Study On Purification, Characterization And Conformation Of A Mannose-Binding Lectin From Ophiopogon Japonicus Lectin

A novel mannose-binding lectin has been purified and characterized from the dry underground rhizomes of a Chinese traditional medicine herb, Ophiopogon japonicus (Thund) Ker-Gawl which belongs to the family of Liliaceae. The purification procedure consisted of extraction, precipitation with 80％ammonium sulfate, and Ion-exchange chromatography on DEAE-Sepharose followed by gel filtration on Sephacryl S-100. The purified Ophiopogon japonicus lectin (OJL) showed a single band of 12kDa on SDS-PAGE under reducing and non-reducing conditions. The molecular mass of the native protein was 24kDa. The results showed that the lectin was composed of two identical subunits of 12kDa each. The hemagglutinating activity of OJL toward rabbit erythrocytes could be exclusively inhibited by mannan and didn't depend on metal ion. The activity of the lectin was not heat-stable and its optimum pH was 4～8. Chemical modification studies demonstrated that Arg were essential for the hemagglutinating activity.The fluorescence spectrum (FLS) of OJL excited at 280nm and 295nm showed a maximum peak at 328nm.The characteristic peak of Tyr did not exist, and it showed that the fluorescence energy of Tyr was transformed to Trp and strength the fluorescence of Trp. When OJL was excited at 295m, the FLS showed a maximum peak at 328nm, theλmax of fluorescence emissionspectrum blue-shifted about 20nm compared with theλmax of free Tyr (348nm).After treatment of OJL with NBS, the result showed that only one tryptophan residue was modified. Modification of Tyr, Ser/Thr and Trp residues had no effect on its hemagglutinating activity. However, total loss of OJL hemagglutinating activity was evident after modificating of the arginine residues by 2, 3-butanedione. That means the arginine residue is indispensable for the hemagglutination activity of OJL. Fluorescence spectra of OJL treating with some reagents show that modification of arginine residues led to a marked decrease in the fluorescence intensity upon excitation at 295nm. And with the gradually increasing concentration of NBS, the intensity of fluorescence was reduced correspondingly.The FLS of OJL with different temperature and pH also showed there was some change. The hemagglutinating activity of OJL was lost by 3mol/L of GuHCl. And 67％of the hemagglutinating activity still was hold when 8mol/L of urea existed. The study on fluorescence quenching showed that the fluorescence from Trp residues was quenchedmainly by acrylamide. 93.46％of Trp was quenched by acrylamide; while 41.25％and 55.56％of intrinsic fluorescence were quenched by CsCl and KI, respectively.The lectin also showed significant antifungal activity toward Magnaporthe grisea Barr and Maize Curvularia lunata when the concentration of OJL was over 0.5 mg/ml.