Fluorescence and circular dichroism studies on human serum low density lipoprotein particles and lipid depleted derivatives

Low density lipoprotein from human serum, derivatives derivitives prepared free of neutral lipids and total lipids, have been studied by fluorescence and circular dichroism methods. Removal of the neutral lipids had little effect on the tryptophan fluorescence at neutral pH. However, by the criteria of circular dichroism, over the range of 200 nm to 250 nm, there was a reduction in secondary structure of over 75%. Removal of the remaining phospholipids resulted in a qualitatively different structure by both fluorescence and circular dichroism criteria. Neutral lipids were removed from LDL in a stepwise fashion in order to determine the exact amount of neutral lipid required for the native circular dichroism spectrum. The circular dichroism band intensity was constant until approx. 10% of the total cholesterol (as cholesterol ester) remained. The intensity then abruptly dropped as more cholesterol was removed. It was concluded that the 2 spectroscopic methods report on 2 distinct aspects of LDL structure. The tryptophan fluorescence appears to be sensitive to the presence of phospholipids. The circular dichroism, however, appears to be sensitive to the binding of a small amount of neutral lipid. These findings suggest that a functional and geometric separation of binding sites may exist for these 2 classes of lipids. Such a distinction is predicted by the icosohedral model of the quaternary structure of LDL. In this model, the phospholipids are located on the surface of the particle, in the holes of an icosohedrally symmetric surface network of protein subunits; the neutral lipids are located in the particle core. Finally it was suggested that functional significance may be attached to the findings that relatively few cholesterol ester molecules are needed to maintain the native secondary structure of LDL. This provides a mechanism whereby the amount of bound neutral lipid could be raised or lowered (for transport and transfer to cells) without affecting the protein in any structurally significant manner.