Cytochrome c interaction with membranes. I. Use of a fluorescent chromophore in the study of cytochrome c interaction with artificial and mitochondrial membranes |
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Authors: | J Vanderkooi M Erecińska B Chance |
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Affiliation: | Johnson Research Foundation, Department of Biophysics and Physical Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104 U.S.A. |
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Abstract: | Quenching of 12-(9-anthroyl) stearic acid (AS) fluorescence by cytochrome c occurs through an energy-transfer mechanism and can be used to measure the binding of the cytochrome to artificial and mitochondrial membranes. The quenching of AS3 fluorescence is biphasic ( below 25 msec and above 500 msec) and its extent diminishes at high salt concentration or at high pH and increases in the presence of negatively charged lipids.Addition of cytochrome c to cytochrome c-depleted mitochondria results in binding of the cytochrome to the membrane and quenching of AS fluorescence. The affinity of oxidized cytochrome c for cytochrome c-depleted mitochondria is 1.8 × 106m, while the affinity constant for reduced cytochrome c is 0.5 × 106m. The lower affinity of the reduced cytochrome c for mitochondrial membranes is in accordance with midpoint potential differences between the bound and free forms. |
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Keywords: | To whom correspondence should be addressed. |
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