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

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.

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

“…In these reports, the biomolecule is physically entrapped during the formation of the support. Entrapment can result in a high activity for the immobilized biomolecule by avoiding problems such as improper orientation, steric hindrance, and multi-site attachment that can occur during covalent immobilization [36–38]. In addition, no modification of the protein is required before its immobilization.…”

“…In addition, no modification of the protein is required before its immobilization. However, many of the materials that are currently used for entrapment are not suitable for use under the pressure or flow conditions that are often present in HPLC systems [36,38]. Also, these entrapment methods can lead to slow mass transfer as an applied analyte attempts to interact with a biomolecule that may be held deep within the support [39].…”

“…This method has been utilized in a slurry-based format with HPLC-grade silica to entrap HSA and to create supports and columns that are suitable for use in HPAC [2,36,40]. It has also been demonstrated that this slurry-based method can be adapted for work with AGP [36,38]. This approach retains the ability of entrapment to provide an immobilized protein in a soluble and active form, while also overcoming the slow mass transfer effects and flow or pressure limitations that are often present when using hydrogels or sol-gel entrapment [2,36,38–40].…”

“…It has also been demonstrated that this slurry-based method can be adapted for work with AGP [36,38]. This approach retains the ability of entrapment to provide an immobilized protein in a soluble and active form, while also overcoming the slow mass transfer effects and flow or pressure limitations that are often present when using hydrogels or sol-gel entrapment [2,36,38–40]. However, the extent of immobilization for proteins in this slurry-based method is limited by the overall solution volume, since only those proteins that are present within the pores or near the surface of the support will be entrapped during this process [38].…”

Studies of drug interactions with alpha 1 -acid glycoprotein by using on-line immunoextraction and high-performance affinity chromatography

“…In the zonal elution studies, 5 μM of disopyramide, warfarin, imipramine, propranolol or chlorpromazine, and 20 μM of sodium nitrate (which was used as a non-retained solute) [21,22,24] were prepared in pH 7.4, 0.067 M potassium phosphate buffer. A 20 μL portion of each sample was applied to an antibody microcolumn containing adsorbed AGP (i.e., prepared as described in Sections 2.3–2.4) and a control column at 37 °C and 0.10 mL/min.…”