Purification And Identification Of Tartary Buckwheat Lectin

Buckwheat is an important Polygonaceae grain and is also a traditional medicinal and edible plant. Tartary Buckwheat seeds are rich in proteins, such as, protease inhibitors and specific antigen, which are studied well. In recent years, lectin has been played an important role in biometrics, cancer and immune response, so it becomes the focus of many scholars. But the more relevant buckwheat bioactive ingredients, including lectins and their classification and function in plant lectin in the study have yet to be revealed. In this study, Yunnan Buckwheat seeds are used as materials, a molecular weight of 62 kDa protein was obtained. This research found it belonged to the class of lectin family. On the basis, a series of properties were done.Tartary Buckwheat lectin was extracted and purified by extraction buffer, ammonium sulfate precipitation, dialysis and ion exchange chromatography. Periodic acid-Schiff (PAS) staining was used to identify its protein species, and sulfuric acid-phenol method was used to measure the sugar contents. The hemagglutination activity and sugar binding specificity were tested by hemagglutination reaction and hemagglutination inhibition assays. pNpp and chitin were as substrate to detect phosphatase and chitinase activity, respectively. SOD activity was detected using pyrogallol autoxidation. Plasmid DNA as a substrate, detecting its effect on the topology of the plasmid. Cell-free translation system inhibition experiments to study the effects on protein translation system. Different temperatures and pH effect on the phosphatase activity. HPLC and fluorescence spectrum were used to study the effect of different metal ions on the activity and structure.The results showed TBL was a kind of coagulation proteins from Tartary Buckwheat seeds. Purified TBL indicated a single band via SDS-PAGE, and the molecular weight of TBL was 62 kDa. PAS staining proved TBL was a glycoprotein with a sugar content of 5.8%. Hemagglutination reaction demonstrated that TBL could agglutinate the O type of human blood with titer of 15μg/mL. The hemagglutination activity was inhibited by D-glucose and D-mannose, from which, TBL could be deduced as a mannose-binding lectin. Characterization identification displayed TBL had phosphatase activity with Michaelis constant Km= 9.86×10-3 mol/L and chitinase activity with specific activity:1.38U/mg. Pasmid as substrate, TBL could make supercoiled plasmid DNA form be nicked and linear DNA form. Cell-free translation system inhibition experiments showed that it could prevent protein synthesis. HPLC showed Mn2+ could promote the activity, whereas Cu2+ inhibit.Lectin had a unique role in resistance, antibacterial, anti-cancer and the regulation of immune activity. In this paper, the tartary buckwheat lectin with enzyme functions was obtained, which had O-and N-glycosidic glucosidase activity, could cut plasmid DNA and inhibit protein synthesis. Through functional analysis and contrast with the reported several plant lectins. We deduced that TBL belonged to Ⅱ ribosome inactivating protein. These results will provide a theoretical basis for further study of the function and application of TBL.