Kinetic and equilibrium studies of porphyrin interactions with unilamellar lipidic vesicles

Abstract

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.

Kinetics of Interaction of the Myristoylated Alanine-rich C Kinase Substrate, Membranes, and Calmodulin

“…As discussed elsewhere (36), the relaxation time of the transfer process ( tr ) is dominated by k off if three conditions are met:…”

Reactivity towards singlet oxygen of propofol inside liposomes and neuronal cells

“…[38,39]. These values are (1.35 T 0.05) Â 10 7 M À1 s À1 for DP, which is embedded within the liposomes [31], and (0.8 T 0.04) Â 10 7 M À1 s À1 for the aqueous dye, UP [29,30]. The large quenching rate constant obtained for DP compared to UP implies that PPF must be embedded within the liposome.…”

“…The two photosensitizers, uroporphyrin I (UP, Frontier scientific, USA) and deuteroporphyrin (DP), which was prepared and purified as previously described [28], were used as 1 O 2 source. They were chosen because the former is located in the aqueous (D 2 O) phase [29,30] while the latter is embedded in the liposome membrane [31]. The association constant of DP for DMPC liposomes (diameter, 100 nm) was found to be (12 T 2) Â 10 4 M À1 [31].…”

“…They were chosen because the former is located in the aqueous (D 2 O) phase [29,30] while the latter is embedded in the liposome membrane [31]. The association constant of DP for DMPC liposomes (diameter, 100 nm) was found to be (12 T 2) Â 10 4 M À1 [31]. Then, total incorporation of DP within the liposomes was ensured by using a 2.2 Â 10 À2 M DMPC concentration.…”

ROS: Reactive Oxygen Species

“…In order to get a better insight into the dynamics of these processes our group has developed methods based on membrane models consisting in unilamellar vesicles and kinetic analysis (Kuzelova and Brault 1994;Vever-Bizet and Brault 1993). Figure 3 represents the schema of the passive transport of a molecule through a lipid bilayer.…”