Studies of phenytoin binding to human serum albumin by high-performance affinity chromatography

Jianzhong Chen, Corey Ohnmacht, David S. Hage

Research output: Contribution to journalArticlepeer-review

73 Scopus citations


High-performance affinity chromatography was used to study the binding of phenytoin to an immobilized human serum albumin (HSA) column. This was accomplished through frontal analysis and competitive binding zonal elution experiments, the latter of which used four probe compounds for the major and minor binding sites of HSA injected into the presence of mobile phases containing known concentrations of phenytoin. It was found that phenytoin can interact with HSA at the warfarin-azapropazone, indole-benzodiazepine, tamoxifen, and digitoxin sites of this protein. The association constants for phenytoin at the indole-benzodiazepine and digitoxin sites were determined to be 1.04 (±0.05) × 104 M-1 and 6.5 (±0.6) × 103 M-1, respectively, at pH 7.4 and 37°C. Both allosteric interactions and direct binding for phenytoin appear to take place at the warfarin-azapropazone and tamoxifen sites. This rather complex binding system indicates the importance of identifying the binding regions on HSA for specific drugs as a means for understanding the transport of such substances in blood and in characterizing their potential for drug-drug interactions.

Original languageEnglish (US)
Pages (from-to)137-145
Number of pages9
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Issue number1
StatePublished - Sep 25 2004


  • Human serum albumin
  • Phenytoin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology


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