Hydrophobic/hydrophilic balance of proteins: a major determinant of cholesterol crystal formation in model bile.

Biliary proteins are speculated to play an important role in modulating nucleation of cholesterol crystals from supersaturated gallbladder bile, acting either as pro- or antinucleating agents. However, conflicting results have been obtained in attempts to isolate specifically active proteins. Our hypothesis, therefore, was that this may be a nonspecific effect of proteins, related to their secondary structure or to their overall hydrophobic/hydrophilic balance. We studied the effect of a number of "nonspecific" proteins with different secondary structures on cholesterol crystal formation in model bile. Their relative hydrophobic/hydrophilic indices were experimentally determined by measuring their retention time on a phenyl-agarose column (hydrophobic ligand). The potency of each protein in enhancing or inhibiting crystal formation was ranked according to the lowest protein concentration capable of significantly influencing crystal formation by comparison with control using model bile with cholesterol saturation index (SI) of 1.2 and 1.5. Some of these proteins (chymotrypsin, IgA, myoglobin) significantly enhanced crystal formation, while some (apolipoproteins A-I, A-II, and B) inhibited it, and others (IgG, chymotrypsinogen) showed no effect. The different effects were not related to their secondary structure but to their hydrophobic/hydrophilic index, with the most hydrophilic proteins showing maximal pronucleating potency and vice versa (r = 0.93; and 0.97, P < 0.005 for SI = 1.2 and 1.5, respectively). Pronucleating proteins enhanced, while antinucleating proteins inhibited, the transfer of cholesterol and phospholipid from micellar to vesicular forms. We conclude that the effect of proteins on cholesterol crystal formation in model bile is nonspecific and mainly related to their hydrophobic indices rather than to their secondary structures.