Studies on cytochrome oxidase. III. Improved preparation and some properties.
Studies on cytochrome oxidase. III. Improved preparation and some properties.
复制标题
细胞色素氧化酶的研究。
DOI：
10.1016/s0021-9258(19)63284-9
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发表时间：
1961
期刊：
影响因子：
--
通讯作者：
T. Yonetani
中科院分区：
文献类型：
--
作者：
T. Yonetani
Ever since cytochrome oxidase was first solubilized from heart muscle preparations by Yakushiji and Okunuki (1) and Straub (2) in 1941, the further purification of this enzyme has been the subject of studies by many workers, using extraction with bile salts (3-14), or digestive enzymes such as trypsin (6, 7) and snake venom (15), fractionation with ammonium sulfate (5-14), chromatography(9, 12, 13, 15-17), and treatment with thioglycolate (14). In most instances, cytochrome oxidase is kept in a solution with the aid of bile salts such as cholate and deoxycholate, which are inhibitory to the enzyme activity, especially in highly purified preparations of this enzyme (4, 6, 7, 9). These inhibitory effects have, however, been overcome with nonionic synthetic detergents such as Emasol, Tween, and Triton (18, 19). With the aid of these synthetic detergents a highly concentrated and enzymatically active preparation of cytochrome oxidase made according to a modification of the method of Okunuki et al.(9) has been used in spectrophotometric(20-23) and magnetochemical studies (24, 25). During the course of further purification of the preparation, enzymatically active green crystals and stable lyophilized preparations of cytochrome oxidase were obtained from a highly concentrated preparation. The present paper describes the purification and some properties of these preparations.EXPERIMENTAL PROCEDURE Preparation of Cytochrome Oxidase-A purified preparation of cytochrome oxidase is made from 20 beef hearts according to a slight modification (20) of the method of Okunuki et al.(9). The present preparation differs slightly from that described in two previous papers (9, 20) in that the fraction precipitated in ammonium sulfate solution between 26 and 330/, of saturation is collected, instead of that obtained between 25 and 35% of saturation; this allows better separation of cytochrome oxidase from cytochromes b and cl. All operations are performed at O-3O. The precipitate can be solubilized with 0.05 M phosphate buffer, pH 7.2, containing 0.5% Emasol 41301 (18, 19), if the following criteria are satisfied:(a) complete removal of cytochromes b and cl; this should be examined by the low-temperature spectrometry (26) or the low-temperature spectrophotometry
