1. A spectrophotometric direct-reading assay for measurements of beta-oxidation by intact mitochondria is described. The procedure relies on the ability of ferricyanide to trap reducing equivalents generated by the acyl-CoA dehydrogenases (EC The reduction of ferricyanide was recorded by using a dual-wavelength spectrophotometer. 2. Oxaloacetate or acetoacetate was used to stimulate the rate of beta-oxidation by rotenone-blocked mitochondria. Although both were effective with rat liver mitochondria, oxaloacetate gave about 75% more stimulation. With heart or kidney mitochondria, only oxaloacetate gave marked stimulation. Acetoacetate had no stimulatory effect with heart mitochondria, but a small stimulatory effect on the rate of beta-oxidation by kidney mitochondria. 3. The stoicheiometry of beta-oxidation-dependent reduction of ferricyanide was examined, and good correlations were found between experimental and theoretical amounts of ferricyanide reduced. 4. Ferricyanide appears as efficient a final electron acceptor as O2. With ferricyanide the rate of beta-oxidation by heart mitochondria can be measured without interference from the oxidation of tricarboxylic acid-cycle intermediates.

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