Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin

Jeanethe Anguizola, Erin Debolt, D. Suresh, David S. Hage

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


The primary endogenous ligands of human serum albumin (HSA) are non-esterified fatty acids, with 0.1-2 mol of fatty acids normally being bound to HSA. In type II diabetes, fatty acid levels in serum are often elevated, and the presence of high glucose results in an increase in the non-enzymatic glycation of HSA. High-performance affinity chromatography (HPAC) was used to examine the combined effects of glycation and the presence of long chain fatty acids on the binding of HSA with R-warfarin and l-tryptophan (i.e., probes for Sudlow sites I and II, the major sites for drugs on this protein). Zonal elution competition studies were used to examine the interactions of myristic acid, palmitic acid and stearic acid with these probes on HSA. It was found that all these fatty acids had direct competition with R-warfarin at Sudlow site I of normal HSA and glycated HSA, with the glycated HSA typically having stronger binding for the fatty acids at this site. At Sudlow site II, direct competition was observed for all the fatty acids with l-tryptophan when using normal HSA, while glycated HSA gave no competition or positive allosteric interactions between these fatty acids and l-tryptophan. These data indicated that glycation can alter the interactions of drugs and fatty acids at specific binding sites on HSA. The results of this study should lead to a better understanding of how these interactions may change during diabetes and demonstrate how HPAC can be used to examine drug/solute-protein interactions in complex systems.

Original languageEnglish (US)
Pages (from-to)175-181
Number of pages7
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
StatePublished - May 15 2016


  • Drug-protein binding
  • Fatty acids
  • Glycation
  • High-performance affinity chromatography
  • Human serum albumin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology


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