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

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.

Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications

“…A similar approach has been used to study the interactions of R/S -propranolol and verapamil with high-density lipoprotein (15) and the binding of R/S -propanolol with very low-density lipoprotein (16). HPAC and affinity chromatography have also been employed in examining the binding of various solutes and drugs with the transport proteins α 1 -acid glycoprotein (AGP) or human serum albumin (HSA) (10,17,18), the binding of urediofibrate-like dual agonists with peroxisome proliferator-activated receptors (19), and the interactions of many compounds with immobilized enzymes or lectins (12,20–22). …”

“…The linear relationships that were obtained (Figure 3) made it possible to determine the equilibrium constants for glimepiride at Sudlow site II on each type of HSA that was examined (11,24). The same method has been used to examine the competition of other site-specific probes and drugs during their interactions with the normal or modified HSA and with AGP (17,18,25–27). Similar approaches have been used to investigate additional interactions, including multi-site binding and allosteric effects (8,28–32).…”

Analysis of stereoselective drug interactions with serum proteins by high-performance affinity chromatography: A historical perspective

“…Some of these studies have looked at the effects of temperature and mobile phase composition on the retention and chiral separation of such drugs as atenolol, warfarin, verapamil, methadone, atropine, disopyramide, ketamine, and various profens (e.g., ketoprofen and ibuprofen) [8–10,72–75]. In addition, detailed thermodynamic studies have been carried out with this protein and such drugs as propranolol, carbamazepine, and warfarin [61,62,76–78]. More recent studies have also employed HPAC and affinity columns to examine the kinetics for some of these interactions [79,80].…”

“…It was shown in 2004 how this same data can be used in a more quantitative manner to not only identify the type of interaction that is taking place but to determine the coupling constants and binding parameters that are present for this interaction [111,112]. This quantitative approach has been used to examine the allosteric effects that occur between tamoxifen and warfarin as these drugs both bind to HSA [113], the allosteric interactions that occur between warfarin and S -propranolol on AGP [114], the interactions between ibuprofen and benzodiazepines on HSA [115], and the effect of tolbutamide on the interactions of R -warfarin with HSA [116]. …”

“…However, it is now also common to employ more complex models and equations that may involve non-linear fits [7,22]. Examples of such models include those that involve multi-site binding, non-saturable interactions, and combinations of saturable plus non-saturable interactions, such as have been observed for the interactions of some drugs with modified forms of HSA and with AGP or lipoproteins [6,22,76–78,80–84,108–110,132–134]. This approach has further been employed in the study of other systems, such as drug-receptor interactions [135] and the binding of various solutes with lectins or enzymes [136–139].…”