The Preparation And Evaluation Of Biomimetic Dye Affinity Absorbent

Affinity chromatography is an efficient , rapid ,and high-performance purification and separation method which has been used for identification, purification and separation of numerous of proteins and biomolecules, and based on highly specific molecule recognition or biorecogniton. Dye-affinity chromatography has been considered as one of the important alternatives ,especially for biomimetic dye affinity chromatography which mimick the structure of the substrates, cofacters , or bingding agents . Dye-ligands are able to bind most type of proteins and are commercially available,inexpencive ,and can be easily synthetic and immobilized .In this paper, we prepared dye-affinity sorbents in which chitosan microspheres were used as the support, and three dye ligands ( new synthesized dye,Cibacron Blue F3GA ,and Procion blue MX-R) were incorporated into these microbeads for separation and purification of proteins. The main work followes:1, The new dye (RAG for short) was prepeared by linking azophenylsulfonate to the reactive chlorotriazine ring of Procion Blue MX-R via nucleophilic substitute reaction in alkaline solution. The physical and chemical properties of the dye were characterized by using IR, UV-vis, Raman and so on.2, Chitosan microspheres were prepared by a inverse phrase cross-linking technique, which epichlorohydrin was used as a crosslinking reagent. The effect factors of preparation were studied in detail, and the favorable operation conditions were found at : the chitosan concentration is 2% , the proportion of oil to chitosan solution is 1:1,and the concentrations of epichlorohydrin and the porogen are 0.04mol/L and 0.5% respectively.The physical and chemical properties of the microspheres were characterized by using IR , scan electron microscope. Results indicate that the microspheres prepared here have high porosity and low non-specific adsorption for proteins.3, New synthesized dye (RAG)and Cibacron Blue F3GA and Procion blue MX-R were covalently coupled with Chitosan microspheres via the nucleophilicsubstitution reaction between the chloride of their triazine ring and the hydroxyl groups of the Chitosan molecules under alkaline conditions, and thus two novel dye-affinty sorbent CS-RAG and CS-CBA and CS-PBR were obtained. The ligand density can be easily adjusted by the adding values. It was found that these dye-attached microspheres were stable in acidic and alkaline solution while the ligand leakage was very low.^ CS-RAG carrying RAG 60.21 mg /g bead was used in the Bovine serum albumin adsorption studies. The maximum adsorption was observed at pH5.0The adsorption capacity decreased with an increase of ionic strengh. From adsorption isotherm at 25 °C which followed the Langmuir equation, the maximum adsorption capacity and dissociation constant were determined as respectively. The model of Chase was adopted to study the kinetic of adsorption, and the forward reaction velocity constant k\ was 0.02247 mL/mg.min. High desorption ratios were achieved by using 0.05 mol/LKSCN +0.1 mol/LNaCl(pH4.0).5^ The affinity sorbent CS-RAG carrying RAG 60.21 mg /g bead was used in the adsorption studies of human serum albumin with Norfloxacin . The effect of removing the abundant proteins was studied by detecting the concentration of Norfloxacin with HPLC.