[44] Preparation and properties of reduced coenzyme Q-cytochrome c reductase (complex III of the respiratory chain)

TLDR: This chapter describes the preparation and properties of reduced coenzyme Q-cytochrome c reductase (complex III of the respiratory chain), which can be stored as a solution in the Tris-suerose-histidine buffer for weeks at –20° with little loss in activity.

Abstract: Publisher Summary This chapter describes the preparation and properties of reduced coenzyme Q-cytochrome c reductase (complex III of the respiratory chain) The rate of reduction of cytochrome c by reduced coenzyme Q 2 is estimated from the amount of cytochrome c that is reduced (absorbency change at 550 mμ) in a sample of the assay mixture that has been allowed to react for 10 seconds The enzyme-catalyzed reaction is stopped by the addition of an appropriate stop reagent Three procedures for the preparation of (reduced coenzyme Q) cytochrome c reductase are described All three procedures utilize the S-1 fraction of the procedure described for the preparation of DPNH cytochrome c reductase The purification procedure described in the chapter utilizes the final supernatant solution obtained in the preparation of DPNH-cytochrome c reductase (Reduced eoenzyme Q)-eytoehrome e reduetase can be stored as a solution in the Tris-suerose-histidine buffer for weeks at –20° with little loss in activity The reduced forms of coenzyme Q 1 , eoenzyme Q 2 , and eoenzyme Q 10 all serve as electron-donor substrates with complex III (Reduced coenzyme Q)-cytochrome c reductase is inhibited almost completely by antimycin A at concentrations approaching the concentration of cytochrome c l in the solution of enzyme (15 μg per milligram of protein of complex III) In the assay system described, (reduced coenzyme Q)-cytochrome c reductase is active in the pH range from 60 to 85, with an optimal activity at pH 74