High affinity binding of serum histidine-rich glycoprotein to nickel-nitrilotriacetic acid: The application to microquantification

Shuji Mori, Hideo Kohka Takahashi, Kiyonori Yamaoka, Motoi Okamoto, Masahiro Nishibori

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Histidine-rich glycoprotein (HRG) is a serum protein with possible pluripotent activities. In this study, a method for the quantification of rabbit histidine-rich glycoprotein (rHRG) was developed based upon the high affinity binding profile of rHRG to nickel-nitrilotriacetic acid (Ni-NTA), an improved chelation agent. When the binding profile of Ni-NTA for whole serum proteins was assessed by Western blotting, Ni-NTA exhibited the binding specificity only to rHRG even after washing with 20 mM imidazole, owing to the unusual amounts of histidine residues in rHRG. In the following experiments, the rHRG immobilized onto a microplate with specific antibody was determined spectrophotometrically with peroxidase-labeled Ni-NTA. This method permitted evaluation of rHRG concentrations ranging from 1.0 to 100 ng/ml, and was actually applicable to the monitoring of rHRG in Resource Q-fractionated serum preparations. Also, the co-addition of L-histidine into the incubation mixture significantly diminished the specific binding between rHRG and Ni-NTA. These findings indicate the potential usefulness of this method for the specific measurement of small amounts of rHRG and for understanding the roles of abundant histidine residues in rHRG-metal cation interaction.

Original languageEnglish
Pages (from-to)93-102
Number of pages10
JournalLife Sciences
Issue number1
Publication statusPublished - May 23 2003



  • Histidine
  • Histidine rich glycoprotein
  • Nickel-nitrilotriacetic acid

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

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