Methods for the determination of binding constants by capillary electrophoresis

Rundlett, Kimber L.; Armstrong, Daniel W.

doi:10.1002/1522-2683(200105)22:7<1419::aid-elps1419>3.0.co;2-v

Cited by 182 publications

(150 citation statements)

References 49 publications

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“…39,40 It is also possible that one solute may have binding regions that do not affect the interaction of the second solute with L. As suggested in Ref. 6, it is possible to correct for these effects by using the following equations in place of eq 9 or 2, respectively, (14) ( 15) where x is the retention factor for A due to its additional secondary interactions. 6,25 Note in the case of eq 14 that a correction is only required in the numerator on the left, since the value of x is constant and will cancel out in the denominator when the difference is taken between k and k 0 .…”

Section: Corrections For Secondary Interactionssupporting

confidence: 70%

“…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%

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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: Corrections For Secondary Interactionssupporting

confidence: 70%

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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“…Weak binding is typically analyzed by ACE or FA [14]. According to recent observations [15,16], CZE can be also applied to study weak binding systems, in contrast to some former reports [11,17]. It was found in CZE that the peak height can also be used to evaluate the concentration of free ligand or receptor as in FA.…”

Section: Five Ce Modesmentioning

confidence: 99%

Recent advances in the study of biomolecular interactions by capillary electrophoresis

Ding

et al. 2004

Electrophoresis

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Recent advances in the study of biomolecular interactions by capillary electrophoresisCapillary electrophoresis (CE) has been abundantly used in the study of molecular interactions owing to such advantages as short analysis time, low sample size requirement, high separation efficiency, and flexible applications. The focus of this paper is to review recent studies and advances (mainly from 1998 to now) in biomolecular interactions using CE. Five CE modes: zone migration CE, affinity CE, frontal analysis (FA), Hummel-Dreyer (HD) and vacancy peak (VP) are cited and compared. Quantitative aspects of the thermodynamics and kinetics of biomolecular interaction are reviewed. Several biomolecular binding systems, including protein-protein (polypeptide), protein-DNA (RNA), protein(polypeptide)-carbohydrate, protein-small molecule, DNAsmall molecule, small molecule-small molecule, have been well characterized by CE. CE is shown to be a powerful tool for the determination of the binding parameters of various bioaffinity interactions.

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“…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%

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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Methods for the determination of binding constants by capillary electrophoresis

Cited by 182 publications

References 49 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

Recent advances in the study of biomolecular interactions by capillary electrophoresis

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

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