Detection of heterogeneous drug-protein binding by frontal analysis and high-performance affinity chromatography

Zenghan Tong, K. S. Joseph, David S. Hage

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


This study examined the use of frontal analysis and high-performance affinity chromatography for detecting heterogeneous binding in biomolecular interactions, using the binding of acetohexamide with human serum albumin (HSA) as a model. It was found through the use of this model system and chromatographic theory that double-reciprocal plots could be used more easily than traditional isotherms for the initial detection of binding site heterogeneity. The deviations from linearity that were seen in double-reciprocal plots as a result of heterogeneity were a function of the analyte concentration, the relative affinities of the binding sites in the system and the amount of each type of site that was present. The size of these deviations was determined and compared under various conditions. Plots were also generated to show what experimental conditions would be needed to observe these deviations for general heterogeneous systems or for cases in which some preliminary information was available on the extent of binding heterogeneity. The methods developed in this work for the detection of binding heterogeneity are not limited to drug interactions with HSA but could be applied to other types of drug-protein binding or to additional biological systems with heterogeneous binding.

Original languageEnglish (US)
Pages (from-to)8915-8924
Number of pages10
JournalJournal of Chromatography A
Issue number49
StatePublished - Dec 9 2011


  • Acetohexamide
  • Drug-protein binding
  • Frontal analysis
  • Heterogeneous binding
  • High-performance affinity chromatography
  • Human serum albumin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry


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