| Abstract: |
SUMMARY AND CONCLUSIONLectins naturally occurring glycoproteins that bind carbohydrate residues selectively and non- covalently. Lectins can be found in all kingdoms of life ranging from viruses through bacteria and plants to animals. Simple lectins consist of a small number of subuntis, not necessarily identical, of molecular weight usually below 40 kDa. This class comprises practically all known plant lectins in addition to the galectins, a family of galactose specific animal lectins. Legume lectins represent the largest and most thoroughly studied family of the simple lectins. The members of this protein family consist of two or four subunits (or protomers), either identical or slightly different, each with a single, small carbohydrate- combining site with the same specificity. They also contain one ion each of tightly attached calcium and manganese per subunit, which are required for carbohdrate binding. Mannose specific lectins are widely distributed in higher plants and are belived to play a role in recognition of high mannose glycans of foreign microorganisms or plant predators. In this report we describe the purification and characterization of two lectins present in Egyptian Pisum staivum (PSL) and Lens culinaris (LCL) seeds and report on their antimicrobial activity, the lectin carbohydrate binding specificity, molecular structure, and biochemical properties. The obtained results could be summarized as follow:1. Both PSL and LCL agglutinated all human blood groups (A, AB, B and O), showing no human blood specificity. However, they were distinctly characterized by slight preference towards the O blood group. PSL and LCL were most potent agglutinins for native rabbit erythrocytes, followed by rat and chicken erythrocytes. However, the activity of both lectins couldn’t be detected against sheep erythrocytes.2. The carbohydrate binding specificity of both studied lectins showed that most potent inhibitor was D- mannose following by D- glucose while D- galactose was the weakest one.3. The carbohydrate analysis of both studied lectins hydrolyzates by HPLC represented two moles of glucose bounds to one mole of the protein.4. Examination of minerals by atomic absorption spectroscopic analysis showed that both lectins are a metalloproteins each having 4 atoms each of calcium and manganese per lectin molecule.5. The amino acid analysis of purified PSL and LCL characterized by the absence of sulfur containing amino acids and were distinctly rich in acidic as well as hydroxyl amino acids.6. The apparent rnolecular masses of both studied lectins indicating a homodineric structure of two monomers each consisting of one α chain about 6 kDa and one β chain about 19 kDa. These results are similar to several other reports, indicating that most lectins from the various species Vicia, Pisum, Lens, and Lathyrus belonging to class of two-chain lectins, i.e. the polypeptides are fragmented into a light (α) and heavy (β) chain, where the two chains are bound together to produce one monomer of a molecular weight around 26 kDa.7. Hemagglutination of both studied lectins were markedly affected by pH, while they maintained 100% of their activities at a pH rang 6-8. Decreasing the pH to 5 and 4 lead to decreasing the activity by 10% and 60%, respectively, and the agglutination was completely abolished when lowering the pH below 4. On the other side, increasing the pH above 8, has gradually decreased hemagglutinating activity by 10%, 20%, 60%, and 80% at the pH values 9, 10, 11 and 12, respectively for pea lectin and by 10%, 30% 70%, 90% at the pH values 9,10,11 and 12, respectively for lentil lectin..8. Both studied lectins heat stable below 60οC for 30 min. The activation energy of denaturation (ΔǴ) for PSL and LCL was estimated to be 105.67 and 105.14 kJmol-1.9. To confirm the important role of divalent cations (e.g., calcium and manganese) both studied lectins their hemagglutination activity remarkably decreased after metal removel; however, when Ca 2+ and Mn2+ (50 mM) were added the activity fully restored.10. The purified lectins equally inhibited the growth of the fungi (Fusarium oxysporum, Trichoderma viride, Asparigillus flavus, Cruvularia alternata and Candida ablicans) and the bacteria (Escherichia coli and Bacillus cereus).
|
|
|