SUCCINIC DEHYDROGENASE OF AZOTOBACTER VINELANDII

Abstract

Succinoxidase from A. vinelandii strain O was prepared in "soluble" form by 2 methods (a) direct sonic disruption of the cells (10 kc Raytheon sonic oscillator) or (b) grinding the cells with alumina, separating the enzymatically active particles and disrupting these by sonic oscillation. After centrifugation at 145,000 G for 30 min. 50-75% of the succinic dehydrogenase remained in the supernatant fluid. Succinic dehydrogenase was assayed by 2,6-dichlorophenolindophenol reduction ( [DELTA] OD600) in the presence of CN. Inhibition of the oxidase activity (O2 uptake) required high levels of CN. Inhibition of succinic dehydrogenase by malonate (77%) and pyrophosphate (90%) was found at 0.003 [image]. Pyrophosphate inhibition could be reversed by adding Mg++. Phosphate and chloride ions caused inhibition; antimycin A had little effect. Five fold purification of succinic dehydrogenase was obtained by treating the "soluble" prepn. with (a) alumina C [gamma] and Zn(OH)2 gels to adsorb inactive protein (b) 45% (NH4)2 SO4 to precipitate the enzyme followed by adsorption of inactive protein on Zn(OH)2 gel. Further treatment caused loss of activity which could not be restored by addition of various cofactors. The calculated Q02 CN) at 37[degree] C was 1,700. The enzyme was stable between pH 5.5 and 6.5; max. activity was found at pH 6.5.