Effects of cholesterol on membrane surfaces as studied by high-pressure fluorescence spectroscopy

We have studied the effects of cholesterol on membrane surfaces using fluorescence spectroscopy at high pressure. At atmospheric pressure, the dissociation state of a pH-sensitive fluorophore (6-decanylnaphthol or DECNA) incorporated into several types of membranes showed an apparent increase in dissociation with cholesterol content coming somewhat closer to its dissociation state in solution. Previous studies have shown that when DECNA is free in solution, pressure induces proton dissociation due to the volume decrease that occurs when water electrostricts around the ions. But in phosphatidylcholine (PC) bilayers, proton dissociation is inhibited, either due to the inability of the surface to expand and allow for increased hydration, or other changes in lipid structure. The pressure behavior of DECNA in dioleoyl-PC, dioleoylphosphatidic acid and dioleylphosphatidylglycerol bilayers shows that incorporation of 5-10% cholesterol causes DECNA to behave like it is in a more unrestricted environment. This trend is reversed at higher cholesterol concentrations. These data, together with compressibility measurements, support the model of Sankaram and Thompson [M. Sankaram, T.E. Thompson, Biochemistry 29 (1990) 10676.] whereby in the disordered phase, cholesterol can span the two leaflets causing an increase in the area between the head groups; whereas in the ordered phase, no expansion occurs. Thus, the effect of cholesterol on membrane surfaces depends on its phase diagram.