The Synthesis And Application Of Metal-chelate Affinity Reverse Micelles

Reversed micelles for the protein extraction have been extensively studied. Ionic surfactants were mostly employed to form the extractive reversed micellar systems, and problems remained for the denaturation of proteins and the low selectivity to proteins. The non-ionic surfactant modified with dye ligand can extraction protein without protein denaturation, but it cannot resist the high ionic strength and high concentration of denaturant. The dissertation develops a novel metal-chelate affinity reversed micelles system formulated by nonionic surfactant, which can resist the high ionic strength and high concentration of denaturant and can apply to the protein extaction.The noninonic surfactant of Span 85 is modified with chelate agent TED then chelate Cu²⁺ as a metal-chelate affinity surfactant by a two-phase reaction. A novel reversed micelles formed by the mixture of Span 85 and Span 85-TED-Cu conjugate are extensively characterized in the water content (W0), reverse micelles radius (Z-average size) by water determination and laser scattering technology. The results show that the water content and size of the reversed micelles are significantly increased by the introduction of Cu²⁺ ligands, and the reversed micelles can keep water content stable in wide pH range(5⁹), high ionic strength(≤0.5mol/L) and high concentration of denaturant solution(8mol/L Urea).Apply the metal-chelate affinity reverse micelles in protein extraction. The protein extraction yield associates with the coupled Cu²⁺ concentration, Span85 concentration and alcohol. It could resist wide pH range, high ionic strength and high concentration of denaturant. When use the reverse micelles extract hemoglobin, the extraction yield (E) reaches 70%. The dissertation investigates the effect of alcohol on the affinity-based reversed micelles due to the significant impact of the assistant in reversed micellar system. The addition of alcohol gets higher protein extraction yield, but protein tends to become sediment in the water and reverse micelles interface, which is a drawback for protein refolding.