David S. Hage

High-performance affinity chromatography: a powerful tool for studying serum protein binding

High-performance affinity chromatography (HPAC) is a method in which a biologically-related ligand is used as a stationary phase in an HPLC system. This approach is a powerful means for selectively isolating or quantitating agents in complex samples, but it can also be employed to study the interactions of biological systems. In recent years there have been numerous reports in which HPAC has been used to examine the interactions of drugs, hormones and other substances with serum proteins. This review discusses how HPAC has been used in such work. Particular attention is given to the techniques of zonal elution and frontal analysis. Various applications are provided for these techniques, along with a list of factors that need to be considered in their optimization and use. New approaches based on band-broadening studies and rapid immunoextraction are also discussed.

Recent advances in chromatographic and electrophoretic methods for the study of drug-protein interactions

Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines various chromatographic and electrophoretic methods that have been developed to study such interactions. An overview of each technique is presented along with a discussion of its strengths, weaknesses and potential applications. Formats that are discussed include the use of both soluble and immobilized drugs or proteins, and approaches based on zonal elution, frontal analysis or vacancy peak measurements. Furthermore, examples are provided that illustrate the use of these methods in determining the overall extent of drug-protein binding, in examining the displacement of a drug by other agents and in measuring the equilibrium or rate constants for drug-protein interactions. Examples are also given demonstrating how the same methods, particularly when used in high-performance liquid chromatography or capillary electrophoresis systems, can be employed as rapid screening tools for investigating the binding of different forms of a chiral drug to a protein or the binding of different proteins and peptides to a given pharmaceutical agent.

Review: Glycation of human serum albumin.

Glycation involves the non-enzymatic addition of reducing sugars and/or their reactive degradation products to amine groups on proteins. This process is promoted by the presence of elevated blood glucose concentrations in diabetes and occurs with various proteins that include human serum albumin (HSA). This review examines work that has been conducted in the study and analysis of glycated HSA. The general structure and properties of HSA are discussed, along with the reactions that can lead to modification of this protein during glycation. The use of glycated HSA as a short-to-intermediate term marker for glycemic control in diabetes is examined, and approaches that have been utilized for measuring glycated HSA are summarized. Structural studies of glycated HSA are reviewed, as acquired for both in vivo and in vitro glycated HSA, along with data that have been obtained on the rate and thermodynamics of HSA glycation. In addition, this review considers various studies that have investigated the effects of glycation on the binding of HSA with drugs, fatty acids and other solutes and the potential clinical significance of these effects.

Survey of recent advances in analytical applications of immunoaffinity chromatography

Methods that use immunoaffinity chromatography (IAC) for sample preparation or detection are becoming increasingly popular as tools in the analysis of biological and nonbiological compounds. This paper presents an overview of immunoaffinity chromatography and examines some recent developments of this technique in analytical applications. The emphasis is placed on HPLC-based IAC methods or those that combine IAC with other instrumental techniques; however, novel approaches that employ low-performance IAC columns for chemical quantitation are also considered. Particular applications that are examined include (1) the use of IAC in the direct detection of analytes, (2) the extraction of samples by IAC prior to on- or off-line detection by other methods, (3) the use of IAC in chromatographic-based immunoassays, and (4) the development of postcolumn reactors based on IAC for the detection of analytes as they elute from other types of chromatographic columns. The advantages and limitations for each approach are considered. In addition, a summary is provided of reports in the literature that have used IAC for these various formats.

Characterization of the protein binding of chiral drugs by high-performance affinity chromatography interactions of R- and S-ibuprofen with human serum albumin

Zonal elution and high-performance affinity chromatography were used to study the different binding characteristics of R- and S-ibuprofen with the protein human serum albumin (HSA). This was done by injecting small amounts of R- and S-ibuprofen onto an immobilized HSA column in the presence of a mobile phase that contained a known concentration of R- or S-ibuprofen as a competing agent. These studies indicated that R- and S-ibuprofen had one common binding site on the immobilized HSA column. In addition, S-ibuprofen had at least one other major binding region. The association equilibrium constant for R-ibuprofen with HSA was found to be 5.3 x 10(5) M-1 at pH 6.9 and 25 degrees C. Under the same conditions, the association constants for S-ibuprofen at its two sites were 1.1 x 10(5) M-1 and 1.2 x 10(5) M-1. The S-ibuprofen sites were present in about a 1:1 ratio and appeared to exhibit some allosteric interactions at high S-ibuprofen concentrations. The chromatographic technique used in this work is a general one which can be adapted for use in studying the interactions of other chiral compounds with either HSA or additional proteins.

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase.

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin-azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.

Characterization of the binding and chiral separation of d- and l-tryptophan on a high-performance immobilized human serum albumin column

High-performance affinity chromatography was used to study the separation and binding of D- and L-tryptophan on an immobilized human serum albumin (HSA) column. Frontal analysis and zonal elution studies indicated that both D- and L-tryptophan were binding to single but distinct sites on HSA. L-Tryptophan bound to the indole site of HSA. D-Tryptophan had indirect interactions with the warfarin site of HSA but no interactions with the indole site. The association constants for the binding of D- and L-tryptophan at pH 7.4 and 25 degrees C were 0.4 x 10(4) and 2.7 x 10(4) M-1, respectively. The value of delta G for these sites ranged from -5.2 to -5.7 kcal/mol (1 cal = 4.184 J) and had a significant entropy component. The effects of varying the pH, phosphate concentration, temperature and polarity of the mobile phase on the binding of D- and L-tryptophan to HSA were examined. The role of sample size in determining peak shape and retention was also considered. From these data, general guidelines were developed for the separation of D- and L-tryptophan on immobilized HSA. Under optimized conditions the enantiomers were separated in less than 2 min with baseline resolution.

Immunoaffinity chromatography: an introduction to applications and recent developments

Immunoaffinity chromatography (IAC) combines the use of LC with the specific binding of antibodies or related agents. The resulting method can be used in assays for a particular target or for purification and concentration of analytes prior to further examination by another technique. This review discusses the history and principles of IAC and the various formats that can be used with this method. An overview is given of the general properties of antibodies and of antibody-production methods. The supports and immobilization methods used with antibodies in IAC and the selection of application and elution conditions for IAC are also discussed. Several applications of IAC are considered, including its use in purification, immunodepletion, direct sample analysis, chromatographic immunoassays and combined analysis methods. Recent developments include the use of IAC with CE or MS, ultrafast immunoextraction methods and the use of immunoaffinity columns in microanalytical systems.

Pharmaceutical and biomedical applications of affinity chromatography: Recent trends and developments

Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered.

Characterization of thyroxine—albumin binding using high-performance affinity chromatography: I. Interactions at the warfarin and indole sites of albumin

A high-performance affinity column containing immobilized human serum albumin (HSA) was used to study the binding of thyroxine at the warfarin and indole sites of HSA. Frontal analysis, using R-warfarin and L-tryptophan as probes for these sites, demonstrated that the immobilized HSA had binding behavior equivalent to that observed for HSA in solution. By injecting R-warfarin or L-tryptophan in the presence of excess thyroxine, it was found that thyroxine was binding directly to both types of site. The warfarin and indole sites had relatively strong binding for thyroxine, with association constants at 37 degrees C of 1.4 x 10(5) and 5.7 x 10(5) M-1, respectively. The value of delta G for these sites ranged from -7 to -8 kcal/mol and had a significant entropy component. The techniques used in this study are not limited to thyroxine-HSA interactions, but should also be valuable in examining the site-specific binding of other drugs and hormones to HSA.