Immobilized metal ion affinity capillary electrophoresis of proteins--a model for affinity capillary electrophoresis using soluble polymer-supported ligands.
An affinity capillary electrophoresis method has been developed that employs small ligands covalently bound to a replaceable soluble polymer matrix. The metal chelate iminodiacetate-Cu(II) coupled to polyethylene glycol was used as a model system and the interactions of different model proteins, namely ribonucleases, cytochromes c, chymotrypsin, and kallikrein, were investigated. The method allowed for easy determination of dissociation constants using a modified Langmuir adsorption isotherm equation which is applicable to interactions with fast on/off kinetics. It is shown that the general rules for protein interaction with matrix-bound metal chelates established for immobilized metal ion affinity chromatography (IMAC) are maintained in immobilized metal ion affinity capillary electrophoresis (IMACE). In contrast to gel electrophoresis, IMACE allowed for using similar conditions as in IMAC, especially regarding the high salt concentrations, usually employed with the latter technique. The usefulness of the method for quantification of low-affinity interactions and studying protein surface structure and structure/function relationship using the metal affinity is demonstrated.
Haupt
K
K
Laboratoire d'Interactions Moléculaires et de Technologie de Séparations, Université de Technologie de Compiègne, France.
Roy
F
F
Vijayalakshmi
M A
MA
D000818:Animals; D002918:Chymotrypsin / Q000032:analysis; D003574:Cytochrome c Group / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007610:Kallikreins / Q000032:analysis; D007700:Kinetics; D008024:Ligands; D008670:Metals; D008956:Models, Chemical; D011108:Polymers; D012259:Ribonuclease, Pancreatic / Q000032:analysis; D012260:Ribonucleases / Q000032:analysis*; D012697:Serine Endopeptidases / Q000032:analysis*; D012995:Solubility
D003574:Cytochrome c Group; D008024:Ligands; D008670:Metals; D011108:Polymers; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic; D007610:Kallikreins; D012697:Serine Endopeptidases; D002918:Chymotrypsin