Preparation Of Fenfucan Derivative Affinity Column And Its Application In GABA Receptor Separation

y-Aminobutyric acid (GABA) receptor is the concerned target for manyinsecticides. The structure of GABA receptor is closely related to the insecticideresistance and toxicology. Therefore, isolation/puriifcation of GABA receptors andfutrher study on the spatial structure will shed light on the development ofhigh-efficient, low toxic and safe insecticide as well as overcoming the pesticideresistance. This paper presents a thorough study of affinity separation method forpurifying bighead GABA receptor.This paper outlined the reported GABA receptor isolation/puriifcation methods,and then determined the ligand of affinity chromatography to be fipronil derivatives,basing on the computational analysis result of the organic molecules inhibited toGABA receptor in the literature. The affinity chromatography media was preparedthrough matrix activation, coupling and closure process, at the same time, theapplication conditions of aiffnity chromatography was determinedMatrix activation: Select Sepharose CL-6B as affinity chromatography matrix,1,4-butanediol diglycidyl ether as activator and directly connected on SepharoseCL-6B. The density of epoxy groups contained in activated Sepharose CL-6B was64.26|imol/g gel. The influence of activation temperature，time, pH and otherconditions on the eiffciency of activation was also studied. The optimal activeconditions of Sepharose CL-6B with1,4-butanediol diglycidyl ether as following: themixture of5.0g Sepharose CL-6B,5.0ml0.3mol/L sodium borohydride,10mgNaBH4, and5ml1,4-butanediol diglycidyl ether，at37。C，stirring at170rpm for8h.Coupling and closing: The immobilization of Fipronil derivatives on activatedSepharose CL-6B was realized through the reaction of the terminal primary amine ofFipronil deirvatives with the epoxy group of activated Sepharose CL-6B，and then theresidual epoxy group was closed with1mol/L ethanolamine.The coulping degree ofFipronil derivatives on Sepharose CL-6B reached58.68|imol/g gel.Aiffnity chromatography: The received affinity chromatography media wastransported to a column, and used it for the the separation and purification of GABAreceptor from bighead brain. Atfer affinity chromatography and ion exchange chromatography, the concentration of GABA receptor was4.6jag/mL, and its totalproduct was138|iig, SDS-PAGE image showed two band, molecular weight at44kDand55kD respectively. The two bands may be the a and (3subunits of bighead GABAreceptor, and this result was close to the corresponding subunits of other organismsreported in the literature.The innovation of this paper was successfully received GABA receptor frombighead brain by using affinity chromatography for the ifrst time, and identified themolecular weight of each subunit.