Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography.

Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 µL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research.

Development of enhanced capacity affinity microcolumns by using a hybrid of protein cross-linking/modification and immobilization.

A hybrid method was examined for increasing the binding capacity and activity of protein-based affinity columns by using a combination of protein cross-linking/modification and covalent immobilization. Various applications of this approach in the study of drug-protein interactions and in use with affinity microcolumns were considered. Human serum albumin (HSA) was utilized as a model protein for this work. Bismaleimidohexane (BMH, a homobifunctional maleimide) was used to modify and/or cross-link HSA through the single free sulfhydryl group that is present on this protein. Up to a 75-113% increase in protein content was obtained when comparing affinity supports that were prepared with BMH versus reference supports that were made by using only covalent immobilization. Several drugs that are known to bind HSA (e.g., warfarin, verapamil and carbamazepine) were further found to have a significant increase in retention on HSA microcolumns that were treated with BMH (i.e., a 70-100% increase in protein-based retention). These BMH-treated HSA microcolumns were used in chiral separations and in ultrafast affinity extraction to measure free drug fractions in drug/protein mixtures, with the latter method giving association equilibrium constants that had good agreement with literature values. In addition, it was found that the reversible binding of HSA with ethacrynic acid, an agent that can combine irreversibly with the free sulfhydryl group on this protein, could be examined by using the BMH-treated HSA microcolumns. The same hybrid immobilization method could be extended to other proteins or alternative applications that may require protein-based affinity columns with enhanced binding capacities and activities.

Analytical methods for kinetic studies of biological interactions: A review.

The rates at which biological interactions occur can provide important information concerning the mechanism and behavior of these processes in living systems. This review discusses several analytical methods that can be used to examine the kinetics of biological interactions. These techniques include common or traditional methods such as stopped-flow analysis and surface plasmon resonance spectroscopy, as well as alternative methods based on affinity chromatography and capillary electrophoresis. The general principles and theory behind these approaches are examined, and it is shown how each technique can be utilized to provide information on the kinetics of biological interactions. Examples of applications are also given for each method. In addition, a discussion is provided on the relative advantages or potential limitations of each technique regarding its use in kinetic studies.

Determination of rate constants and equilibrium constants for solution-phase drug-protein interactions by ultrafast affinity extraction.

A method was created on the basis of ultrafast affinity extraction to determine both the dissociation rate constants and equilibrium constants for drug-protein interactions in solution. Human serum albumin (HSA), an important binding agent for many drugs in blood, was used as both a model soluble protein and as an immobilized binding agent in affinity microcolumns for the analysis of free drug fractions. Several drugs were examined that are known to bind to HSA. Various conditions to optimize in the use of ultrafast affinity extraction for equilibrium and kinetic studies were considered, and several approaches for these measurements were examined. The dissociation rate constants obtained for soluble HSA with each drug gave good agreement with previous rate constants reported for the same drugs or other solutes with comparable affinities for HSA. The equilibrium constants that were determined also showed good agreement with the literature. The results demonstrated that ultrafast affinity extraction could be used as a rapid approach to provide information on both the kinetics and thermodynamics of a drug-protein interaction in solution. This approach could be extended to other systems and should be valuable for high-throughput drug screening or biointeraction studies.

Analysis of biomolecular interactions using affinity microcolumns: a review.

Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods.