Equilibrium and kinetic studies of the solubilization of phospholipid-cholesterol bilayers by C12E8. The influence of the lipid phase structure

The influence of the lipidic phase structures on their susceptibility to so lubilization by the nonionic detergent C12E8 has been explored. To this aim , a phase diagram for dimyristoylphosphatidylcholine/ cholesterol in excess water has been constructed in which phase boundaries were derived from hig h-sensitivity differential scanning calorimetry. Six different lamellar pha ses can be obtained with this system, namely crystalline (L-c'), gel (L-bet a' or L-beta), rippled (P-beta'), liquid crystalline or fluid (L-alpha), ge llike liquid ordered (L-o beta), and fluidlike liquid ordered (L-o alpha). The solubilization of samples in each of these phases by C12E8 has been stu died through changes in suspension turbidity under equilibrium conditions a nd also using a stopped-flow time-resolved technique. We find that variatio ns in temperature and cholesterol content within a single phase can affect the equilibrium and kinetic parameters of detergent solubilization somewhat , particularly in the former case. However, much larger variations in the e quilibrium and kinetics parameters of C12E8 solubilization are noted betwee n different phases, particularly those phases containing cholesterol. In ge neral, the presence of cholesterol potentiates the solubilization of DMPC v esicles at lower temperatures and inhibits their solubilization at higher t emperatures. Moreover, in the more fluid phases (L-alpha for DMPC alone or L-o beta and L-o alpha for DMPC-cholesterol mixtures), vesicle turbidity wa s not affected by detergent concentration until concentration near the solu bilization concentration was reached, at which point an increase in turbidi ty attributed to vesicle lysis and reassembly occurs prior to vesicle solub ilization. In contrast, this effect is markedly reduced in the various gel phases of DMPC alone, where vesicle turbidity generally decreases monotonic ally with detergent concentration. Cholesterol-induced liquid crystalline-l ike liquid ordered phases (L-o alpha), that are presumed to coexist with th e L-alpha phase in animal cell membranes, are much more resistant to solubi lization than the predominant fluid disordered L-alpha phase, present in su ch membranes.