Studies of Verapamil Binding to Human Serum Albumin By High-Performance Affinity Chromatography

Authors

Rangan Mallik, University of Nebraska - Lincoln
Michelle J. Yoo, University of Nebraska - Lincoln
Sike Chen, University of Nebraska - Lincoln
David S. Hage, University of Nebraska - LincolnFollow

Date of this Version

12-1-2008

Citation

Published in final edited form as: J Chromatogr B Analyt Technol Biomed Life Sci. 2008 December 1; 876(1): 69–75. doi:10.1016/j.jchromb. 2008.10.022.

Comments

Copyright Elsevier Inc. Used By Permission.

Abstract

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g., the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 10⁴ M⁻¹ and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37°C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e., the warfarin-azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (±0.1) × 10⁴ M⁻¹. Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with L-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body.