Study On Purification And Properties Of Lectin From Gill Of Bighead Carp (Aristichthys Nobilis)

Bighead carp (Aristichthys nobilis) is one of freshwater fish food resources with very large population. The bighead carp has a tremendous growth rate and disease resistant. Adults can be quite large, making it a lucrative aquaculture fish. In recent years, the research of utilization of bighead carp has made considerable development. Bighead carp industry is gradually moving towards healthy development. In order to explore the feasibility of the comprehensive utilization of gills from bighead carp, enhance the value of this processing waste, this article was described the purification and characterization of a lectin from gill of bighead carp. The main research contents are as follows:The chemical composition analysis of the gill and body of bighead carp showed that the protein content was nearly equal to the body. Therefore, the gill of bighead carp was a available scraps for comprehensive utilization.The optimal extraction process of the lectin from gill of bighead carp as follows: the buffer system as 22.81 mmol/L of Tris-HCl buffer (pH 8.5), solid-liquid ratio 1:6, extraction time 16.43 h. Under these conditions, the actual extraction of specific activity was 8125.49 9647.06 HU/mg.A lectin with fucose-specific was purified from the crude extraction of gill from bighead carp. The purification procedure consisted of separation on a DEAE-Sepharose FF ion exchange column, followed by gel filtration chromatography on Sephacryl S-200 HR and fast protein liquid chromatography on Superdex 200 10/300 GL columns. The purified lectin was designated as GANL (gill of Aristichthys nobilis lectin). The purification fold of this procedure was 21.2 times, the protein recovery was 3.6%. The purified lectin showed single protein band under SDS-PAGE.The studies of physical and chemical properties review that for the six tested animal erythrocytes and human A, B and O-type red blood cells, GANL only agglutinated rabbit native erythrocytes and human O-type red blood cells. The hemagglutination activity showed strong blood supply animal species specificity. Hemagglutination activity of GANL was not inhibited by any of the monosaccharides, disaccharide and glycoproteins tested except for fucose, showing a strong sugar-binding specificity. Our results suggest that the optimal temperature for GANL is close to 50℃. We also found that some original activity was maintained even after heating to 90℃for 30 min, suggesting that this lectin protein is extremely thermostable. GANL was unstable under acid condition but stable under alkaline conditions, the optimal pH was 8⁹. We found that some original activity was maintained following incubation at pH 11 for 1 h. The hemagglutination activity of GANL was not dependent on Ca²⁺, Mn²⁺ and Mg²⁺. The hemagglutination activity of GANL was significantly affected by reducing agentβ-mercaptoethanol, detergents SDS and trypsin hydrolysis, succinyl-modified. In contrast, The hemagglutination activity of GANL was not significantly affected by DMSO and trifluoroacetic acid.GANL was a glycoprotein with a native relative molecular weight of 2.20×10⁵, composed with six identical subunit with relative molecular weight of 3.55×10⁴. The carbohydrate content of GANL was 13.4 %. The linkage of carbohydrate and protein was O-glycosidic bond between sugar and Thr. Cys-S was detected in the lectin molecule (0.81%). The structure of GANL was complex, showing a ladder-like protein band under non-reducing SDS-PAGE. The molecules of GANL containing theα-helix 34.8 %,β-fold 12.1 %, random coil 33.0 %,β-corner 24.5 %.The studies suggested that GANL only agglutinated Haemetococcus pluvialis among the three microalgae tested. The agglutination activity showed a cell desity dependent mannar. In the seven microorganisms tested, GANL only agglutinated two kinds of Gram-negative fish pathogenic micro-organisms, Vibrio anguillarum and Vibrio harveyi. GANL had no agglutinating effect on others bacteria and fungi. Microbial agglutination of GANL also showed stronger specificity. GANL can only inhibit the growth of V. anguillarum and V. harveyi. This growth inhibition can be converted by 0.2 mol/L of fucose suggested that the microbial growth inhibition related with CRDs.GANL exerted potent antitumor activity against the HeLa cell line,SKOV3 cell line and HepG2 cell line. The antitumor activity of GANL on HeLa cell line has a dose-dependent manner. The IC₅₀ was 11.86μg/mL,while the dose-dependent manner on SKOV3 and HepG2 was not obvious.GANL was weakly mitogenic towards the murine splenocytes in comparison with ConA, a standard plant mitogen. However, we observed significant mitogenic activity towards murine splenocytes that were previously incubated with ConA. GANL had no significant mitogenic effect towards murine splenocytes previously incubated with LPS, in comparison with GANL alone. Therefore, we speculated that GANL have mitogenic effect towards T cells, but no effect on B cells.