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Ultrafast affinity extraction was used to study hormone-protein interactions in solution, using testosterone and its transport proteins human serum albumin (HSA) and sex hormone binding globulin (SHBG) as models. Both single column and two-dimensional systems based on HSA microcolumns were utilized to measure the free fraction of testosterone in hormone/protein mixtures at equilibrium or that were allowed to dissociate for various lengths of time. These data were used to determine the association equilibrium constants (Ka) or global affinities (nKa') and dissociation rate constants (kd) for testosterone with soluble HSA and SHBG. This method was also used to measure simultaneously the free fraction of testosterone and its equilibrium constants with both these proteins in physiological mixtures of these agents. The kd and Ka values obtained for HSA were 2.1-2.2 s(-1) and 3.2-3.5 × 10(4) M(-1) at pH 7.4 and 37 °C. The corresponding constants for SHBG were 0.053-0.058 s(-1) and 0.7-1.2 × 10(9) M(-1). All of these results gave good agreement with literature values, indicating that this approach could provide information on a wide range of rate constants and binding strengths for hormone-protein interactions in solution and at clinically relevant concentrations. The same method could be extended to alternative hormone-protein systems or other solutes and binding agents.

High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2-11.8×10(5)M(-1) at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9-16×10(3)M(-1)). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In diabetes, the elevated levels of glucose in the bloodstream can result in the nonenzymatic glycation of proteins such as human serum albumin (HSA). This type of modification has been shown to affect the interactions of some drugs with HSA, including several sulfonylurea drugs that are used to treat type II diabetes. This study used high-performance affinity chromatography (HPAC) to examine the interactions of glipizide (i.e., a second-generation sulfonylurea drug) with normal HSA or HSA that contained various levels of in vitro glycation. Frontal analysis indicated that glipizide was interacting with both normal and glycated HSA through two general groups of sites: a set of relatively strong interactions and a set of weaker interactions with average association equilibrium constants at pH 7.4 and 37 °C in the range of 2.4-6.0 × 10(5) and 1.7-3.7 × 10(4) M(-1), respectively. Zonal elution competition studies revealed that glipizide was interacting at both Sudlow sites I and II, which were estimated to have affinities of 3.2-3.9 × 10(5) and 1.1-1.4 × 10(4) M(-1). Allosteric effects were also noted to occur for this drug between the tamoxifen site and the binding of R-warfarin at Sudlow site I. Up to an 18% decrease in the affinity for glipizide was observed at Sudlow site I ongoing from normal HSA to glycated HSA, while up to a 27% increase was noted at Sudlow site II. This information should be useful in indicating how HPAC can be used to investigate other drugs that have complex interactions with proteins. These results should also be valuable in providing a better understanding of how glycation may affect drug-protein interactions and the serum transport of drugs such as glipizide during diabetes.

Ultrafast affinity extraction and a multi-dimensional affinity system were developed for measuring free drug fractions at therapeutic levels. This approach was used to compare the free fractions and global affinity constants of several sulfonylurea drugs in the presence of normal human serum albumin (HSA) or glycated forms of this protein, as are produced during diabetes. Affinity microcolumns containing immobilized HSA were first used to extract the free drug fractions in injected drug/protein mixtures. As the retained drug eluted from the HSA microcolumn, it was passed through a second HSA column for further separation and measurement. Items that were considered during the optimization of this approach included the column sizes and flow rates that were used, and the time at which the second column was placed on-line with the HSA microcolumn. This method required only 1.0 μL of a sample per injection and was able to measure free drug fractions as small as 0.09-2.58% with an absolute precision of ±0.02-0.5%. The results that were obtained indicated that glycation can affect the free fractions of sulfonylurea drugs at typical therapeutic levels and that the size of this effect varies with the level of HSA glycation. Global affinity constants that were estimated from these free drug fractions gave good agreement with those predicted from previous binding studies or determined through a reference method. The same approach could be utilized with other drugs and proteins or modified binding agents of clinical or pharmaceutical interest., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha 1 -acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered. [ABSTRACT FROM AUTHOR]

In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha 1 -acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110–830 ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10–20 min when using only 3–10 μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The authors would like to call the reader's attention to the following corrections in this article. In the description given for the process of preparing glycated human serum albumin under "In vitro glycation of HSA", the concentrations of D-glucose that were employed were 15 mM and 30 mM. [ABSTRACT FROM AUTHOR]

One-site immunometric assays that utilize affinity microcolumns were developed and evaluated for the analysis of protein biomarkers. This approach used labeled antibodies that were monitored through on-line fluorescence or near-infrared (NIR) fluorescence detection. Human serum albumin (HSA) was utilized as a model target protein for this approach. In these assays, a fixed amount of labeled anti-HSA antibodies was mixed with samples or standards containing HSA, followed by the injection of this mixture onto an HSA microcolumn to remove excess antibodies and detect the non-retained labeled antibodies that were bound to HSA from the sample. The affinity microcolumns were 2.1 mm i.d. × 5 mm and contained 8–9 nmol of immobilized HSA. These microcolumns were used from 0.10 to 1.0 mL/min and gave results within 35 s to 2.8 min of sample injection. Limits of detection down to 0.10–0.28 ng/mL (1.5–4.2 pM) or 25–30 pg/mL (0.38–0.45 pM) were achieved when using fluorescein or a NIR fluorescent dye as the label, with an assay precision of ±0.1–4.2%. Several parameters were examined during the optimization of these assays, and general guidelines and procedures were developed for the extension of this approach for use with other types of affinity microcolumns and protein biomarkers. [ABSTRACT FROM AUTHOR]