Nitrate reductase (NaR) is a metalloprotein and contains one each of Flavin (as FAD), heme iron, molybdopterin and belongs to a super family of enzymes including xanthine oxidase, nitrite reductase and DMSO reductase. NaR was a soluble enzyme and purified from Zea mays (HQPM-4) by ammonium sulfate precipitation and gel filtration on blue dextran by affinity chromatography and gel filteration by 12.32 fold with 53.19 yield. It was necessary to investigate better conditions i.e. pH, temperature to stabilize the enzyme activity as a result of small quantities of tissue and low amount of enzyme contained in the leaves. The purified enzyme (NaR) after Gel filteration had a specific activity of 454.61Units/mg. The blue-Sepharose method and further gel filteration method using sephaedex gel offers the advantages of a rapid purification of plant NR to a high specific activity with reasonable recovery of total activity. The different kinetic parameters of enzyme (NaR) such as pH, incubation temp., time of incubation and effect the substrate (nitrate) concentration were optimized. We found that, the optimum stability of nitrate reductase (NADH) activity was at a pH 7.5. Affinity chromatographic method has been developed to purify the enzyme from corn leaves and gel filteration method gives the highest purity.
