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

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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.

On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent

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Acknowledgments

This work was supported by the National Institutes of Health under grants R01 GM044931 and R01 DK069629. The authors also thank J. Vargas-Badilla for the information on the column-to-column reproducibility.

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  1. Chemistry Department, University of Nebraska, 704 Hamilton Hall, Lincoln, NE, 68588, USA

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

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  1. Jeanethe Anguizola
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  2. Cong Bi
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  3. Michelle Koke
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  4. Abby Jackson
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  5. David S. Hage
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Correspondence to David S. Hage.

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Conflict of interest

The authors declare no conflict of interest in the work that is presented in this study. The general concept of the entrapment approach that was used in this work is described in U.S. Patent 8,268,570.

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Anguizola, J., Bi, C., Koke, M. et al. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography. Anal Bioanal Chem 408, 5745–5756 (2016). https://doi.org/10.1007/s00216-016-9677-7

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  • DOI: https://doi.org/10.1007/s00216-016-9677-7

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