The temperature and pH optimum for reduction of cytochrome c was 25 degrees C and 7.4 respectively. The Km values for the binding of NADPH and cytochrome c were 9.4 and 3.2 microM, respectively. In this paper, we present some results of the purification and partial characterization of microsomal NADPH-cytochrome P450 reductase from Gentiana triflora flowers.