Succinic oxidase preparation

Trifiuoroacetylating all of the lysine residues of cytochrome c leaves the Soret and visible spectrum of the molecule intact but destroys all activity with the succinate oxidase preparation (147). The blocking groups can be gently hydrolyzed away again to recover the intact protein with all lysines free and with 100% activity. 

This activity contrasts to the small (3%) KCN-insensitive respiratory activity observed when NADH was used as substrate with the mitochondria (Table I). To calculate the contribution of each oxidase to the total oxygen uptake, the titration method of Bahr and Bonner (25) was used, ( -uptake of submitochondrial particles (membranes) prepared from juice vesicles of Hamlin oranges harvested in September and January was measured in the presence and absence of ImM KCN titrated with a series of SHAM concentrations using the malate, succinate, glutamate substrate mixture (Table II). 

Upon purification, the K -stimulated phosphatase activity is always copurified with the (K )-ATPase activity [63-65]. Mitochondrial markers, such as cytochrome c oxidase, succinate dehydrogenase, monoamino-oxidase, and the ribo-somal marker RNA are largely removed by the purification procedure. The same is true for the anion-sensitive ATPase and 5 nucleotidase activities, but some (Na — K )-ATPase activity is still present in highly purified (K" -I-H )-ATPase preparations. Purification is also characterised by a lowering of the K -insensitive Mg ATPase activity, but even in the purest preparations some Mg -ATPase activity (4% of (K -I- H )-ATPase activity) is still present. This may represent an impurity or an inherent property of the enzyme. 

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. 

On the other hand, if the oxidase became inert either because it was inhibited by some reagent or because the reaction took place anaerobically, the rate of reduction of cytochrome a was much greater in the presence of cytochrome c than in its absence, when succinate was added to the above preparation. In this case it may be that the sequence was as follows ... 

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