TRANSVERSAL DISTRIBUTION OF ACYL-LINKED PYRENE MOIETIES IN LIQUID-CRYSTALLINE PHOSPHATIDYLCHOLINE BILAYERS - A FLUORESCENCE QUENCHING STUDY

Quenching of the fluorescence of pyrene-labeled phospholipids by dibromolipids was used to determine the chain length dependence of the bilayer depths of the pyrenyl moieties. Six 1-palmitoyl-2-(pyrenyl-12-acyl)-phosphatidylcholines (Pyr(n)PC) were examined, with end-labeled pyrenyl chains varying in length, n, from 4 to 14 carbons. These lipids were incorporated, at a concentration of 0.3 mol %, into bilayers composed of various mixtures of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and of one of three 1-palmitoyl-2-(x,y-dibromostearoyl)phosphatidylcholine quencher lipids (Brx,yPC; x,y = 6,7; 9,10; or 11,12). Parallel experiments were carried out with bilayers containing 50 mol % cholesterol. Quenching in these systems is dynamic, as demonstrated by the identical dependence of steady-state fluorescence intensities and excited state lifetimes of Pyr(8)PC on the mole fraction of BT6,7PC Stern-Volmer analysis of the Brx,yPC mole fraction dependence of Pyr(n)PC fluorescence yielded apparent quenching constants, of the Br K-sv, which show a systematic relation with both the length of the pyrenyl acyl chain and the position of the bromine atoms. The quenching data were further analyzed by plotting K-sv as a function of n (defined above), or b (the average of the two bromine positions for each Pyr(n)PC), or n - b (the separation between pyrenes and bromines), In all cases, the data were fit by Gaussian functions yielding estimates of the centers and the apparent 1/e half-widths of the transversal distributions of the pyrenyl moieties in methylene units (mu). Both in the absence and in the presence of cholesterol, the position of each Pyr(n)PC Gaussian center is equal to the sum of n plus a constant d approximate to 2.5 mu, corresponding to the distance from the effective center of the pyrenyl moiety to its point of attachment to the acyl chain. However, consistent with the well-documented cholesterol-induced conformational ordering of the acyl chains, addition of 50 mol % cholesterol to the bilayers results in a considerable reduction of the lie half-widths of the distributions, from approximate to 9 mu in its absence to approximate to 5 mu. The good agreement between measured and predicted equilibrium depths of the pyrenyl moieties indicates that these Pyr(n)PC lipid analogues mimic their natural precursors more closely than others labeled with more polar fluorophores, which show a marked tendency to partition to the membrane surface.