Use of a fluorescent cholesterol derivative to measure lateral mobility of cholesterol in membranes

Abstract

N1-Cholesterylcarbamoyl-N8-(4-nitrobenzo-2-oxa-1,3-diazole)-3,6-dioxaoctyl-1,8-diamine (NBD-Chol), a new fluorescent derivative of cholesterol, was incorporated into L-alpha-dimyristoylphosphatidylcholine (Myr2PtdCho)-based liposomes. The lateral mobility of this derivative, as well as that of N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine (NBD-PtdEtn), was measured by fluorescence recovery after photobleaching techniques. In Myr2PtdCho liposomes, the diffusion coefficients (D) of the two probes are the same within experimental error below (D, approximately equal to 2 X 10(-10) cm2 X sec-1) and above (D, approximately equal to 2 X 10(-8) cm2 X sec-1) the main phase transition temperature of the bulk lipid (Tm). There is, however, a distinct difference between the mobilities of the derivatives at concentrations of added cholesterol between 5 and 20 mol % at temperatures below the main phase transition. Under these conditions, the diffusion coefficient of NBD-Chol is approximately twice that of NBD-PtdCho, a result consistent with the idea that cholesterol undergoes a lateral phase separation in these membranes at concentrations less than 20 mol %. At cholesterol concentrations greater than 20 mol % or temperatures above the Tm, the D values of the two probes are identical. The lateral mobility of a cholesterol derivative has thus been monitored directly in cholesterol-containing membranes.