KINETIC AND EQUILIBRIUM STUDIES OF PORPHYRIN INTERACTIONS WITH UNILAMELLAR LIPIDIC VESICLES

The interaction of deuteroporphyrin with dimyristoylphosphatidylcholin e unilamellar vesicles of various sizes (ranging from 38 to 222 nm) ha s been studied using a stopped flow with fluorescence detection. Besid e the kinetics of porphyrin incorporation into vesicles, the transfer of porphyrin from vesicles to human serum albumin has been investigate d both experimentally and theoretically. The effects of both vesicle a nd albumin concentrations indicate that the transfer proceeds through the aqueous phase. It is governed by the rate of incorporation of porp hyrin into the outer vesicle hemileaflet (k(on)), by the exit to the b ulk aqueous medium (k(off)), and by the association (k(as)) and dissoc iation (k(dis)) constants relative to albumin. In both systems studied , a slower transbilayer flip-flop accounts for the biphasic character of the kinetics. This model is strongly supported by the effects of ve sicle size, temperature, and cholesterol. The dependence of k(on) on t he vesicle size indicates that the incorporation is diffusion controll ed. The constant k(off) is found to be closely coupled to the phase st ate of the bilayer. The transbilayer flip-flop rate constant is approx imately the same in both directions (similar to 0.4 s(-1) at 32 degree s C and pH 7.4). It is strongly affected by the presence of cholestero l in vesicles and by the temperature, with a sharp enhancement around the phase transition. With the exception of very small vesicles obtain ed by sonication, no influence of the vesicle size on the flip-flop ra te was observed. An accelerating effect of tetrahydrofuran, used to im prove the solubility of porphyrin, has been noted. Steady-state measur ements and kinetics results were in excellent agreement. The interest of systems involving albumin as a scavenger to extract important rate constants, is emphasized.