Kuzelová K, Brault D
Laboratoire de Biophysique, Inserm U.201, CNRS U.A. 481, Muséum National d'Histoire Naturelle, Paris, France.
Biochemistry. 1994 Aug 16;33(32):9447-59. doi: 10.1021/bi00198a010.
The interaction of deuteroporphyrin with dimyristoylphosphatidylcholine unilamellar vesicles of various sizes (ranging from 38 to 222 nm) has been studied using a stopped flow with fluorescence detection. Beside the kinetics of porphyrin incorporation into vesicles, the transfer of porphyrin from vesicles to human serum albumin has been investigated both experimentally and theoretically. The effects of both vesicle and albumin concentrations indicate that the transfer proceeds through the aqueous phase. It is governed by the rate of incorporation of porphyrin into the outer vesicle hemileaflet (kon), by the exit to the bulk aqueous medium (koff), and by the association (kas) and dissociation (kdis) 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 vesicle size, temperature, and cholesterol. The dependence of kon on the vesicle size indicates that the incorporation is diffusion controlled. The constant koff is found to be closely coupled to the phase state of the bilayer. The transbilayer flip-flop rate constant is approximately the same in both directions (approximately 0.4 s-1 at 32 degrees C and pH 7.4). It is strongly affected by the presence of cholesterol in vesicles and by the temperature, with a sharp enhancement around the phase transition. With the exception of very small vesicles obtained by sonication, no influence of the vesicle size on the flip-flop rate was observed. An accelerating effect of tetrahydrofuran, used to improve the solubility of porphyrin, has been noted. Steady-state measurements and kinetics results were in excellent agreement. The interest of systems involving albumin as a scavenger to extract important rate constants, is emphasized.
