Study On The Extraction, Purification And Characterization Of Lectin From The Canavalia Ensiformis Seed

Lectins are a kind of proteins that can specifically and reversibly bind with carbohydrates, but usually without any enzymatic or antibiotic activity. They were widely distributed in plants, animals and microorganisms. In recent years, the research on lectins are more and more popular.A convenient, sensitive and reliable protocol was established to determine the hemagglutination activity of Concanavalin A (ConA) which features sensitization and fixation of the effector red blood cells by glutaraldehyde crosslinking. When the human erythrocyte was treated with 1.75 U/mL trypsin, its agglutinability was increased by 8 times. In order to obtain good results, samples should be crushed to 60 mesh, ratio (W/V) of sample to aetheris was 1:8, lasting for 8 h.The optimum extractive condition of ConA was determined through single factor test and Response surface methodology: buffer/material ratio 8.88:1, extraction time 8.65 h, the best extracting solution was pH 7.4, 0.01mol/L isotonic phosphate buffered saline (PBS) (contained 0.17mol/L NaCl), Under these conditions, the hemagglutinating activity of ConA was 167.61 HU/mg. Then fraction with 70% (NH4)2SO4, the hemagglutinating activity of ConA reached to 237.43 HU/mg.A man/glc-specific (ConA) was purified from seeds of Canavalia ensiformis (L.) DC) by a procedure including extraction, fraction with (NH4)2SO4, ion-exchange chromatography on DEAE-52 column and followed by gel filtration on Sephadex G-100. The purified ConA showed a single band on SDS polyacrylamide gel electrophoresis. The agglutinating activity of the purified ConA showed an activity increase of 50.15 fold and an activity recovery of 57.06%. The molecular weight of the subunit was 29 600 dalton.ConA was a kind of lectin stable to heat treatment. It was stable at below 60℃, but it was completely inactive at above 100℃for 20min. ConA had higher lectin activity at pH of 5.8 ~ 8.3, but its lectin activity was inactive at pH above 11.9. ConA can agglutinate A, B, O, AB types of human blood and the erythrocytes of rabbit, chicken, duck, pigeon, common carp and crucian carp, without specificity of erythocyte agglutination. The agglutinating activity of ConA vanished after demetalization by dialysis against EDTA. Followed by dialysis against NaCl, full activity was restored by addition of Ca2+, Mn2+. Amino acid analysis showed that it contained 17 kinds of amino acids, in which the contents of aspartic acid and glutamicacid were relatively high. 276nm is the optimum UV absorption of ConA in physiological saline. The variation of ConA conformation in different solution conditions were followed by intrinsic fluorescence.The fluorescence spectrum of natural ConA excited at 280nm and 295nm showed a maxium peak both at 334nm. The characteristic peak of Tyr did not exist, and it showed that the fluorescence energy of Tyr was transferred to Trp and strength the fluorescence of Trp; pH value, denaturant urea could all reduce the fluorescence strength of ConA, but could not lead to changes in the profile of flurescence spectra and the position of emission peak, which suggested that the structure of ConA was stable.