Poly-(L)-Histidine Immobilized Cryogels for Lysozyme Purification

Abstract

Immobilized metal ion affinity chromatography is one of the methods used for the adsorption of proteins. In this study, poly(glycidyl methacrylate) cryogel discs were prepared by free radical polymerization. The metal chelating groups were polymeric chain of poly-(l)-histidine (mol wt5000) having poly-imidazole ring sequence. Then, Cu(II), Zn(II), and Ni(II) ions were separately chelated on the poly-(l)-histidine immobilized poly(glycidyl methacrylate) cryogel discs to be used in immobilized metal ion affinity chromatography separation of lysozyme. The swelling test, Fourier transform infrared spectroscopy, Brunauer-Emmett-Teller, and scanning electron microscopy were performed to characterize both poly(glycidyl methacrylate) and poly-(l)-histidine immobilized poly(glycidyl methacrylate) cryogel discs. The effects of the pH, lysozyme concentration, adsorption time, and ionic strength on the adsorption capacity were studied. These parameters were varied between 4.0 and 8.0 for pH, 0.0 and 2.0mg/ml for initial lysozyme concentration, 0 and 120min for adsorption time, and 0.0 and 1.0 mu M for ionic strength. The maximum lysozyme adsorption capacity of the Cu(II), Zn(II), and Ni(II) ions chelated poly-(l)-histidine immobilized poly(glycidyl methacrylate) cryogel discs was 36.4, 26.8, and 17.3mg/g cryogel, respectively. Desorption of lysozyme from cryogel discs was easily achieved by 1.0M NaCI solution. Repeated adsorption-elution processes showed that these cryogel discs were suitable for repeatable lysozyme adsorption. Adsorption isotherms fitted to Langmuir model and adsorption kinetics suited to pseudo-second order model. Thermodynamic parameters (i.e. H degrees, S degrees, G degrees) were also calculated from Langmuir isotherms at different temperatures.