Interaction of proteins with immobilized Cu2+: Quantitation of adsorption capacity, adsorption isotherms and equilibrium constants by frontal analysis
✍ Scribed by Makonnen Belew; Tai-Tung Yip; Lennart Andersson; Jerker Porath
- Book ID
- 104144817
- Publisher
- Elsevier Science
- Year
- 1987
- Tongue
- English
- Weight
- 755 KB
- Volume
- 403
- Category
- Article
- ISSN
- 1873-3778
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✦ Synopsis
The interaction of lysozyme, ovalbumin, bovine and pig serum albumins with Cu2+ immobilized on Chelating Sepharose Fast Flow or TSK gel chelate-SPW 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 pmol/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 . 104; ovalbumin, 1.5 . 105; BSA, 1.7 . 105; PSA, 3.7 a lo5 and imidazole, 8 . lo3 M-r. 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 l/3 to l/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,