Nonspecific high affinity binding of bile salts to carboxylester lipases.

The interactions with bile salts of carboxylester lipases (EC 3.1.1.13) from human pancreatic juice and pig pancreas were characterized by physical methods. Bile salts cause a decrease in the fluorescence intensity of the proteins at the emission maximum of 333-335 nm. The concentration dependence of this decrease shows saturation behavior, is relatively nonspecific with respect to bile salt conjugation or the presence of the 7 alpha-hydroxyl group, and is consistent with a 1:1 interaction between enzyme and bile salt. Direct measurement of the binding of [3H]cholate by equilibrium dialysis supports the stoichiometry. Other detergents also bind, causing fluorescence changes, but with much lower affinities. Binding of taurocholate to the monomeric pig enzyme is enhanced by increasing ionic strength, indicating the predominance of hydrophobic interactions. In the range of pH 5.5-6.8, binding is pH-independent with dissociation constants of 3-20 microM. At higher pH, affinity is greatly reduced and the fluorescence spectrum changes, indicating the importance of a protonated group for efficient interaction. Occupancy of the bile salt binding site partially stabilizes the enzyme against inactivation by heat but not trypsin. However, circular dichroism spectra do not indicate that bile salt binding is accompanied by any change in secondary structure. The monomeric pig enzyme binds to the argon/water interface in the presence of bile salts and binding of taurocholate to diisopropylphosphoryl-enzyme is similar to that measured with native enzyme. These results suggest that surface binding and catalysis occur at sites distinct from the bile salt binding site of the enzyme. Stabilization of the monomeric pig enzyme against denaturation at high energy surfaces occurs concomitantly with occupancy of the bile salt binding site. Overall, the data suggest that an important role of bile salts in vivo is to stabilize these enzymes at lipid-water interfaces.