Mikrokriyojeller ile Transferrin Saflaştırılması
Abstract
Within the scope of the thesis, two different, immunoaffinity and molecularly imprinted, microcryogels based on affinity chromatography in order to purify transferrin from plasma were prepared. Poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) [P(HEMA-GMA)] was selected as a matrix for immunoaffinity microcryogel, and anti-transferrin (anti-Tf) antibodies were immobilized to microcryogels at various initial concentrations through the epoxy groups in the microcryogel structure without the need for any activating agents. For the molecularly imprinted microcryogel, poly(2-hydroxyethyl methacrylate-N-methacryloyl-(L)-tryptophan) [P(HEMA-MATrp)] was chosen as a matrix. The N-methacryloyl-L-tryptophan (MATrp) monomer, the polymerizable derivative of the L-tryptophan amino acid used as a hydrophobic ligand, was synthesized. The synthesized monomer was characterized by nuclear magnetic resonance, Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy studies. Subsequently, P(HEMA-MATrp) microcryogels were produced by free radical polymerization of 2-hydroxyethyl methacrylate (HEMA) and MATrp monomers at -16°C in the presence (imprinted microcryogel) and absence (non-imprinted microcryogel) of human serum transferrin (hsTf) using N,N′-methylenebis(acrylamide) as a cross-linker. Microcryogels were characterized with swelling tests, surface area measurements with Brunauer–Emmett–Teller method, FTIR, Raman spectroscopy, helium pycnometer, optical microscope and scanning electron microscope. Optimization studies were then carried out to determine the optimal adsorption conditions of microcryogels. As a result of these studies, it was found that the optimum pH value for both microcryogels was 6.0 and the maximum adsorption capacities at this pH value for immunoaffinity and imprinted microcryogels were found to be 9.82 mg/g and 2.11 mg/g, respectively. Langmuir adsorption isotherms and pseudo-second-order kinetic models for both immunoaffinity and imprinted microcryogels are consistent with the adsorption process, which means that the favorable adsorption is monolayer and chemically controlled. The selectivity of imprinted microcryogel was found to be 2.10 and 2.24-fold higher for human serum albumin and myoglobin, respectively, when compared to non-imprinted microcryogel. The purities of hSTf purified from the artificial plasma with the immunoaffinity P(HEMA-GMA) microcryogel and the imprinted P(HEMA-MATrp) microcryogel were examined by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). According to the results of reusability studies of the microcryogels, after the tenth use of the same microcryogel, the maximum hsTf adsorption capacities of immunoaffinity microcryogel and imprinted microcryogel have been calculated a decrease about 20% and 10%, respectively.