Preparation of magnetic metal-affinity sorbents and its use in a continuous flow system for cytochrome c adsorption

A magnetic metal-chelate affinity adsorbent utilizing N-methacryloyl-(l_)-histidine methyl ester (MAH) as a metal-chelating ligand was prepared. MAH was synthesized using methacryloyl chloride and L-histidine methyl ester. Magnetic beads with an average diameter of 50-100 urn were produced by suspension polymerization of ethylene glycol dimethacrylate (EGDMA) and MAH carried out in a dispersion medium. Specific surface area of the magnetic beads was found to be 80 $m^ 2$/g. Elemental analysis of the magnetic beads for nitrogen was estimated as 70 umol MAH/g polymer. Magnetic beads were loaded with the $Cu^ {2+}$ ions directly via MAH for the adsorption of cytochrome c continuous flow system. The cytochrome c adsorption on the mag-poly(EGDMA-MAH) beads was 42 mg/g. $Cu^ {2+}$ complexing increased the cytochrome c adsorption significantly. The maximum cytochrome c adsorption capacity of the $Cu^ {2+}$-chelated beads (carrying 68 umol $Cu^ {2+}$ per gram of polymer) was found to be 197 mg/g at pH 8.0 in phosphate buffer. Cytochrome c adsorption decreased with increasing temperature. Cytochrome c molecules could be reversibly adsorbed and desorbed five times with the magnetic adsorbents without noticeable loss in their cytochrome c adsorption capacity. Cytochrome c adsorption decreased with increasing magnetic field. The binding isotherm was determined by scatchard initially followed by application of Hill equation to the data obtained, then binding constant and $n _H$ Hill coefficient were estimated.

PDF

___

1.M.N. Gupta, S. Jain, I. Roy, Biotechnol. Prog. 18(2002)78.
2.G. Tishchenko, J. Dybal, K. Meszarosova, Z. Sedlakova, M. Bleha, J. Chromatogr. A. 954 (2002) 115.
3.V. Gaberc-Porekar, V. Menart, J. Biochem. Biophys. Method 49 (2001) 335.
4.A. Denizli, F. Denizli, E. Pişkin, J. Biomater. Sci., Polym. Ed. 10(1999)305.
5.F. Bianchi, R.Rausseaux-Prevost, P.Sautiere, J. Rausseaux J.Chromatogr. 518 (1990) 123-134
6.R. Chicz, F. Regnier, Anal. Chem. 61 (1989) 1742-1749.
7.N.K. Harakasetal., Biotechnol. Progr.4(1988) 149-158.
8.S.Y. Suen, Y.C. Liu, C.S. Chang, J. Chromatogr., B. 797 (2003) 305.
9.E.S. Hemdan, Y.J. Zhao, E. Sulkowski, J. Porath, Proc. Natl. Acad. Sci.U. S. A. 86 (1989) 1811.
10.B. Xue, Y. Sun, J. Chromatogr., A. 921 (2001) 109.
11.B. Akkaya, L. Uzun, F. Candan, A. Denizli. Mat. Sci. Eng. C, 27(2007) 180-189
12.R. Gutierrez, E.M.M. Valle, M.A. Galan. Sep. Purif. Rev. 36 (2007) 71-111.
13.Xüe, B., & Sun, Y, J. ChromatogrA, 921 (2001) 109-119
14.E.B. Altıntaş, L. Uzun, A. Denizli, China Particuology, 5 (2007) 174-179
15.B. Garipcan, A. Denizli, Macromol. Biosci. 2 (2002)135.
16.S. Akgöl, A. Denizli, J. Mol. Çatal., AChem. 2fi (2004) 7.
17.A. Denizli, B. Salih, E. Pişkin, J. Chromatogr.; A. 731 (1996)57.
18.H.M. Swartz, J.R. Bolton, D.C. Borg, BologiCa| 1 Applications of Electron Spin Resonance ? Wiley, New York, 1972.
19.D. Böhm, B. Pittermann, Chem. Eng. Technol 23(2004)309.
20.R.J. Todd, R.D. Johnson, F.H. Arnold, j. Chromatogr. A 662 (1994) 13-26.
21.V.D. Noto, L.D. Via, P. Zatta, Coordin. Chem. Rev. 228(2002) 343-362.
22.D. Böhm, B. Pittermann, Chem. Eng. Technol. 23 (2000) 4.