Characterization of binding by sulfonylureas with normal or modified human serum albumin using affinity microcolumns prepared by entrapment.

Modification of proteins can occur during diabetes due to the formation of advanced glycation end-products (AGEs) with reactive dicarbonyls such as glyoxal (Go) and methylglyoxal (MGo). Human serum albumin (HSA) is a serum protein that binds to many drugs in blood and that is known to be modified by Go and MGo. This study examined the binding of various sulfonylurea drugs with these modified forms of HSA by using high-performance affinity microcolumns prepared by non-covalent protein entrapment. Zonal elution experiments were employed to compare the retention and overall binding constants for the drugs with Go- or MGo-modified HSA vs normal HSA. The results were compared to values from the literature, such as measured or estimated using affinity columns containing covalently immobilized HSA or biospecifically-adsorbed HSA. The entrapment-based approach provided estimates of global affinity constants within 3-5 min for most of the tested drugs and with typical precisions of ±10-23%. Each entrapped protein microcolumn was stable for over at least 60-70 injections and one month of use. The results obtained with normal HSA agreed at the 95% confidence level with global affinity constants that have been reported for the given drugs in the literature. It was found for HSA that had been modified with clinically-relevant levels of either Go or MGo that an increase in the global affinity constant of up to 2.1-fold occurred for some of the tested drugs. The information acquired in this study can be used in the future to adapt this entrapment-based approach to study and evaluate interactions between other types of drugs and normal or modified binding agents for clinical testing and biomedical research.

Preparation of entrapment-based microcolumns for analysis of drug-humic acid interactions by high-performance affinity chromatography.

Reversible interactions between drugs and humic acid in water can be an important factor in determining the bioavailability and effects of these pharmaceuticals as micropollutants in the environment. In this study, microcolumns containing entrapped humic acid were used in high-performance affinity chromatography (HPAC) to examine the binding of this agent with the drugs tetracycline, carbamazepine, ciprofloxacin, and norfloxacin. Parameters that were varied to optimize the entrapment of humic acid within HPLC-grade porous silica included the starting concentration of humic acid, the mass ratio of humic acid vs silica, and the method of mixing the reagents with the support for the entrapment process. The highest retention for the tested drugs was obtained when using supports that were prepared using an initial humic acid concentration of 80 mg mL-1 and a humic acid vs silica mass ratio of 600 mg per g silica, along with preincubation of the humic acid with hydrazide-activated silica before the addition of a capping agent (i.e., oxidized glycogen). Characterization of the humic acid support was also carried out by means of TGA, FTIR, SEM, and energy-dispersive X-ray spectroscopy. The binding constants measured by HPAC for the given drugs with entrapped Aldrich humic acid gave good agreement with values reported in the literature under similar pH and temperature conditions for this and other forms of humic acid. Besides providing valuable data on the binding strength of various drugs with humic acid, this work illustrates how HPAC may be used as an analytical tool for screening and characterizing the interactions of drugs and man-made contaminants with humic acid or related binding agents in water and the environment.

Analysis of the binding of warfarin to glyoxal- and methylglyoxal-modified human serum albumin by ultrafast affinity extraction.

Ultrafast affinity extraction (UAE) and affinity microcolumns containing immobilized human serum albumin (HSA) were employed to evaluate the effect of advanced stage glycation on HSA and its binding to warfarin, a common site-specific probe for Sudlow site I of this protein. The modification of HSA by glyoxal (GO) and methylglyoxal (MGO) was considered, where GO and MGO are known to be important in the formation of many types of advanced glycation end products. Free drug fractions were measured by UAE for warfarin in solutions containing normal HSA or HSA that had been modified by GO or MGO at levels seen in serum during diabetes. The free fractions measured with the GO-modified HSA gave association equilibrium constants that ranged from 2.42-2.63 × 105 M-1 at pH 7.4 and 37 °C. These values were not significantly different from a value of 2.33 (±0.15) × 105 M-1 that was determined by the same method for warfarin with normal HSA. Similar studies using MGO-modified HSA gave association equilibrium constants for warfarin in the range of 3.07-3.31 × 105 M-1, which were 1.32- to 1.42-fold higher than the value seen for normal HSA (differences that were significant at the 95% confidence level). These results will be valuable in future binding studies based on affinity chromatography or other methods that employ warfarin as a probe to examine drug interactions at Sudlow site I of HSA and modified forms of this protein. This work also illustrates how UAE can be used, with analysis times of only minutes, to detect and measure small changes in the binding by drugs with unmodified or modified forms of a soluble binding agent or protein.

Entrapment of Proteins Within Columns for High-Performance Affinity Chromatography.

Entrapment is a noncovalent immobilization method that enables a large biological binding agent, such as a protein, to be put within a support without modifying the structure of the binding agent. This chapter describes an on-column entrapment method that can be used with proteins and HPLC-grade silica to prepare columns for high-performance liquid chromatography. In this method, a protein is trapped within a dihydrazide-activated silica support by using oxidized glycogen as a capping agent. This method allows the protein to be placed within the support in a soluble form and with little or no loss of activity. The approach and reagents needed for this method are described in this chapter, along with some applications reported for columns that have been made using on-column protein entrapment.

Affinity monolith chromatography: A review of general principles and recent developments.

Affinity monolith chromatography (AMC) is a liquid chromatographic technique that utilizes a monolithic support with a biological ligand or related binding agent to isolate, enrich, or detect a target analyte in a complex matrix. The target-specific interaction exhibited by the binding agents makes AMC attractive for the separation or detection of a wide range of compounds. This article will review the basic principles of AMC and recent developments in this field. The supports used in AMC will be discussed, including organic, inorganic, hybrid, carbohydrate, and cryogel monoliths. Schemes for attaching binding agents to these monoliths will be examined as well, such as covalent immobilization, biospecific adsorption, entrapment, molecular imprinting, and coordination methods. An overview will then be given of binding agents that have recently been used in AMC, along with their applications. These applications will include bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, and dye-ligand or biomimetic affinity chromatography. The use of AMC in chiral separations and biointeraction studies will also be discussed.

Affinity chromatography: A review of trends and developments over the past 50 years.

The field of affinity chromatography, which employs a biologically-related agent as the stationary phase, has seen significant growth since the modern era of this method began in 1968. This review examines the major developments and trends that have occurred in this technique over the past five decades. The basic principles and history of this area are first discussed. This is followed by an overview of the various supports, immobilization strategies, and types of binding agents that have been used in this field. The general types of applications and fields of use that have appeared for affinity chromatography are also considered. A survey of the literature is used to identify major trends in these topics and important areas of use for affinity chromatography in the separation, analysis, or characterization of chemicals and biochemicals.

Development of an on-line immunoextraction/entrapment system for protein capture and use in drug binding studies by high-performance affinity chromatography.

An on-line purification and entrapment system was developed that could extract a protein from a sample such as serum and entrap this protein within a small column for use in high-performance affinity chromatography. Human serum albumin (HSA) was employed as a model protein for this work. Immunoextraction columns containing polyclonal anti-HSA antibodies were developed to capture and isolate HSA from applied samples. This was followed by the use of a strong cation-exchange column to recapture and focus HSA as it eluted from the immunoextraction columns. The recaptured HSA was entrapped within 1.0 cm × 2.1 mm I.D. columns containing hydrazide-activated silica and in the presence of oxidized glycogen as a capping agent. The binding and elution properties of HSA on the various components of this system were examined and optimized. The entrapped columns produced by this system were then evaluated for their use in binding studies with several sulfonylurea drugs. The HSA columns created by this approach typically contained 0.3-0.6 nmol HSA and were stable over several weeks and more than 50-60 sample injections. Drug binding constants could be determined with these columns in 8 min or less by zonal elution and gave good agreement with literature values. The same system could be used for the capture and entrapment of other proteins by utilizing antibodies against the given target for immunoextraction.

Optimization of protein entrapment in affinity microcolumns using hydrazide-activated silica and glycogen as a capping agent.

Several approaches were compared for the entrapment of proteins within hydrazide-activated silica for use in affinity microcolumns and high performance affinity chromatography. Human serum albumin (HSA) and concanavalin A (Con A) were used as model proteins for this work. Items considered in this study included the role played by the solution volume, amount of added protein, and use of slurry vs. on-column entrapment on the levels of solute retention and extent of protein immobilization that could be obtained by means of entrapment. The levels of retention and protein immobilization were evaluated by injecting warfarin or 4-methylumbellipheryl α-D-mannopyranoside as solutes with known binding properties for HSA or Con A. Altering both the solution volume and amount of added protein led to an increase of up to 17-fold in the extent of protein immobilization for HSA in slurry-based entrapment; on-column entrapment provided an additional 3.6-fold increase in protein content vs. the optimized slurry method. Similar general trends were seen for Con A. The protein contents obtained by entrapment for HSA or Con A (i.e., up to ~87 and 46 mg/g silica, respectively) were comparable to or higher than levels reported for the covalent immobilization of these proteins onto silica. The retention of warfarin on the entrapped HSA was at least 1.7-fold higher than has been obtained under comparable support and mobile phase conditions when using covalent immobilization. These results indicated that entrapment can be an attractive alternative to covalent immobilization for proteins such as HSA and Con A, with this approach serving as a potential means for obtaining good solute binding and retention in work with affinity microcolumns or related microscale devices.

Analysis of solute-protein interactions and solute-solute competition by zonal elution affinity chromatography.

Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets.