Studies On The Immobilization Of Ricinus Communis Agglutinin And Its Application

Ricinus Communis Agglutinin?RCA?is a macromolecular protein with biological activity from the castor beans,only accounts 0.005%-0.015% of castor bean.It specifically binds with the sugar chain terminal sequence Gal?1-4GlcNAc.After entering the animal body,it makes hemoglobin occurr agglutination.It is one of the ideal probes for study the molecule containing sugar chain due to it specifically binds to sugar.In this study,the characteristics of RCA were used to explore its application as a probe after immobilization in separation and purification of sugar proteins in blood,in the chemical modification of enzymes and so on.After activating the inert carrier gel Sepharose 4B by epichlorohydrin,RCA purified from the castor bean were immobilized on Sepharose 4B to make the RCA-Sepharose 4B affinity column.The coupling rate of RCA to Sepharose 4B carrier gel was 1.916 mg/g or 1.725 mg/g.The RCA-Sepharose 4B affinity column was used to probe the associated protein in the rabbit plasma,results showed that there were at least two kinds of polypeptide could interact with the RCA in rabbit plasma.SDS-PAGE showed the molecular weights of these two polypeptides were 97 KDa and 120 KDa respectively.Further experiments have confirmed that these polypeptides contained the sugar,and they could really interact with RCA in vitro.This study demonstrated that RCA-Sepharose 4B affinity column was suitable for separating and purifying glycoprotein from a complex multi-component in biological sample,and could also be used as a probe to explore the protein interacted with RCA.The egg white lysozyme?Lysozyme EC3.2.1.17,LYZ?was modified by using the galactose of terminal amine.The separation between the modified lysozyme?Gal-LYZ?and unmodified lysozyme was very difficult because that the molecular changes of enzyme was a little after modified,the difference was not obvious.The RCA-Sepharose 4B affinity column was used to uccessfully separated the both enzymes in this study,which extended the application of immobilized RCA in biochemical research,and proofed that the immobilized RCA could be a good tool in the research of chemically modified enzyme.The enzymatic activity of obtained LYZ was 3122.7 U/mg and the recovery reached to 80.64%,enzyme yields was 1.789 mg/ml.Results showed that the absorption and fluorescence spectra of LYZ after galactose-modified had changed,which explained the structural change after enzymatic modification.The optimum temperatures for the enzymatic activity before and after modifying of LYZ were 41 ? and 37 ?,the optimum pH were 6.2 and 6.6,respectively.Cu²⁺,Mn²⁺,Ba²⁺,Zn²⁺,Na+ all could activate the LYZ before and after modification,K+ showed inhibition to the both enzymes.Mg²⁺ activated the LYZ but showed a slight inhibition to Gal-LYZ.Ca²⁺,Fe²⁺ activated the Gal-LYZ and showed inhibition to the LYZ.Generally,the activity of Gal-LYZ has greatly reduced,which indicated that the galactose have occupied the substrate receiving fracture of the modified LYZ surface,so the enzymatic activity reduced.