Isolation of lectins from hemolymph of decapod crustaceans by adsorption on formalinized erythrocytes

Abstract

Hemolymph of decapod crustaceans contains lectins of important specificity. An isolation procedure, based on adsorption of hemolymph lectins on red blood cells (RBC) fixed with formaldehyde, is described. Hemolymph is let to clot for 3 h at 22–28°C (RT) and for 24 h at 5°C; centrifuged at 13 000 g for 30 min; filtered through 5-μm filters; diluted with an equal volume of 50 mM NaCl, 100 mM CaCl 2; supplemented with protease as well as phenoloxidase inhibitors; centrifuged at 13 000 g for 20 min. Formalinized RBC (FRBC) are mixed with diluted hemolymph to a suspension of about 20% v/v FRBC. After incubation for 30 min at RT, FRBC are washed five times with 150 mM NaCl, 10 mM CaCl 2. The lectins adsorbed on FRBC are desorbed using either 100–500 mM of carbohydrate solutions in 0.9% NaCl or 50 mM Tris–HCl buffer, pH 8.0 containing 100 mM NaCl and 20 mM entylenediaminetetraacetate (EDTA). The procedure is efficient in isolating the hemolymph lectins of the decapods Liocarcinus depurator and Potamon potamios.