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

Shuji Mori, Masahiro Nishibori, Kiyonori Yamaoka, Motoi Okamoto

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)191-196
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume383
Issue number2
DOIs
Publication statusPublished - Nov 15 2000

Keywords

  • Acid fuchsin
  • Affinity purification
  • Characterization
  • HRG
  • Metal ion requirement

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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