Affinity techniques capillary electrophoresis

A wide variety of techniques have been applied to measure drug-macromolecule binding constants and to characterize drug interactions at the binding site, namely, biochemical binding assays [20], high-performance affinity chromatography, capillary electrophoresis, and fluorescence spectroscopy [21]. 

Affinity capillary electrophoresis (ACE), reviewed by Shimura and Kasai,42 is a method for studying receptor-ligand binding in free solution using CE. The technique depends upon a shift in the electrophoretic mobility of the receptor upon complexation with a charged ligand. Pure receptor preparations or accurate concentration values are not required because only migration times are measured. 

Tanaka, Y., and Terabe, S. (1997). Separation of the enantiomers of basic drugs by affinity capillary electrophoresis using a partial filling technique and ai-acid glycoprotein as chiral selector. 

Capillary zone electrophoresis (CZE) is the most common electrophoretic separation technique due to its simplicity of operation and its flexibility. It is the standard mode for drug analysis, identification of impurities, and pharmacokinetic studies. Other separation modes, such as capillary isotachopho-resis (CITP), micellar electrokinetc chromatography (MEKC), capillary electrochromatography (CEC), capillary gel electrophoresis (CGE), capillary isoelectric focusing, and affinity capillary electrophoresis (ACE), have then-advantages in solving specific separation problems, since the separation mechanism of each mode is different. 

Capillary electrophoresis offers a set of important advantages that make it a premier technique for the investigation of enantioselective effects in the affinity interactions between chiral drugs and cyclodextrins. The most important advantage of CE is the inherently high separation efficiency offered by this technique. As already known, the most important contributors to peak resolution (R) are a separation selectivity (a) and an efficiency (N). A relationship between these parameters in CE is described by the following equation (2) ... 

A Amini, D Westerlund. Evaluation of association constants between drug enantiomers and human aracid glycopotein by applying a partial-filling technique in affinity capillary electrophoresis. Anal Chem 70 1425-1430, 1998. 

NHH Heegaard, HD Mortensen, P Roepstorff. Demonstration of a heparinbinding site in serum amyloid P component using affinity capillary electrophoresis as an adjunct technique. J Chromatogr A 717 83—90, 1995. 

J Heintz, M Hernandez, FA Gomez. Use of a partial-filling technique in affinity capillary electrophoresis for determining binding constants of ligands to receptors. J Chromatogr A 840 261-268, 1999. 

Among the electrophoretic methods of chiral resolution, various forms of capillary electrophoresis such as capillary zone electrophoresis (CZE), capillary isotachophoresis (CIF), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), affinity capillary electrophoresis (ACE), and separation on microchips have been used. However, in contrast to others, the CZE model has been used frequently for this purpose [44]. On the other hand, drawbacks associated with the electrophoretic technique due to lack of development of modem chiral phases have limited the application of these methods. Moreover, the electrophoretic techniques cannot be used at the preparative scale, which represents an urgent need of chiral separation science. 

Among others, Wieboldt et al. [57] used a similar technique, immunoaffi-nity ultrafiltration, in conjunction with ion spray HPLC/MS, on a small benzodiazepine library tested for binding to a polyclonal sheep IgG antibody. Dunayevskiy et al. [58] used gel filtration together with CE/MS and LC/MS on a small library of drugs tested for binding to human serum albumin, Chu and coworkers et al. [59-61] reported an application of affinity capillary electrophoresis (ACE)-MS to some tri- and tetrpeptide libraries (>1000 compounds)... 

Capillary affinity electrophoresis (CAE) or affinity capillary electrophoresis (ACE) — An electrophoretic separation technique (- electrophoresis), in which -> analytes are separated in a capillary, with the -> supporting (background) electrolyte containing substances capable of specific, often biospecific, interactions with the analytes. Ref [i] Riekkola ML, Jonsson jA, Smith RM (2004) Pure Appl Chem 76 443... 

Although affinity capillary electrophoresis (ACE) in its classical mode (one of the reagent is dissolved in a BGE, another is injected) is the most widely used technique in the literature, other capillary electrophoretic methods exist which are even more favorable concerning the information about binding parameters obtainable the Hummel-Dreyer (HD) method, frontal analysis (FA), the vacancy peak (VP) method, and vacancy affinity capillary electrophoresis (VACE) (see, e.g., Refs. 49-57). All the methods need as a precondition that the equilibrium between the reactants (say, protein P, drug D, and complex formed PD) is established rapidly compared to the dislocation of the electropho-retically migrating zones. The experimental setup of the HD and the ACE methods is identical, and so is the setup for the VP and the VACE methods. FA differs from all the other techniques. 

The next four chapters discuss the basic principles underlying operation and method development of the most common electrodriven analytical techniques CE, capillary isoelectric focusing (cIEF), capillary gel electrophoresis (CGE), and affinity capillary electrophoresis (ACE). Weinberger presents a comprehensive approach for method development in CE with an emphasis on small-molecule applications. This is followed by Kilar s chapter describing the principles of and method development in cIEF, as well as recent innovations... 

The majority of reports have used electrospray ionization mass spectroscopy (ESI-MS) as an analytical detection method because of its sensitivity and the soft namre of its ionization procedure, which generally only leads to the detection of the molecular ions of the positive library members. Many separation techniques have been coupled to ESI-MS, including affinity chromatography (49), size exclusion chromatography (50, 51), gel filtration (52), affinity capillary electrophoresis (53-58), capillary isoelectric focusing (59), immunoaffinity ultrafiltration (60), and immunoaffinity extraction (61). ESI-MS has also been used alone (62) to screen a small carbohydrate library. Other examples reported alternative analytical techniques such as MALDI MS, either alone (63, 64) or in conjunction with size exclusion methods (65), or HPLC coupled with immunoaffinity deletion (66). 

Orru, S., Pagnozzi, D., and Pucci, R, Interaction proteomics, Biosci. Rep. 25, 45-56, 2005 Zanders, E.D., Ed., Chemical Genomics Reviews and Protocols, Humana Press, Totowa, NJ, 2005 Schou, C. and Heegaard, N.H., Recent applications of affinity interactions in capillary electrophoresis. Electrophoresis 27, 44-59, 2006 Niwayama, S., Proteomics in medicinal chemistry. Mini Rev. Med. Chem. 6, 241-246, 2006 Nedelkov, D. and Nelson, R.W., Eds., New and Emerging Proteomics Techniques, Humana Press, Totowa, NJ, 2006. See also Activity-Based Proteomics. 

Affinity Capillary Electrophoresis-Mass Spectrometry. Affinity capillary electrophoresis was originally used for the determination of the binding constants of small molecules to proteins (47-49). This solution-based technique is rapid and requires only small amounts of ligands. Affinity constants are measured based on the mobility change of the ligand on interaction with the receptor present in the electrophoretic buffer (50). By combining affinity capillary electrophoresis with on-line mass spectrometric detection and... 

Affinity capillary electrophoresis (ACE) constitutes a versatile microana-lytical technique that allows the estimation of affinity constants of analytes through the study of interactions such as protein-hgand, protein-antibody, and antibody-antigen. In ACE, PF techniques (whose optimization is not an easy task) can also be used to minimize the amount of sample needed. Chemometric methodology has also been applied for the optimization of the PF technique in ACE. 

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