Fluorescence properties of lipoprotein B and apolipoprotein B

Abstract

The fluorescence properties of apolipoprotein B (ApoB) in various media, including aqueous solutions of three different pH, 6 m urea, 6 m guanidine-HCl and native lipoprotein B (LP-B) particles have been compared by measuring the accessibility of trytophan side chains to iodide ions. The modified Stern-Volmer plots ( FΔF vs. 1/[KI]) for LP-B demonstrate heterogeneity of quenching rates at pH 9.0, with a total accessibility of fluorescence to iodide of 43%. At pH 7.3, the total accessibility of LP-B fluorescence to iodide is only 20%. Quenching at pH 2.7 follows a pure Stern-Volmer mechanism. A straight line at this pH intercepting y-axis at 1.0 indicates 100% accessibility of tryptophan residues in LP-B. These results suggest that there are at least three different groups of tryptophan residues present per intact LP-B particle and that each group is situated in a different environment. One group, showing an enhanced quenching rate, is probably near the charged domain; another group, showing a slower quenching rate, is in a relatively hindered environment, and a third group is probably buried in a more hydrophobic environment, inaccessible to iodide at neutral or high pH. But at pH 2.7, all tryptophan residues appear to become situated closer to the surface of the LP-B particle. For isolated ApoB at pH 7.3 and 9.0 in aqueous buffer, about 30% of the fluorescence is relatively easily accessible; another 40% is less easily accessible and the remaining 30% is inaccessible to iodide. These inaccessible tryptophan residues are most likely located in a more hydrophobic matrix and probably in the β-pleated sheet region of ApoB. Similarly to LP-B at pH 2.7, all of the tryptophan residues of ApoB are exposed to the aqueous surface except that one third of them are quenched at a faster rate than the rest. At pH 7.3, in the presence of urea or guanidine-HCl, all of the fluorescence of ApoB is exposed to the aqueous surface, suggesting the presence of random and nonrigid conformation in these media. These results suggest that the conformation of ApoB in aqueous media is pH sensitive. This is true whether the ApoB is present in intact LP-B or as the isolated apolipoprotein. Furthermore, upon removal of lipids from LP-B and passing the ApoB into a denaturing environment, the apolipoprotein loses its ordered structure. When passing ApoB from denaturing agents back to aqueous buffers of neutral or basic pH. ApoB is able to reorient itself to gain an ordered structure, not necessarily identical to that in LP-B, but parallel to it.