Capillary electrophoresis frontal analysis: Principles and applications for the study of drug‐plasma protein binding

Østergaard, Jesper; Heegaard, Niels H. H.

doi:10.1002/elps.200305526

Cited by 118 publications

(123 citation statements)

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“…A number of alternative, instrumental methods based on HPLC, affinity capillary electrophoresis and biosensors have been described to provide more rapid and convenient tools for drug-protein binding studies. [10][11][12][13][14][15][16] However, these techniques have been used mainly for the analysis of individual drugs or to study direct drug-drug competition and have previously provided only qualitative information on allosteric effects between drugs.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Studies of Allosteric Effects by Biointeraction Chromatography: Analysis of Protein Binding for Low-Solubility Drugs

Chen

Hage

2006

Anal. Chem.

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A new chromatographic method was developed for characterizing allosteric interactions between an immobilized binding agent and low solubility compounds. This approach was illustrated by using it to characterize the interactions between tamoxifen and warfarin during their binding to the protein human serum albumin (HSA), with β-cyclodextrin being employed as a solubilizing agent for these drugs. It was confirmed in this work through several experiments that warfarin had a single binding site on HSA with an association equilibrium constant of 2-5 × 10 5 M -1 (average, 3.9 × 10 5 M -1 ) at 37°C, in agreement with previous reports. It was also found that tamoxifen had a single major binding site on HSA, with an association equilibrium constant of 3-4 × 10 7 M -1 (average, 3.5 × 10 7 M -1 ) at 37°C. When warfarin was used as a mobile phase additive in competition studies with tamoxifen, this had a positive allosteric effect on tamoxifen/HSA binding, giving a coupling constant of 2.3 (± 0.3). Competitive studies using tamoxifen as a mobile phase additive indicated that tamoxifen had a negative allosteric effect on warfarin/HSA binding, providing a coupling constant of 0.79 (± 0.03). A unique feature of the technique described in this report was its ability to independently examine both directions of the warfarin/tamoxifen allosteric interaction. This approach is not limited to warfarin, tamoxifen and HSA but can also be used to study other solutes and binding agents.

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Section: Introductionmentioning

confidence: 99%

Quantitative Studies of Allosteric Effects by Biointeraction Chromatography: Analysis of Protein Binding for Low-Solubility Drugs

Chen

Hage

2006

Anal. Chem.

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“…Noncovalent interactions between molecules, such as protein-DNA and protein-drug, are of great importance to their proper functions in living systems [13]. Binding constants usually are the appropriate measurement of the interactions.…”

Section: Introductionmentioning

confidence: 99%

“…Methods to determine binding constants include spectroscopy, microdialysis, calorimetry and separation techniques such as CE, etc. [13][14][15][16][17]. All methods have advantages and disadvantages.…”

Section: Introductionmentioning

confidence: 99%

“…The newly-developed CE methods have advantages, such as accuracy and small reagent consumption. Several CE methods are used for binding constant determination, including affinity CE [18], partial filling technique [19,20], frontal analysis (FA) [13], vacancy peak method [21] and Hummel-Dreyer method [22]. Among these FA is relatively simple to perform and produces robust binding constant values.…”

Section: Introductionmentioning

confidence: 99%

“…The principle of FA in CE is based on the separation of ligand from protein and proteinligand complex [13,23]. A larger volume of sample solution than normal is introduced into the capillary.…”

Section: Introductionmentioning

confidence: 99%

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Frontal analysis in microchip CE: A simple and accurate method for determination of protein–DNA dissociation constant

et al. 2007

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Equilibrium constants, such as the dissociation constant (K d ), are a key measurement of noncovalent interactions that are of importance to the proper functioning of molecules in living systems. Frontal analysis (FA) is a simple and accurate capillary electrophoresis (CE) method for the determination of K d . Microchip CE coupled with laser-induced fluorescence (LIF) detection was used to determine K d of protein-DNA interactions using the FA method. A model system of immunoglobulin E (IgE) and the IgE-binding aptamer was selected to demonstrate the capability of FA in microchip CE. Because the fluorescence emission was dependent on the dye migration velocity, the velocity of the free aptamer was adjusted to be the same as that of the aptamer-IgE complex by setting up individual separation voltage configurations for the free and bound aptamers. The ratio of the free and bound aptamers in the equilibrium mixture was directly measured from the heights of their plateaus detected at 1.0 cm from the intersection of the microchip, and no internal standard was needed. The K d of the IgE-aptamer pair was determined as 6 ± 2 nM which is consistent with the reported results (8 nM).

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Affinity Capillary Electrophoresis as a Tool to Characterize Intermolecular Interactions

Kiessig¹,

Stettler²,

Fuhrimann³

et al. 2012

Supramolecular Chemistry

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The application of affinity capillary electrophoresis (ACE), a submode of capillary zone electrophoresis, to investigate the interactions between ligands and their substrates is described in this chapter. Using ACE, it is possible to characterize noncovalent molecular interactions (complexation and partition equilibria) of different binding strengths. Resulting association constants ( K ) provide a measured value of the affinity of a ligand molecule to a substrate. Starting with the background of high‐performance capillary electrophoresis (HPCE), in general, and ACE, various possibilities are given how ACE can be used in capillaries or chips. A brief mathematical description of the context between measured parameters (ionic mobility, peak area, or peak height) and evaluated binding parameters (as binding constants and stoichiometry number), as well as various ACE modes and constellations of intermolecular interacting molecules suitable for a wide range of applications are provided. Finally, selected applications are described in detail in terms of the experimental setup and estimation of the binding strength by a suitable mathematical model.

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Capillary electrophoresis frontal analysis: Principles and applications for the study of drug‐plasma protein binding

Cited by 118 publications

References 76 publications

Quantitative Studies of Allosteric Effects by Biointeraction Chromatography: Analysis of Protein Binding for Low-Solubility Drugs

Quantitative Studies of Allosteric Effects by Biointeraction Chromatography: Analysis of Protein Binding for Low-Solubility Drugs

Frontal analysis in microchip CE: A simple and accurate method for determination of protein–DNA dissociation constant

Affinity Capillary Electrophoresis as a Tool to Characterize Intermolecular Interactions

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