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 dibro molipids was used to determine the chain length dependence of the bila yer depths of the pyrenyl moieties. Six palmitoyl-2-(pyrenyl-12-acyl)- phosphatidylcholines (Pyr(n)PC) were examined, with end-labeled pyreny l 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 lmitoyl-2-(x,y-dibromostearoyl)phosphatidylcholin e quencher lipids (Brx,yPC; x,y = 6,7; 9,10; or 11,12). Parallel exper iments were carried out with bilayers containing 50 mol % cholesterol. Quenching in these systems is dynamic, as demonstrated by the identic al dependence of steady-state fluorescence intensities and excited sta te 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 fluoresce nce yielded apparent quenching constants, of the Br K-sv, which show a systematic relation with both the length of the pyrenyl acyl chain an d the position of the bromine atoms. The quenching data were further a nalyzed 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 (th e separation between pyrenes and bromines), In all cases, the data wer e fit by Gaussian functions yielding estimates of the centers and the apparent 1/e half-widths of the transversal distributions of the pyren yl moieties in methylene units (mu). Both in the absence and in the pr esence of cholesterol, the position of each Pyr(n)PC Gaussian center i s equal to the sum of n plus a constant d approximate to 2.5 mu, corre sponding to the distance from the effective center of the pyrenyl moie ty to its point of attachment to the acyl chain. However, consistent w ith the well-documented cholesterol-induced conformational ordering of the acyl chains, addition of 50 mol % cholesterol to the bilayers res ults in a considerable reduction of the lie half-widths of the distrib utions, 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.