Partial characterization and possible uses of lectin from the internal organs of black sea cucumber (Holothuria scabra Jaeger)

A sialic acid specific lectin with mitogenic and allelopathic activity was isolated from the internal organs of Holothuria scabra by protein extraction using 0.01 M Tris buffer solution containing 0.15 M NaCl, pH 7.5, ammonium sulfate fractionation (0-60% saturation) and gel chromatography using Sephadex G-200. Inhibition studies showed that fetuin prevent agglutination and was used as ligand in affinity chromatography to isolate the lectin. The purity of both eluates was determined by polyacrylamide gel electrophoresis in a non-denaturing condition. The isolated lectin was found to be non-blood type specific and non-blood group specific since it agglutinated all types of human blood as well as animal erythrocytes. The agglutinability of the lectin increased with addition of calcium and trypsin. Optimum activity of the lectin was achieved between 20 deg C - 50 deg C and at pH 6-8. The native molecular weight was estimated to be 355 kD using Sephadex G-200. SDS-PAGE of both the affinity and gel filtration eluates yield five sub-units. For affinity chromatography, the eluates had sub-units of molecular masses of 114, 93, 80, 69 and 49 kD. On the other hand, G-200 eluates obtained sub-units of molecular masses of 120, 98, 80, 65 and 51 kD. The purified lectin was found to exhibit mitogenic activity and seed germination inhibiting activity and a mild toxicity against Artemia salina nauplii. Increase in cell size were observed 3 days after incubation of the lymphocytes with the lectin. The lectin prevented the germination of radish seeds and has 43% mortality on A. salina after 24 hours incubation. No antimicrobial and insecticidal activities were observed. The lectin is a glycoprotein with 1.328% total sugar. Galactose, glucose and oligosaccharides were detected in the hydrolyzate. The purified lectin can be used as modifier in carbon paste electrode and the prepared electrode (HSIOLMCPE) was found to detect mercury. The optimized conditions for mercury (2) analysis by differential pulse adsorptive stripping voltammetry (DPAdSV) were the following: pH 5-9, 20% modifier composition, 4 minutes accumulation time and 5 minutes deposition time. Regeneration of the electrode was achieved by soaking the electrode for 30 minutes in 0.1 M EDTA. Analysis for mercury content in laboratory waste sample using the prepared electrode gives comparable results with that of the standard AAS method