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[45] Cytochrome oxidase from beef heart mitochondria

1967; Academic Press; Linguagem: Inglês

10.1016/0076-6879(67)10048-7

1557-7988

David C. Wharton, Alexander Tzagoloff,

Photosynthetic Processes and Mechanisms

This chapter discusses the cytochrome oxidase from beef heart mitochondria. Cytochrome c oxidase is mostly assayed by the spectrophotometric method. The rate of oxidation of ferrocytochrome c is measured by following the decrease in the absorbency of its α-band at 550 mμ. The activity of cytochrome c oxidase may be defined in terms of the first-order velocity constant. The oxidation-reduction components of isolated cytochrome c oxidase are cytochrome a, cytochrome a3, and copper. Cytochrome c oxidase can also be assayed by measuring oxygen uptake either manometrically or polarographically. Two procedures for purifying cytochrome c oxidase from beef heart mitochondria are described. The enzyme obtained by procedure I is less pure based on its activity and spectrum. Preparations of cytochrome oxidase are stored best in 0.25M sucrose (pH 7.0-7.5) at –15° at a protein concentration of 1 mg/ml. The composition of cytochrome oxidase prepared by procedure II is tabulated. Ferrocytochrome c donates electrons directly to cytochrome oxidase. The reaction as measured spectrophotometrically obeys first-order reaction kinetics. Cytochrome oxidase is inhibited by cyanide, azide, hydroxylamine, and sodium sulfide.