Temperature, pressure and cholesterol effects on bilayer fluidity; a comparison of pyrene excimer/monomer ratios with the steady-state fluorescence polarization of diphenylhexatriene in liposomes and microsomes

Pyrene excimer/monomer (E/M) ratios have been compared with the steady-state fluorescence polarization (P) of diphenylhexatriene (DPH) in multilamellar liposomes of dilaurylphosphatidylcholine and rat liver microsomes. The purpose was to use the well-understood properties of DPH to reveal the nature of bilayer fluidity which pyrene manifests as an E/M ratio. Reducing the temperature (from 37 degrees C to 8 degrees C), increasing the hydrostatic pressure (from 0.1 to 70 MPa), and, in liposomes, cholesterol enrichment (up to 0.30 mole fraction) separately decreased the E/M ratios and increased P. The pyrene membrane/buffer partition coefficient was affected by temperature but not by pressure, and in the case of cholesterol enrichment, it was assumed to be unaffected. Plots of P as a function of the E/M ratio showed the two to be closely correlated (r = 0.99 in liposomes and 0.96 in microsomes), independent of the treatment used to reduce fluidity. The apparent activation volume and enthalpy for excimer formation was calculated and compared with published data. Pyrene E/M ratios probably reflect the intermolecular volume (fluidity) of the outer region of the bilayer, which is reduced by a decrease in temperature and an increase in pressure and cholesterol. DPH reports the bilayer interior, which is similarly ordered by the experimental treatments. The regional distinction between the two probes, however, accounts for the divergence of E/M ratios and P, which has been reported in membranes enriched with fluidizing fatty acids.