Abstract
Riboflavin synthetase, which catalyzes formation of the vitamin from 6, 7-dimethyl-8-ribityllumazine, was purified about 700-fold from an extract of spinach by two separate procedures of the followings; (1): extraction with a phosphate buffer, the first fractionation with ammonium sulfate, protamine sulfate treatment, the second fractionation with ammonium sulfate, treatment with CM-cellulose, and two-time chromatographies on DEAE-cellulose, and (2): extraction with a phosphate buffer, two-time ammonium sulfate precipitations, and chromatographies on DEAE-cellulose and on DEAE-Sephadex. The specific activities of the first and second enzyme preparations were of 15.1 and 16.2 mμmoles riboflavin formed per hour per mg protein at 37°, respectively. The purified enzyme retained initial activity at 0-4° for 24 hours, and was stable for one week at 0-4° under the saturation with ammonium sulfate. Freezing caused complete loss of the activity. Cysteine, ascorbate and Na2SO3 protected the enzyme against the inactivation. The optimal activity was exhibited at pH 7.5. The apparent activation energy was 15, 000 calories per mole between 45° and 25°. Michaelis constant was calculated as 4.5×10-5M at pH 7.5. Acetate, acetylacetone, acetoine, glucose, succinate, malonate, glycollate, oxalate, threonine and glycine (10-2M each) did not influence the reaction rate. But, glyoxal and pyruvate (10-2M each) was markedly inhibitory. Ascorbafe, cysteine and Na2SO3 enhanced the rate. p-Chloromercuribenzoate, HgCl2 and CuSO4 (10-3M each) blocked completely the reaction. KCN, EDTA, o-phenanthlorine and 8-hydroxyquinoline (10-3M each) had no effect on the rate. Stoichiometry of the reaction was determined by spectrophotometry: one molecule of the vitamin was yielded from two molecules of the substrate.
