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Background and purpose: Nitric oxide (NO) is produced by nitric oxide synthase (NOS) from L-arginine and has an important role in a variety of physiological and pathological processes in biological systems. In this study, kinetic activities of isolated NOS were evaluated from sheep kidney. Materials and methods: In this reasearch homogenization, ammonium sulfate precipitation and column chromatography on DEAE-32 Cellulose and 2', 5'-ADP-agarose of 500 grams of sheep kidney were used to purify isolated nitric oxide. In all steps of purification process the amount of protein and its activity was assayed using Bradford and Griess reactions. Results: The molecular weight of sheep kidney on SDS-PAGE electrophoresis was 54 KD. Specific activity was 0.6 units/mg protein and recovery of purification was 0.9% (relative purity was 20 times more). Optimum temperature and pH were 30°C and 7.4 and incubation of purified enzyme at thermal interval of 10 to 30°C for 15 minute showed a stable enzyme activity. At 75 µM concentration of L-arginine substrate, the highest level of NOS activity was seen. The Vmax and Km of enzyme were 250 nmol/min/mg protein and 5.32 ?M, respectively. Conclusion: NOS enzyme from sheep kidney was purified with relative purity of 20 times and their optimum temperature, pH, suitable substrate concentration, Vmax and Km were 30°C, 7.4, 75 µM, 250 nmol/min/mg proteins and 5.32 µmol, respectively. [ABSTRACT FROM AUTHOR]