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 (DMPC)-based liposomes. The lateral mobility of this derivative, as well as that of N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine (NBD-PE), was measured by fluorescence recovery after photobleaching techniques. In DMPC liposomes, the diffusion coefficients (D) of the 2 probes are the same within experimental error below (D, .apprxeq. 2 .times. 10-10 cm2.cntdot.sec-1) and above (D .apprxeq. 2 .times. 10-8 cm2.cntdot.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 conditons, the diffusion coefficient of NBD-Chol is .apprx. 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 2 probes are identical. The lateral mobility of a cholesterol derivative was thus monitored directly in cholesterol-containing membranes.