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.

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.

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.

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.

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.

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.

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.

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.

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.