Review Affinity capillary electrophoresis: A physical-organic tool for studying interactions in biomolecular recognition Ian J. Colton, Jeffrey D. Carbeck, Jianghong Rao, Professor George M. Whitesides * Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA
email: George M. Whitesides (gwhitesides@gmwgroup.harvard.edu)
*Correspondence to George M. Whitesides, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA (Tel: +617-495-9431; Fax: +617-495-9857)
Abstract Affinity capillary electrophoresis (ACE) is a technique that is used to measure the binding affinity of receptors to neutral and charged ligands. ACE experiments are based on differences in the values of electrophoretic mobility of free and bound receptor. Scatchard analysis of the fraction of bound receptor, at equilibrium, as a function of the concentration of free ligand yields the dissociation constant of the receptor-ligand complex. ACE experiments are most conveniently performed on fused silica capillaries using a negatively charged receptor (molecular mass < 50 kDa) and increasing concentrations of a low molecular weight, charged ligand in the running buffer. ACE experiments that involve high molecular weight receptors may require the use of running buffers containing zwitterionic additives to prevent the receptors from adsorbing appreciably to the wall of the capillary. This review emphasizes ACE experiments performed with two model systems: bovine carbonic anhydrase II (BCA II) with arylsulfonamide ligands and vancomycin (Van), a glycopeptide antibiotic, with D-Ala-D-Ala (DADA)-based peptidyl ligands. Dissociation constants determined from ACE experiments performed with charged receptors and ligands can often be rationalized using electrostatic arguments. The combination of differently charged derivatives of proteins - protein charge ladders - and ACE is a physical-organic tool that is used to investigate electrostatic effects. Variations of ACE experiments have been used to estimate the charge of Van and of proteins in solution, and to determine the effect of the association of Van to Ac2KDADA on the value of pKa of its N-terminal amino group.
Review Affinity capillary electrophoresis Geza Rippel, Hugo Corstjens, Hugo A. H. Billiet, Dr. Johannes Frank * Delft University of Technology, Kluyver Laboratory of Biotechnology, Department of Biochemical Engineering, Delft, The Netherlands
email: Johannes Frank (frank@stm.tudelft.nl)
*Correspondence to Johannes Frank, Delft University of Technology, Kluyver Laboratory of Biotechnology, Department of Biochemical Engineering, Julianalaan 67, NL-2628 BC Delft, The Netherlands (Tel: +31-15-278-2332; Fax: +31-15-278-2355)
Abstract The application of affinity capillary electrophoresis (ACE) to the study of molecular interactions is reviewed. ACE appears to be a sensitive, versatile and convenient tool to obtain reliable data on binding constants and stoichiometries of interacting systems using the Hummel-Dreyer method and variants thereof. A powerful feature is the possibility to analyze simultaneously the affinity of a large number of compounds for the same ligand, making it a promising tool for the screening of large combinatorial libraries.
-------------------------------------------------------------------------------- Received: 12 June 1997
Review Methods for the estimation of binding constants by capillary electrophoresis Kimber L. Rundlett, Professor Daniel W. Armstrong * Department of Chemistry, University of Missouri-Rolla, Rolla, MO, USA
email: Daniel W. Armstrong (mrichard@umr.edu)
*Correspondence to Daniel W. Armstrong, Department of Chemistry, University of Missouri-Rolla, Rolla, MO 65401, USA (Tel: +573-341-4429; Fax: +573-341-6166)
Keywords Equilibrium constants ?Molecular association ?Linear plotting ?Nonlinear regression ?Errors
Abstract Capillary electrophoresis (CE) has developed into a particularly effective means to determine apparent equilibrium constants for molecular association in solution (e.g., to micelles, cyclodextrins, antibiotics, proteins, RNA, DNA. etc.). The various experimental, graphical and mathematical approaches for determining association constants are reviewed. In CE, association constants can be calculated because there is a relationship between substrate concentration and the measured electrophoretic mobility of the solute. Most of the approaches for obtaining association constants by CE are conceptually and mathematically related to one another. Likewise, they are analogous to many spectroscopic techniques that are used for obtaining association constants. The advantages, limitations and proper use of the various CE approaches are examined.
-------------------------------------------------------------------------------- Received: 18 July 1997
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