Cytochrome oxidase purification

Gudat, J. C., Singh, J., and Wharton, D. C. (1973). Cytochrome oxidase from Pseudomonas aeruginosa I. Purification and some properties. Biochim. Biophys. Acta 292, 376-390. 

Horio, T., Higashi, T., Matsubara, H., Kusai, K., Nakai, M., and Okunuki, K. (1958). High purification and properties of Pseudomoruis cytochrome oxidase. Biochim. Biophys. Acta 29, 297-302. 

Kuronen, T., and Ellfolk, N. (1972). A new purification procedure and molecular properties of Pseudomonas cytochrome oxidase. Biochim. Biophys. Acta 275, 308-318. 

Parr, S. R., Barber, D., Greenwood, C., Phillips, B. W., and Melling, J. (1976). A purification ptocedure for the soluble cytochrome oxidase and some other respiratory proteins from Pseudomonas aeruginosa. Biochem. J. 157, 423-430. 

Sawhney, V., and Nicholas, D. J. D. (1978). Sulphide-linked nitrite reductase from Thio-bacillus denitrificans with cytochrome oxidase activity Purification and properties. J. Gen. Microbiol. 106, 119-218. 

The purification and characterization of individual cytochromes (simple or complex) and other redox centres in electron-transfer chains leads to a study of their interactions with each other, with the ultimate objective of reconstituting parts of the chain. Approaches to this problem will be illustrated724 with reference to the reaction of cytochrome c with ubiquinol cytochrome c reductase (complex III, cytochrome bcx) and cytochrome oxidase (complex IV) as shown in equation (2l). 

Kai M, Yano T, Tamegai H, Fukumori Y, Yamanaka T (1992) Thiobacillus ferrooxidans cytochrome c oxidase purification, and molecular and enzymatic features. J Biochem 112 816— 821... 

Saul RL, Kabir SH, Cohen Z, Bruce WR, Archer M (1981) Reevaluation of nitrate and nitrite levels in the human intestine. Cancer Res 41 2280-2283 Sawhney V, Nicholas DJD (1978) Sulphide-linked nitrite reductase from Thiobacillus denitrifi-cans with cytochrome oxidase activity purification and properties. J Gen Microbiol 106 119-128... 

Mitochondrial cytochrome b was solubilized for the first time by Cooperstein and his associates, and attempts to purify cytochrome b have been made. The enzyme has now been prepared free of cytochrome oxidase, succinic dehydrogenase, and cytochromes c and a. However, the criteria of purity are not always reliable. The best purification procedures have yielded preparations without enzymic activity. In the absence of cationic detergents, the protein tends to aggregate, but with such detergents a minimum molecular weight of 28,000 has been obtained. 

Keilin and Hartree tested the catalytic effect of the two cytochrome preparations with (1) cytochrome oxidase, cytochrome c, and ascorbic acid, and (2) cytochrome oxidase, cjriochrome c, succinic dehydrogenase, and succinic acid without finding any difference per iron atom in the effect of the two cytochrome preparations. This, in our view, indicates that certain inert impurities (in these two systems) are removed on the final purification, rather than that 20% of the cytochrome molecule is split off in the purification. 

Purification of Cytochrome Oxidase and Cytochrome b from the Radioactively Labeled Mitochondrial Membranes... 

With respect to question (1), the O -iibertragendes Ferment of Warburg (1932) or cytochrome z of Keilin and Hartree has been considered to be identical with cytochrome oxidase. However, even at present, only a little is known of the properties of cytochrome oxidase, because isolation and purification of this component is very difficult. In regard to question (2), it is supposed that cytochrome b functions as a terminal oxidase in CN- and CO-insensitive respiration since it is an autoxidizable component, but is unable to combine with CO and CN . Many papers support the fact that reduced cytochrome a is an electron donor for cytochrome z, but we cannot agree with this conception in view of the evidence described below. As for cytochrome Cj, discovered by Yakushiji and Okunuki (1940), it was indicated that reduced cytochrome c can readily be oxidized by oxidized cytochrome c and that the heme moiety of insoluble cytochrome Cj is the same as that of solube cytochrome c, but the protein moiety of the former is different. Concerning question (3), it has been inferred that electrons released from the... 

Although, as is well-known, cholate inhibits cytochrome oxidase (Wainio ct a/., 1948 Smith, 195Sa), it was used as a reagent for extraction or solubilization of cytochrome a from beef heart muscle (Yakushiji and Okunuki, 1940), because it is not known whether any detergents other than cholate or deoxycholate are effective for extraction of cytochrome a from these source materials. Therefore, it is very important to test the inhibition of activity in the cytochrome a preparation by cholate and to examine whether the activity lost during purification can be recovered by other types of detergents. It was found that Emasol 4130, a synthetic nonionic detergent, reactivated purified cytochrome a (Yonetani, 1959). 

Fig. 19. Absorption spectrum of P. cytochrome oxidase. Approximately 1.3 mg. of the sample at the final stage of purification was dissolved in 1 ml. of 0.1 Af phosphate buffer (pH 6.0). The absorption spectrum was measured at room temperature (22°C.). Solid line, oxidized form broken line, dithionite-reduced form. To obtain the molecular extinction the ordinate was multiplied by 23.5 x 10 (2S0-S00 mi ) or 7.2 X 10 (500-700 m,i).

Bergh AF, Strobel HW. 1992. Reconstitution ofthe brain mixed function oxidase system purification of NADPH-cytochrome P450 reductase and partial purification of cytochrome P450 from whole rat brain. J Neurochem 59 575-581. 

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