An affinity monolith based on silica and containing immobilized alpha(1)-acid glycoprotein (AGP) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of AGP in the silica monolith was 18% higher than that obtained with silica particles and 61% higher than that measured for a GMA/EDMA monolith. The higher surface area of the silica monolith gave materials that contained 1.5- to 3.6-times more immobilized protein per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the AGP silica monolith were evaluated by injecting two chiral analytes onto this column (i.e., R/S-warfarin and R/S-propranolol). In each case, the AGP silica monolith gave higher retention plus better resolution and efficiency than AGP columns containing silica particles or a GMA/EDMA monolith. The AGP silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with AGP as a chiral stationary phase.
A method is described for the entrapment of proteins in hydrazide-activated supports using oxidized glycogen as a capping agent. This approach is demonstrated using human serum albumin (HSA) as a model binding agent. After optimization of this method, a protein content of 43 (± 1) mg HSA/g support was obtained for porous silica. The entrapped HSA supports could retain a low mass drug (S-warfarin) and had activities and equilibrium constants comparable to those for soluble HSA. It was also found that this approach could be used with other proteins and binding agents that had masses between 5.8 and 150 kDa. KeywordsImmobilization methods; Entrapment; High-performance affinity chromatography; Drug-protein binding; Frontal analysis; Human serum albumin; Glycogen Affinity chromatography uses a biologically-related binding agent as a stationary phase to undergo specific interactions with a target analyte (1-3). The selectivity of this approach is dependent on the way in which the binding agent is attached to the support (4). It is common for a binding agent such as a protein to be covalently immobilized through amine, sulfhydryl, carboxyl or carbonyl groups (4-6). However, covalent immobilization can produce multisite attachment or improper orientation of the binding agent, which may cause an apparent change in this agent's activity. These issues can be minimized in some cases by coupling the binding agent to the support through functional groups that occur in only a few locations in its structure, such as the use of the carbohydrate chains in antibodies or α 1 -acid glycoprotein (AGP) for their site-selective attachment to hydrazide-activated supports (4,6).Another approach to avoid these immobilization effects is to use the physical entrapment (or encapsulation) of the binding agent within a support. This approach has been of interest for many years in work with materials such as sol gels and has generally been based on the physical containment of binding agents in a highly cross-linked polymer network (7-9). However, past approaches have used materials with pressure or flow rate restrictions that are not easily amenable for use with HPLC. Also, a general entrapment approach is still needed that can be used with common HPLC supports such as porous silica. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThis report used the entrapment method shown in Figure 1(a), as based on the use of a hydrazide-activated support and oxidized glycogen (~2.5 × 10 5 kDa) as a capping agent. The ...
A new method for the immobilization of α 1 -acid glycoprotein (AGP) in HPLC columns was recently described for applications such as drug binding studies. Part of this earlier work used self-competition zonal elution studies to measure association equilibrium constants between immobilized AGP and R-or S-propranolol. It was later found that analysis of these data by a common equation derived for linear elution conditions gave erroneous values for experiments actually conducted under nonlinear conditions. This report discusses the nature of this error and uses frontal analysis to estimate the true binding strength between R-and S-propranolol and HPLC columns containing immobilized AGP.A recent paper examined the binding of R-and S-propranolol to a new type of α 1 -acid glycoprotein (AGP) column (1). Part of this earlier work used self-competition zonal elution studies for association equilibrium constant measurements, an approach which has since been noted to contain some errors that are not uncommon in the literature but that do need to be corrected to better estimate the true binding strength of R-and S-propranolol with AGP.The column used in Ref.(1) was prepared through the controlled and mild oxidation of AGP, followed by the immobilization of this protein to hydrazide-activated silica. Part of this paper evaluated binding of this AGP column to R-and S-propranolol by using a self-competition zonal elution experiment (i.e., the "perturbation method", "step and pulse method" or "system peak" method) (2-4). In this approach, a small sample of R-or S-propranolol was injected as a pulse onto the AGP column while a known, fixed concentration of the same compound was applied in the mobile phase as a competing agent. The resulting shift in the retention factor (k') for the observed peak was measured as a function of the mobile phase concentration of the analyte [A]. Plots of 1/k' versus [A] appeared to give a linear response for mobile phases containing 0 to 5 µM propranolol.In these experiments (1), association equilibrium constants were obtained by fitting the data to an equation from the literature that pertains to an analyte at infinite dilution, or "linear elution conditions" (see Eq. 1 in Ref. 1 and equivalent equations in Refs. 5 and 6). However, for an analyte at a finite concentration, this same equation can introduce errors when obtaining *Author for correspondence: Phone 402-472-2744; FAX 402-472-9402; Email, dhage@unlserve.unl.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author...
Interactions of the drug carbamazepine with the serum protein α1-acid glycoprotein (AGP) were examined by high-performance affinity chromatography (HPAC). Frontal analysis studies with an immobilized AGP column and control column indicated carbamazepine had both low affinity interactions with the support and high affinity interactions with AGP. When a correction was made for binding to the support, the association equilibrium constant measured at pH 7.4 and 37°C for carbamazepine with AGP was 1.0 (± 0.1) × 105 M−1, with values that ranged from 5.1 to 0.58 × 105 M−1 in going from 5 to 45°C. It was found in competition studies that these interactions were occurring at the same site that binds propranolol on AGP. Temperature studies indicated that the change in enthalpy was the main driving force for the binding of carbamazepine to AGP. These results provide a more complete picture of how carbamazepine binds to AGP in serum. This report also illustrates how HPAC can be used to examine biological interactions and drug-protein binding in situations in which significant interactions for an analyte are present with both the chromatographic support and an immobilized ligand.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.