Kinetics of phloretin binding to phosphatidylcholine vesicle membranes.

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Abstract
The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a Kd of 8.0 .mu.M and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4.degree. C within 4 .mu.s producing a monoexponential, concentration-dependent relaxation process with time constants in the 30-200 .mu.s time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24.degree. C yielded a binding rate constant of 2.7 .times. 108 M-1s-1 and an unbinding constant of 2900 s-1; .apprx. 66% of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and 3 additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin, yet a Kd equal to 24 .mu.M, bound to PC vesicles with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made 1 to 6 times more viscous than water by addition of sucrose or glycerol to the buffer. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kbinding .alpha. 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kbinding .alpha. [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid.

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