Determination of the equilibrium constant for the binding of ferricytochrome c to phospholipid vesicles and the effect of binding on the reduction rate of cytochrome c

The rate of reduction of cytochrome c by 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine was examined as a function of binding to liposomes prepared from mixed soybean phospholipids, asolectin, and from various purified phospholipids. Binding of cytochrome c to asolectin liposomes caused an increase in the rate of reduction by the pteridine derivative from 2900 to 16 000 M^?1 · s^?1 at pH 7. At low ionic strength (0.003 M) the binding stoichiometry between cytochrome c and asolectin vesicles is 15 ± 2 phosphospolipid/cytochrome c (mole ratio), determined by monitoring the change in reduction rate of cytochrome c by pteridine as cytochrome c is bound to the vesicles. A stoichiometry of 14 phospholipid/cytochrome c was obtained from gel filtration studies. Equilibrium association constants for the binding of cytochrome c to sites on the asolectin vesicles varied from 2.2 · 10⁶ to 1.8 · 10³ M^?1 between 0.02 and 0.10 M ionic strength, respectively. In general, liposomes prepared from purified phospholipids resulted in less binding of cytochrome c per mole of phospholipid and lower reduction rates than those prepared from asolectin.