Abstract
The criteria for elution of proteins from hydroxyapatite columns were examined as a function of (1) protein isoelectric point (22 proteins with isoelectric points between 3.5 and 11.0); (2) ionic nature of eluant (Na salts of PO4, F−, Cl−, SCN−, ClO4−, and CaCl2); and (3) structural differences between related proteins. It was found that proteins can be classified into three groups: (1) basic proteins, which elute at similar, moderate molarities of PO4, F−, Cl−, SCN−, and ClO4−, and low (<0.003 m) Ca2+; (2) acidic proteins which elute at about equal moderate molarities of PO4 and F−, but do not elute with Ca2+ and usually not with Cl−; (3) neutral proteins, which elute with PO4, F−, and Cl−, but show a strong anion specificity, and do not elute with Ca2+ or SCN−. Furthermore, individual specific polar groups are not in general crucial to binding or desorption, and variations in structure, other than major loosening, do not influence strongly the pattern of protein-hydroxyapatite interaction.
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