LOCALIZATION OF TOLBUTAMIDE BINDING SITES ON HUMAN SERUM-ALBUMIN USING TITRATION CALORIMETRY AND HETERONUCLEAR 2-D NMR

The sulfonylureas are a class of oral hypoglycemic agents used to trea t type II diabetes mellitus, and tolbutamide is a ''first generation'' member of this family. It is a nonpolar, weakly acidic drug that bind s to serum albumin in the circulation. In the present study, we have e xamined the interactions of tolbutamide with human serum albumin by is othermal titration calorimetry and heteronuclear multiple-quantum cohe rence NMR spectroscopy, Calorimetric titrations revealed that tolbutam ide binds to albumin at three independent sites with the same or compa rable affinity. This result was independently confirmed by NMR experim ents which resolved three resonances at H-1 chemical shifts of 2.07, 2 .11, and 2.14 ppm, corresponding to [methyl-C-13]tolbutamide bound to three discrete binding sites. The binding affinity quantitated by calo rimetry (K-d = 21 +/- 9 mu M at pH 7.4, 37 degrees C) was approximatel y 5 times lower than the most frequently reported value, Tolbutamide t itrations of albumin complexed with three other drugs whose binding si res have been localized by X-ray crystallography (salicylate, clofibri c acid, and triiodobenzoic acid) demonstrated direct competition for c ommon binding sites. NMR experiments with samples containing [methyl-C -13]tolbutamide and these competing drugs permitted assignment of the resonances at 2.07 and 2.14 ppm to tolbutamide bound to the aspirin si tes in albumin subdomains IIIA and IIA, respectively. These findings p ermit the first assignment of tolbutamide binding sites to specific lo cations on the albumin molecule within the context of the recently pub lished crystal structure of human serum albumin, In addition, this inf ormation provides a molecular basis for predicting unfavorable drug in teractions involving tolbutamide in patients with type II diabetes.