One-step purification of rabbit histidine rich glycoprotein by dye-ligand affinity chromatography with metal ion requirement

A simple method for purification of the histidine rich glycoprotein (rHRG) from rabbit sera was developed. The rHRG was purified by one-step affinity chromatography using the triphenylmethane dye 'acid fuchsin' as a specific ligand, which gave an overall yield above 80%. Interestingly, the binding of rHRG to the ligand required the divalent transition-metal ions such as Zn²⁺, Ni²⁺, and Co²⁺ at pH 9.5. In the presence of 0.5 mM ZnCl₂, the binding was enhanced 15 times compared with that in the absence of ZnCl₂. Bound rHRG was efficiently eluted from the affinity absorbent with 100 nM imidazole or histidine. Purified rHRG was homogeneous with an M(r) of 94 kDa when analyzed by SDS-PAGE, whereas isoelectric focusing revealed microheterogeniety with pI values ranging from 6.3 to 6.8. Blotting analysis with lectins specific for carbohydrate moieties and treatment with glycosidases demonstrated that rHRG is a highly N-glycosylated protein with diverse carbohydrate structures. (C) 2000 Academic Press.