Characterization of the binding of sulfonylurea drugs to HSA by high-performance affinity chromatography.

Abstract

Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high-affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (+/-0.2) x 10(5) and 3.5 (+/-3.0) x 10(2) M(-1) for acetohexamide and values of 8.7 (+/-0.6) x 10(4) and 8.1 (+/-1.7) x 10(3) M(-1) for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high-affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug-binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (+/-0.1) x 10(5) and 4.3 (+/-0.3) x 10(4) M(-1), respectively, at 37 degrees C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (+/-0.2) x 10(4) and 5.3 (+/-0.2) x 10(4) M(-1), respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions.