Application of affinity capillary electrophoresis for the determination of binding and thermodynamic constants of enediynes with bovine serum albumin

El-Shafey, Ahmed; Zhong, Huijuan; Jones, Graham B.; Krull, Ira S.

doi:10.1002/1522-2683(200203)23:6<945::aid-elps945>3.0.co;2-f

Cited by 22 publications

(10 citation statements)

References 20 publications

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“…Pre-equilibrium mixture analysis [12,[15][16][17][18]: The complex is formed by pre-incubating the analyte and the binding probe, and then the resulting equilibrium mixture is analyzed under non-equilibrium separation conditions based on electrophoresis. Mobility-shift analysis [19][20][21][22]: In this approach, the separation buffer contains the binding ligand while the sample contains the analyte; following injection, the analyte-ligand complex continuously undergoes dynamic dissociation/association cycles during CE separation; the migration time of the analyte is shifting with the concentrations of the ligand in the separation buffer. Pre-equilibriummixture analysis is preferred for relatively strong binding systems in which the complex has a slow-off rate and can be detected before it dissociates to a significant extent.…”

Section: Introductionmentioning

confidence: 99%

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

et al. 2008

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The use of traditional capillary electrophoresis (CE) to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombinbinding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with a limit of detection for thrombin of 5 nM at a 3-fold signal-to-noise ratio.

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Section: Introductionmentioning

confidence: 99%

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

et al. 2008

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“…Important advantages are small sample size, relatively short analysis time, ease of automation, no errors due to protein leakage through membrane, no membrane adsorption of the drug, and no osmotic or Donnan effects [5]. Several modes of CE have been utilized for the quantitative assessment of serum albumin-drug interactions in terms of degree of binding or binding constants: affinity capillary electrophoresis (ACE) [7][8][9][10][11][12][13][14][15], Hummel-Dreyer (HD) [15][16][17][18], vacancy affinity capillary electrophoresis (VACE) [15,19], vacancy peak (VP) [15,16], dye displacement technique [20,21], frontal analysis continuous capillary electrophoresis (FACCE) [22], and frontal analysis (FA) [15,16,[23][24][25][26][27][28][29][30][31].…”

Section: Introductionmentioning

confidence: 99%

Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions

et al. 2002

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Capillary electrophoresis frontal analysis was applied to 12 low molecular weight compounds including 8 drug substances displaying a range of different properties with respect to binding affinity, binding location, structure, lipophilicity, charge at physiological pH, and electrophoretic mobility. It was found that capillary electrophoresis frontal analysis can be used as a general method to study and quantify drug-human serum albumin interactions. The binding parameters obtained were consistent with literature values. Dextran was in some cases added to the run buffer to improve separation of the drug and human serum albumin plateau peaks. Results indicate that mobility differences between free and complexed human serum albumin give rise to only minor errors. Capillary electrophoresis frontal analysis was also found applicable to the study of human serum albumin drug displacement reactions. Low sensitivity of the UV-detection system was found to be the major limitation of capillary electrophoresis frontal analysis. The method is simple, and minimal effort has to be put into method development, which makes it well suited for screening in early drug development.

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“…Same to the other metal ions or metal compounds, organotin compounds could achieve equilibrium within the ACE separation time [22,23]. Usually, the interaction between drugs and biomolecules could be considered as 1:1 molar ratio in CE analysis [24][25][26]. Comparing to the organotin compound concentration, the maximum protein concentration is not high.…”

Section: Resultsmentioning

confidence: 99%

Characterization of interactions between organotin compounds and human serum albumin by capillary electrophoresis coupled with inductively coupled plasma mass spectrometry

Sun

Liu

et al. 2012

Talanta

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Inductively coupled plasma mass spectrometry Human serum albumin Affinity capillary electrophoresis Nonequilibrium capillary electrophoresis assays of equilibrium mixtures assays a b s t r a c t Thermodynamic data such as the binding constants are vital parameters describing interactions between exotic trace compounds and biomolecules in biochemical property modeling. In this study, the stability constants of organometallic compound and protein complexes were studied by using capillary electrophoresis coupled with inductively coupled plasma mass spectrometry (CE-ICP-MS), considering its low detection limits and low sample demand. Four organotin compounds (trimethyltin (TMT), tripropyltin (TPrT), tributyltin (TBT), triphenyltin (TPhT)) and human serum albumin (HSA) were used as model organometallic compounds and protein, respectively. Affinity capillary electrophoresis (ACE) and nonequilibrium capillary electrophoresis assays of equilibrium mixtures (NECEEM) were performed and compared by using ICP-MS as the detector to determine the binding constants of organotin compounds and HSA in 1:1 molar ratio assumption. Constant measurements of the two methods were both simple, however, ACE assays were more accurate and more appropriate for the constant determination of the organotin-HSA complexes, considering the errors of the NECEEM method. A good precision of the binding constants (log K b ) using the ACE method was proved by different mathematical calculations, and the values were 6.13 ± 0.51 (TMT), 5.72 ± 0.38 (TPrT), 5.68 ± 0.34 (TBT), 6.05 ± 0.38 (TPhT) respectively for each of the organotin-HSA complexes, showing non-covalent interaction between organotin compounds and HSA. Meanwhile, this study also confirms the suitability of CE-ICP-MS method for further studies on organometallic complexation.

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Application of affinity capillary electrophoresis for the determination of binding and thermodynamic constants of enediynes with bovine serum albumin

Cited by 22 publications

References 20 publications

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions

Characterization of interactions between organotin compounds and human serum albumin by capillary electrophoresis coupled with inductively coupled plasma mass spectrometry

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