On the Elucidation of the pH Dependence of the Oxidation-Reduction Potential of Cytochrome c at Alkaline pH
Karl G. Brandt 1, Paul C. Parks 1, Georg H. Czerlinski 1, and George P. Hess 1
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1 From the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania
Rate constants were measured for the reversible oxidation-reduction reaction between ferricytochrome c and ferrohexacyanide.
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Kinetic measurements in the pH range 7.0 to 9.4 were made by the temperature jump technique, and comparative values of Keq were determined by a spectrophotometric titration method based on absorption difference at 550 mµ between ferri- and ferrocytochrome c. Also, stopped flow experiments at 550 mµ were performed at pH 9.5. In all of these experiments, excess sodium ferrocyanide (8 x 10-3 m) was mixed with varying concentrations (5 x 10-6 to 1.6 x 10-4 m) of ferricytochrome c at a constant ionic strength of 0.2.
The quotient k(ox)k/(red) is essentially pH-independent in the pH region investigated; k(red) was found to be 2.4 x 104 m-1 sec-1, and k(ox), 0.87 x 107 m-1 sec-1.
In the pH region investigated, the over-all equilibrium constant for the reaction, Keq, increased by a factor of 4 with increasing pH.
The stopped flow experiments indicated that a slow process with a half-time of several seconds is responsible for the difference between k(ox)/k(red) and Keq values at alkaline pH values.
The experiments indicate that pH has no effect on the electron transfer process itself. The pH dependence of the oxidation-reduction potential of cytochrome c at alkaline pH is evidently due to the effect of pH on a slow isomerization of ferricytochrome c, with increasing pH decreasing the concentration of cytochrome c which participates in the electron transfer process.
Submitted on March 14, 1966
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