Affinity capillary electrophoresis: a physical-organic tool for studying interactions in biomolecular recognition.
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.
Colton
I J
IJ
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Carbeck
J D
JD
Rao
J
J
Whitesides
G M
GM
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D004136:Dinitrobenzenes / Q000276:immunology / Q000378:metabolism; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008970:Molecular Weight; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D014640:Vancomycin / Q000737:chemistry / Q000378:metabolism
D004136:Dinitrobenzenes; D004151:Dipeptides; D007074:Immunoglobulin G; D008024:Ligands; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D013449:Sulfonamides; D014640:Vancomycin; D002256:Carbonic Anhydrases