On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography

Jeanethe Anguizola, Cong Bi, Michelle Koke, Abby Jackson, David S. Hage

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. [Figure not available: see fulltext.]

Original languageEnglish (US)
Pages (from-to)5745-5756
Number of pages12
JournalAnalytical and Bioanalytical Chemistry
Volume408
Issue number21
DOIs
Publication statusPublished - Aug 1 2016

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Keywords

  • Affinity microcolumn
  • Alpha-acid glycoprotein
  • Drug-protein binding
  • Entrapment
  • High-performance affinity chromatography
  • Immobilization method

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

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