Interaction of proteins with immobilized Cu2+. Quantitation of adsorption capacity, adsorption isotherms and equilibrium constants by frontal analysis.
The interaction of lysozyme, ovalbumin, bovine and pig serum albumins with Cu2+ immobilized on Chelating Sepharose Fast Flow or TSK gel chelate-5PW was studied by frontal analysis at various initial concentrations of these solutes. The chromatographic data so obtained served as a basis for evaluating some relevant affinity chromatography parameters by adapting previously reported equations to this system. The TSK-based adsorbent had lower adsorption capacity for all the model proteins compared to the agarose-based adsorbent, due primarily to its lower porosity which has a marked influence on the accessibility of the immobilized ligand to the proteins. On the other hand, the TSK-based adsorbent offers almost ideal conditions for studying adsorption equilibria under column chromatographic conditions. The adsorption capacity of these adsorbents for the model proteins ranges from about 0.6 to 7 mumol/ml, equivalent to 40-100 mg/ml, of adsorbent. The following equilibrium constants for the interaction of the proteins with immobilized Cu2+ were obtained: lysozyme, 1.8.10(4); ovalbumin, 1.5.0(5); BSA, 1.7.10(5); PSA, 3.7.10(5) and imidazole, 8.10(3) M-1. Despite the comparatively low affinity of imidazole for the adsorbent, it is an effective competing ligand, at comparatively high concentrations, for adsorbed proteins primarily because all adsorption sites are available to it. The results obtained suggest that about 1/3 to 1/2 of the potential adsorption sites on the model proteins are involved in forming coordination complexes with Cu2+ immobilized to covalently bound iminodiacetate groups on insoluble gel matrices.
Belew
M
M
Institute of Biochemistry, University of Uppsala, Sweden.
Yip
T T
TT
Andersson
L
L
Porath
J
J
D000327:Adsorption; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D003300:Copper / Q000032:analysis*; D007093:Imidazoles / Q000302:isolation & purification; D008024:Ligands; D009113:Muramidase / Q000032:analysis; D010047:Ovalbumin / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012709:Serum Albumin / Q000032:analysis; D013552:Swine; D013816:Thermodynamics
D007093:Imidazoles; D008024:Ligands; D011506:Proteins; D012709:Serum Albumin; D003300:Copper; D010047:Ovalbumin; D009113:Muramidase