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

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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
Volume73
Issue number1
DOIs
Publication statusPublished - May 23 2003

Fingerprint

Rabbits
Serum
Histidine
Blood Proteins
nickel nitrilotriacetic acid
histidine-rich proteins
Chelation
Washing
Peroxidase
Cations
Western Blotting
Metals
Antibodies
Monitoring
Experiments

Keywords

  • Histidine
  • Histidine rich glycoprotein
  • Nickel-nitrilotriacetic acid

ASJC Scopus subject areas

  • Pharmacology

Cite this

High affinity binding of serum histidine-rich glycoprotein to nickel-nitrilotriacetic acid : The application to microquantification. / Mori, Shuji; Takahashi, Hideo Kohka; Yamaoka, Kiyonori; Okamoto, Motoi; Nishibori, Masahiro.

In: Life Sciences, Vol. 73, No. 1, 23.05.2003, p. 93-102.

Research output: Contribution to journalArticle

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