Graduate Thesis Or Dissertation
 

The effect of alkaline pH on the succinate oxidase system of sub-mitochondrial particles from beef heart muscle

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  • The spectrophotometric method for the determination of the acid nonextractable flavin in the proteolytic digest of the trichloroacetic acid precipitate from the heart muscle preparation has been examined for the interference of the degraded hemeproteins. The proteolytic digest was chromatographically separated into the heme and flavin fractions. Spectrophotometric determinations made on the separated fractions have indicated that the heme interference is negligible for the heart muscle preparation. The validity of this method has thus been demonstrated. In the course of the proteolysis, cytochrome c₁ and c are evidently degraded to hemepeptides. The spectral behavior of the hemepeptides has been presented. Based on the fluorescent characteristics of the acid non-extractable flavin, a fluorometric method has been devised. This method is approximately 100 times more sensitive than the spectrophotometric assay. Results from these two methods are in good agreement. It is suggested that the fluorometric method may be more suitable for samples containing high concentrations of pigments adsorbing in the 450-530 mu region of the spectrum. Alkaline inactivation of the succinate oxidase activity of the heart muscle preparation in the absence of substrate and presence of oxygen has been used by Keilin and King to prepare a particle which supplies all of the respiratory components except succinate dehydrogenase for reconstitution of succinate oxidase. The succinate dehydrogenase protein, as measured by its flavin coenzyme, was found to be dissociated from the particulate heart muscle preparation by alkaline treatment. The dissociation occurred at the same rate as the inactivation of the succinate oxidase. It was therefore concluded that the original site of binding of the dehydrogenase to the particle is available to bind active succinate dehydrogenase during reconstitution of the system. The inactivation of succinate oxidase under the experimental conditions used was found to follow zero order kinetics. The apparent zero order rate constant varied as the first power of the initial enzyme concentration and the second power of the hydroxyl ion concentration. The temperature dependence of the apparent zero order rate constant was complex. A mechanism is presented which is consistant with the observed kinetics. The equilibrium dissociation of the succinate oxidase system by alkaline treatment in the presence of succinate and absence of oxygen was studied using the acid nonextractable flavin content as a measure of the succinate dehydrogenase concentration. The percent of the dehydrogenase which was soluble was a linear function of the hydroxyl ion concentration and the concentration of the soluble dehydrogenase was proportional to the total concentration bound and soluble, confirming the report of King. The equilibrium constant for the dissociation decreased with increasing temperature and buffer concentration.
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