Quantitative studies of allosteric effects by biointeraction chromatography: Analysis of protein binding for low-solubility drugs

Jianzhong Chen, David S. Hage

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

A new chromatographic method was developed for characterizing allosteric interactions between an immobilized binding agent and low-solubility compounds. This approach was illustrated by using it to characterize the interactions between tamoxifen and warfarin during their binding to the protein human serum albumin (HSA), with β-cyclodextrin being employed as a solubilizing agent for these drugs. It was confirmed in this work through several experiments that warfarin had a single binding site on HSA with an association equilibrium constant of (2-5) × 105 M-1 (average, 3.9 × 105 M-1) at 37 °C, in agreement with previous reports. It was also found that tamoxifen had a single major binding site on HSA, with an association equilibrium constant of (3-4) × 107 M -1 (average, 3.5 × 107 M-1) at 37 °C. When warfarin was used as a mobile-phase additive in competition studies with tamoxifen, this had a positive allosteric effect on tamoxifen/HSA binding, giving a coupling constant of 2.3 (±0.3). Competitive studies using tamoxifen as a mobile-phase additive indicated that tamoxifen had a negative allosteric effect on warfarin/HSA binding, providing a coupling constant of 0.79 (±0.03). A unique feature of the technique described in this report was its ability to independently examine both directions of the warfarin/tamoxifen allosteric interaction. This approach is not limited to warfarin, tamoxifen, and HSA but can also be used to study other solutes and binding agents.

Original languageEnglish (US)
Pages (from-to)2672-2683
Number of pages12
JournalAnalytical Chemistry
Volume78
Issue number8
DOIs
StatePublished - Apr 15 2006

ASJC Scopus subject areas

  • Analytical Chemistry

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