Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene reveal the effect of cholesterol on the microheterogeneity of erythrocyte membrane

Abstract

Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene reveal the effect of cholesterol on the microheterogeneity of erythrocyte membrane The fluorescence decay of 1,6 diphenyl-l,3,5-hexatriene (DPH) has been used to characterize aspects of the erythrocyte membrane structure related to the microheterogeneity of the lipid bilayer. The DPH decay has been studied using frequency domain fluorometry and the data analyzed either by a model of discrete exponential components or a model that assumes a continuous distribution of lifetime values. The main intensity fraction was associated with a lifetime value centered at about 11 ns in the erythrocyte membrane, but a short component of very low fractional intensity had to be considered to obtain a good fit to the data. The lifetime value of the long component was insensitive to temperature, while the width of the distribution decreased with increasing temperature. In multilamellar liposomes prepared from phospholipids extracted from the erythrocytes, the long lifetime component showed a temperature dependence. The depletion of 27% of the cholesterol in the erythocyte membrane induced a broadening of the distribution, suggesting a homogenizing effect of cholesterol. This effect has also been detected in egg phosphatidylcholine at a very low cholesterol/phospholipid molar ratio. The role of cholesterol on membrane heterogeneity is discussed in relation to the effect of cholesterol on water penetration.

Effect of cholesterol on membrane microheterogeneity: a study using 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime distributions

“…Since the DPH lifetime is single exponential in isotropic solvents at room temperature and is directly related to the dielectric constant of its surroundings [18], the homogeniTing effect of cholesterol can also induce a decrease of microenvironments heterogeneity. The results using EPC liposomes are in agreement with our previous observations [8] of higher values for the main lifetime component in erythrocyte membranes with respect to the phospholipid extracts. Moreover, in erythrocyte membranes depleted of cholesterol we also observed an increase of the distribution width.…”

“…A similar effect has been shown in cholesterol-depleted erythrocytes, in which there is a segregation of protein rich domains [7]. In previous studies [8] using DPH fluorescence decay we have shown that, in the erythrocyte membrane, cholesterol depletion induces an increase of membrane microheterogeneity, as evaluated from the lifetime distribution width. We proposed that the origin of the lifetime distribution is due to different dielectric microenvironments in which the DPH molecules are located [9].…”

“…The study of the effect of cholesterol on saturated and unsaturated phosphatidylcholine by using the changes of the fluorescence properties of DPH has enabled us to define an homogenizing effect of cholesterol due to a decrease in water permeability on unsaturated phosphatidylcholine in analogy with the observation made on natural membrane [8]. This effect is shown by the changes of the average lifetime values both using double-exponential or distributional analysis.…”

“…Theoretical considerations and experimental evidence suggest that biological membrane are organized into domains of specific composition and properties [8,13]; however, this structurai aspect of membrane organization is at present difficult to define on a molecular basis due to methodological and technical limitations.…”

“…To investigate the role of cholesterol in controlling the degree of membrane heterogeneity we studied the modification induced by this molecule on liposomes of unsaturated and saturated phospholipids using the changes produced on the fluorescence decay of DPH The decay was measured using multifrequency phase fluorometry, which we have shown to be a suitable technique to describe membrane heterogeneity by means of the distribution analysis of complex excited state decay [8,91.…”