FLUORESCENCE STUDY OF THE MOTIONAL STATES OF CORE AND SURFACE-LIPIDS IN NATIVE AND RECONSTITUTED LOW-DENSITY LIPOPROTEINS

Low density lipoproteins (LDL) consist of an apolar core of cholesterol esters and triglycerides surrounded by a monolayer of phospholipid, cholesterol, and a single molecule of apolipoprotein B (apoB-100). To determine the influence of core and surface constituents on the surface order of LDL, we have measured core and surface order parameters for native LDL, and reconstituted LDLs (rLDL) whose apolar core lipids were extracted and replaced with either cholesterol oleate (CO) or triolein (TO). Order parameters were measured by fluorescence depolarization of diphenylhexatriene (DPH), which is located primarily in the core, and of trimethylammoniumdiphenylhexatriene (TMA-DPH), which is anchored at the water-phospholipid interface. DPH order parameters for LDL reconstituted with TO (r-[TO]LDL) were much lower than those for LDL reconstituted with CO (r-[CO]LDL), consistent with the physical properties of TO, a nonviscous liquid at all temperatures studied, and CO, which exists in a liquid crystalline or viscous liquid state at the temperatures studied. Although core cholesterol esters in r[CO]LDL and native LDL undergo distinct order-disorder transitions, these transitions were not detected by DPH. This is most likely due to the difference between the time scale for end-over-end tumbling of cholesterol esters and the fluorescence lifetime of DPH. Despite the fact that the core lipids of r-[CO]LDL were much more ordered than those of r-[TO]LDL, surface order parameters for both lipoproteins were similar. We conclude that the motional states of the core and surface lipids are relatively independent. Surface order parameters for native LDL were higher than those for reconstituted LDLs. This was attributed to the presence of unesterified cholesterol in the surface of native LDL, and its absence in reconstituted LDL. Finally, the outer surface of r-[CO]LDL was shown to be more ordered than that of unilamellar vesicles. We suggest that this is due to the presence of apoB-100 and neutral lipid molecules in the highly curved surface of LDL which reduce the motional freedom of surface phospholipids.