Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies

Cong Bi, Abby Jackson, John Vargas-Badilla, Rong Li, Giana Rada, Jeanethe Anguizola, Erika Pfaunmiller, David S. Hage

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37 °C for carbamazepine with AGP was found to be 1.0 (±0.5) × 105 M-1, which agreed with a previously reported value of 1.0 (±0.1) × 105 M-1. Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

Original languageEnglish (US)
Pages (from-to)188-196
Number of pages9
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume1021
DOIs
StatePublished - May 15 2016

Keywords

  • Alpha-acid glycoprotein
  • Drug-protein binding
  • Entrapment
  • High-performance affinity chromatography
  • Human serum albumin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

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