Nickel and copper complexes of a chelating methacrylate sorbent in the purification of chitinases and specific immunoglobulin G1 by immobilized metal ion affinity chromatography.
The isolation of the isoforms of endo- and exochitinases of Clostridium aminovalericum T1 and of the horseradish peroxidase (HRP)-specific immunoglobulin G1 from natural sources by immobilized metal ion affinity chromatography was studied. The effect of Cu2+ and Ni2+ complexes of iminodiacetic acid incorporated in porous glycidyl methacrylate-co-ethylene dimethacrylate and in agarose (Sepharose Fast Flow) beads on separation of the target polypeptides was analyzed. It was found that the Cu2+ complexes bound both the HRP-specific IgG1 and some isoforms of chitinases more strongly than the Ni2+ complexes. From the former complexes, both target polypeptides were eluted by a stepwise imidazole concentration gradient of 5-100 mM. The lower strength of Ni2+ complex binding with the HRP-specific IgG1 resulted in its easy elution with a pH gradient of 5.5-5 while some isoforms of chitinases required imidazole for their elution. The "fraction elution degree" of a target polypeptide (i.e., the ratio of its amounts in each eluate fraction and in the combined fractions) was used for the evaluation of the sorption selectivity and binding affinity of the separating components to the studied metal complexes.
Tishchenko
Galina
G
Department of Polymer Membranes. Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Square 2, 162 06 Prague 6, Czech Republic. tiscenko@imc.cas.cz
Hodrová
Blanka
B
Simůnek
Jirí
J
Bleha
Miroslav
M
D002614:Chelating Agents / Q000737:chemistry*; D002688:Chitinases / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008689:Methacrylates / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry*
D002614:Chelating Agents; D007074:Immunoglobulin G; D008689:Methacrylates; D003300:Copper; D009532:Nickel; D002688:Chitinases