Chiral Separation Mechanisms in Protein-Based HPLC Columns. 1. Thermodynamic Studies of (R)- and (S)-Warfarin Binding to Immobilized Human Serum Albumin

Loun, Bounthon; Hage, David S.

doi:10.1021/ac00093a043

Cited by 255 publications

(355 citation statements)

References 27 publications

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“…This agrees with previous studies reporting that both warfarin enantiomers have a single binding region on HSA under the conditions used in this study. 39,40 More detailed information could be obtained from Figure 4(a) by combining these results with previous measurements examining the binding of warfarin with β-cyclodextrin. For instance, the total concentration of β-cyclodextrin in Figure 4(a) was 2.2 mM, giving the value for C S in eq 7.…”

Section: Self-competition Studies With Warfarinmentioning

confidence: 94%

“…This result is similar to previous values of 2.4-4.1 × 10 5 M -1 that have been reported for this interaction in the absence of any solubilizing agent. 40,45,46 By using the reciprocal of the slope, the binding capacity of the immobilized HSA column for warfarin could also be obtained from Figure 4(a). This gave an effective concentration of 33 (± 1) μM for the warfarin binding sites in the column.…”

Section: Self-competition Studies With Warfarinmentioning

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: Self-competition Studies With Warfarinmentioning

confidence: 94%

Section: Self-competition Studies With Warfarinmentioning

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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“…Warfarin (3-(␣-acetonylbenzyl)-4-hydroxycoumarin) binds to drug site I with high affinity (K d Ϸ 3 M) (13,20,21). A crystallographic study in 1992 confirmed subdomain IIA as the binding locus for a single molecule of warfarin but was unable to give specific details on the binding interactions because of the limited resolution of the data (10).…”

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confidence: 99%

Crystal Structure Analysis of Warfarin Binding to Human Serum Albumin

Petitpas¹,

Bhattacharya²,

Twine³

et al. 2001

Journal of Biological Chemistry

768

605

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Human serum albumin (HSA) is an abundant transport protein found in plasma that binds a wide variety of drugs in two primary binding sites (I and II) and can have a significant impact on their pharmacokinetics. We have determined the crystal structures at 2.5 Å-resolution of HSA-myristate complexed with the R-(؉) and S-(؊) enantiomers of warfarin, a widely used anticoagulant that binds to the protein with high affinity. The structures confirm that warfarin binds to drug site I (in subdomain IIA) in the presence of fatty acids and reveal the molecular details of the protein-drug interaction. The two enantiomers of warfarin adopt very similar conformations when bound to the protein and make many of the same specific contacts with amino acid side chains at the binding site, thus accounting for the relative lack of stereospecificity of the HSA-warfarin interaction. The conformation of the warfarin binding pocket is significantly altered upon binding of fatty acids, and this can explain the observed enhancement of warfarin binding to HSA at low levels of fatty acid.

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“…These results could be attributed to the fact that the analytes on molecular level have lower adsorption as temperature increases and therefore they migrate rapidly through the column [20]. According to the Van't Hoff equation [20][21][22][23]:…”

Section: Effect Of Temperaturementioning

confidence: 99%

HPLC determination of enantiomeric thiazolidine-2-carboxylic acid on chiral stationary phase with pre-column derivatization

Zhang

Peng

Zeng

et al. 2013

Acta Chromatographica

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Summary.A new high-performance liquid chromatography (HPLC) method has been developed and validated for determination of enantiomeric purity of thiazolidine-2-carboxylic acid within a short run time of less than 10 min. The method was based on pre-column derivatization of thiazolidine-2-carboxylic acid with aniline, and complete separation of enantiomers has been achieved on a Chiralcel OD-H analytical column (250 × 4.6 mm) using n-hexane-isopropanol (85:15 v/v) as mobile phase at a flow rate of 1.0 mL min −1 under UV and optical rotation (OR) detection. Detection wavelength was set at 254 nm. Then the effects of mobile phase and temperature on enantioselectivity were further evaluated. The method was validated with respect to precision, accuracy, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The recoveries were between 98.5 and 101.3% with percentage relative standard deviation less than 1.16%. The LOD and LOQ for the aniline derivatives of (+)-thiazolidine-2-carboxylic acid were 4.9 and 16.4 μg mL −1 and for the aniline derivatives (−)-thiazolidine-2-carboxylic acid were 5.1 and 17.2 μg mL −1 , respectively.

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Chiral Separation Mechanisms in Protein-Based HPLC Columns. 1. Thermodynamic Studies of (R)- and (S)-Warfarin Binding to Immobilized Human Serum Albumin

Cited by 255 publications

References 27 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

Crystal Structure Analysis of Warfarin Binding to Human Serum Albumin

HPLC determination of enantiomeric thiazolidine-2-carboxylic acid on chiral stationary phase with pre-column derivatization

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