Studies of binding by sulfonylureas with glyoxal- and methylglyoxal-modified albumin by immunoextraction using affinity microcolumns.

Diabetes is characterized by elevated levels of blood glucose, which can result in the modification of serum proteins. The modification of a protein by glucose, or glycation, can also lead to the formation of advanced glycated end-products (AGEs). One protein that can be modified through glycation and AGE formation is human serum albumin (HSA). In this study, immunoextraction based on polyclonal anti-HSA antibodies was used with high-performance affinity microcolumns to see how AGE-related modifications produced by glyoxal (Go) and methylglyoxal (MGo) affected the binding of HSA to several first- and second-generation sulfonylureas, a class of drugs used to treat type II diabetes and known to bind to HSA. With this approach, it was possible to use a single platform to examine drug interactions with several preparations of HSA. Each applied protein sample could be used over 20-50 experiments, and global affinity constants for most of the examined drugs could be obtained in less than 7.5 min. The binding constants measured for these drugs with normal HSA gave good agreement with global affinities based on the literature. Both Go- and MGo-related modifications at clinically relevant levels were found by this method to create significant changes in the binding by some sulfonylureas with HSA. The global affinities for many of the drugs increased by 1.4-fold or more; gliclazide and tolazamide had no significant change with some preparations of modified HSA, and a small-to-moderate decrease in binding strength was noted for glibenclamide and gliclazide with Go-modified HSA. This approach can be adapted for the study of other drug-protein interactions and alternative modified proteins by altering the antibodies that are employed for immunoextraction and within the affinity microcolumn.

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.