Nov, 2000
One-step purification of rabbit histidine rich glycoprotein by dye-ligand affinity chromatography with metal ion requirement
Archives of Biochemistry and Biophysics
- ,
- ,
- ,
- Volume
- 383
- Number
- 2
- First page
- 191
- Last page
- 196
- Language
- English
- Publishing type
- Research paper (scientific journal)
- DOI
- 10.1006/abbi.2000.2032
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.
- Link information
- ID information
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- DOI : 10.1006/abbi.2000.2032
- ISSN : 0003-9861
- Pubmed ID : 11185553
- SCOPUS ID : 0034669369