Immobilization of α1-acid glycoprotein for chromatographic studies of drug-protein binding

Hai Xuan, David S. Hage

Research output: Contribution to journalArticle

79 Citations (Scopus)

Abstract

A new method for preparing immobilized α1-acid glycoprotein (AGP) for use in drug-protein binding studies was developed and optimized. In this approach, periodate was used under mild conditions to oxidize the carbohydrate chains in AGP for attachment to a hydrazide-activated support. The final conditions chosen for this oxidation involved the reaction of 5.0 mg/mL AGP at 4°C and pH 7.0 with 5-20 mM periodic acid for 10 min. These conditions helped maximize the immobilization of AGP without significantly affecting its activity. This method was evaluated by using it to attach AGP to silica for use in high-performance affinity chromatography and self-competition zonal elution studies. In work with R- and S-propranolol, only one type of binding site was observed for both enantiomers on the immobilized AGP, in agreement with previous studies using soluble AGP. The association equilibrium constants measured for the immobilized AGP with R- and S-propranolol at pH 7.4 and 37°C were 2.7 × 106 and 4.2 × 106 M-1, respectively, with linear van't Hoff plots being obtained between 5 and 37°C. Work performed with other drugs also gave good agreement between the behavior seen for immobilized AGP and that for soluble AGP. The same immobilization method described in this work could be used to attach AGP to other materials, such as those used for surface plasmon resonance or alternative biosensors.

Original languageEnglish (US)
Pages (from-to)300-310
Number of pages11
JournalAnalytical Biochemistry
Volume346
Issue number2
DOIs
StatePublished - Nov 15 2005

Fingerprint

Protein Binding
Immobilization
Glycoproteins
Acids
Pharmaceutical Preparations
Propranolol
Affinity chromatography
Periodic Acid
Surface Plasmon Resonance
Enantiomers
Equilibrium constants
Surface plasmon resonance
Biosensing Techniques
Affinity Chromatography
Biosensors
Silicon Dioxide
Binding Sites
Carbohydrates
Association reactions
Oxidation

Keywords

  • Affinity chromatography
  • Drug-protein binding
  • Immobilization methods
  • Propranolol
  • α-Acid glycoprotein

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Immobilization of α1-acid glycoprotein for chromatographic studies of drug-protein binding. / Xuan, Hai; Hage, David S.

In: Analytical Biochemistry, Vol. 346, No. 2, 15.11.2005, p. 300-310.

Research output: Contribution to journalArticle

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