High affinity binding of serum histidine-rich glycoprotein to nickel-nitrilotriacetic acid: the application to microquantification.
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.
Department of Pharmacology, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1, Shikata-cho, Okayama 700-8558, Japan. morimori@md.okayama-u.ac.jp
Shuji
S
Mori
Hideo Kohka
HK
Takahashi
Kiyonori
K
Yamaoka
Motoi
M
Okamoto
Masahiro
M
Nishibori
D000818:Animals; D000906:Antibodies / Q000032:analysis; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D007202:Indicators and Reagents; D008832:Microchemistry; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000097:blood*; D009942:Organometallic Compounds / Q000097:blood*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D011817:Rabbits; D000099053:Histidine-Rich Glycoprotein
D000906:Antibodies; D007202:Indicators and Reagents; D009942:Organometallic Compounds; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; C088321:nickel nitrilotriacetic acid; D009571:Nitrilotriacetic Acid