CHARACTERIZATION OF THE CORE AND SURFACE OF HUMAN PLASMA-LIPOPROTEINS - A STUDY BASED ON THE USE OF 5 FLUOROPHORES

The physical properties of the core and the surface of five classes of human plasma lipoproteins were investigated using five fluorescent Probes. The location of the fluorescence probes in the lipoprotein assembly was determined using collisional quenching and resonance energy transfer. The fluorophores monitor different regions of the lipoproteins, as shown by fluorescence quenching. Diphenylhexatriene (DPH) and methyl trans-parinaric acid (MTPA), which are apolar molecules, are localized mainly in the lipoprotein core. Their distribution into the surface is dependent upon the volume ratio of the hydrophobic part of the envelope and the core. The polar fluorophores, trimethylaminodiphenylhexatriene (TMADPH), hydroxycoumarin (HC) and trans-parinaric acid (TPA) are anchored in the glycerol skeleton region of the surface monolayer with the fluorophore group of HC in the headgroup region of the phospholipids. We determined the temperature-dependent steady-state fluorescence anisotropy (r) of these fluorophores in the four major classes of lipoproteins: VLDL, LDL, HDL2, HDL3 and in abnormal HDL from abetalipoproteinemia patients (HDL(ab)). The hydrophobic probes, DPH and MTPA, reported the r values in the lipoproteins in the following order: LDL > HDL2 > HDL3 much greater than VLDL. This order correlates with the triglyceride-to-cholesterol ester (TG/CE) ratio in the core of lipoproteins. The polar probes HC, TPA and TMADPH reported the r value in a different order: HDL2, HDL3 greater-than-or-equal-to LDL much greater than VLDL. This is compatible with the decreasing order of the protein to lipid ratio in the envelope of these lipoproteins. HDL(ab) was investigated by three fluorescent probes: DPH, TMADPH and HC. The anisotropy of DPH in HDL(ab) was larger than that of either HDL2 or HDL3 in normal donors, probably due to the smaller TG/CE ratio in HDL(ab). The lower r values reported by HC and TMADPH for HDL(ab) are not fully understood and may be related to other factors such as acyl chains composition. The characterization of lipoproteins by fluorescence depolarization using probes of known location in the lipoprotein assembly is very sensitive and may be used to report deviation from the norm.