Erman, James E.
M.S. (Master of Science)
Department of Chemistry
Cytochrome c; Cytochromes; Hydrogen-ion concentration; Peroxidase
The fluorescence quenching technique provides a method for studying the binding between horse heart cytochrome c and protoporphyrin cytochrome c peroxidase. The effect of pH on the binding was studied. The number of binding sites have long been in contention. Based on a 1 : 1 complex formation model, three cases for analyzing the binding using the fluorescence quenching technique were investigated in the range pH 4.0 to pH 8.0. Two models were established. Under the experimental condition, the one binding site model worked well. The second binding site was not observed by using fluorescence quenching. The equilibrium dissociation constants, which are a measure of the binding, were determined. They were less than 1 nM above pH 5.2, the isoelectric point of the enzyme, in 10 mM phosphate buffer, and increased significantly below pH 5.2 in acetate buffer. Binding between cytochrome c and porphyrin cytochrome c peroxidase was much stronger above the isoelectric point than that below the isoelectric point. The effect of apocytochrome c peroxidase on the binding with horse heart cytochrome c was also investigated. It was found that apoCcP and pCcP have the same affinity when binding with cytochrome c. Methods of calibration of extinction coefficients of porphyrin cytochrome c peroxidase for different pH, ionic strength, and wavelength were established.
Lin, Li, "A fluorescence quenching study of the pH dependence of binding between horse heart cytochrome c and cytochrome c peroxidase" (1995). Graduate Research Theses & Dissertations. 304.
x, 117 pages
Northern Illinois University
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