Thermosensitive copolymer of N-vinylcaprolactam and 1-vinylimidazole: molecular characterization and separation by immobilized metal affinity chromatography

Thermosensitive copolymer of N-vinylcaprolactam (VCL) and 1-vinylimidazole (VI), a new reagent for metal–chelate affinity separation of proteins, was characterized by static and dynamic light scattering and 1H NMR spectrometry. The copolymer of Mw=11 700, Mw/Mn=1.1 and VI-unit molar fraction of 11% was prepared by free radical co-polymerization of the monomers followed by dialysis of the product. The copolymer solution in 10 mM Na-phosphate buffer containing 0.15 M NaCl, pH 8.5, at 25°C turned out to be close to θ-conditions. Its diffusion coefficient in these conditions was 6×10−7 cm2 s−1 indicating the hydrodynamic diameter of 8 nm. The copolymer was separated by immobilized metal affinity chromatography on Cu2+-IDA-Sepharose into the non-adsorbing fraction (5 molar% VI-units) and the adsorbing fraction further eluted with EDTA and thermally precipitated at 48°C (8.5 mol% VI-units). The combination of IMAC and thermal precipitation of the copolymer enables isolation of its high-affinity, relatively homogeneous fraction most promising for bioseparations, including displacement chromatography of proteins.