Isolamento, caracterização e atividade biológica da lectina de sementes de variedade brasileira de feijão-lima (Phaseolus lunatus var. cascavel)

• Ano de defesa: 2015
• Autor(a) principal: E-Lacerda, Rodrigo Rodrigues
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal da Paraíba; Brasil; Biologia Celular e Molecular; Programa de Pós-Graduação em Biologia Celular e Molecular; UFPB
• Palavras-chave em Português: Phaseolus lunatus; Caracterização; Antioxidante; Antitumoral; Gastroprotetor; Characterization; Antioxidant; Antitumor; Gastroprotective; CIENCIAS BIOLOGICAS::BIOLOGIA GERAL
• Link de acesso: https://repositorio.ufpb.br/jspui/handle/tede/9437

Lectins are proteins with the ability to interact specifically and reversibly with carbohydrates. They are present in all forms of life, performing vital functions for the maintenance of these beings. The legume lectins have numerous biological activities that can be used for therapy. The aim of this study was to isolate a lectin of Phaseolus lunatus seeds var. cascavel (PLUN), as well as to characterize it and check anti-hemolytic, antioxidant, antitumor and gastroprotective activities. The presence of lectins and an indication of their carbohydrate inhibitor was determined using the hemagglutination activity against human type ABO red blood cells. Protein concentration was determined by Bradford method using bovine serum albumin as standard. The isolation of PLUN was performed by size exclusion chromatography on Sephadex G-100. The weight and the purity of lectin was estimated by EGPA (polyacrylamide gel electrophoresis) under reducing and non-reducing conditions. The characterization was performed PLUN checking their resistance to pH and temperature variations, the presence front integrity of urea, DTT (dithiothreitol), beta-mercaptoethanol, EDTA (ethylenediaminetetra acetic acid), as well as the action of the trypsin and chymotrypsin enzymes . The characterization of glycoproteins was carried out by electrophoretic and staining for determination of total carbohydrates. The anti-hemolytic activity was performed using human ABO red blood cells, the antioxidant activity was determined by the molybdenum reduction methods (total antioxidant) and capture of the DPPH (2,2-diphenyl-1-picryl-hidrazila), and ABTS (2,2' sulfonic -azinobis-3-ethylbenzthiazoline-6 acid), antitumor activity was determined by the MTT (3- [4,5-dimethyl-thiazol-2-yl] -2,5-diphenyltetrazolium bromide) against cells melanoma A375 and the gastroprotective the protection induced gastropathy ethanol. The hemagglutinating activity indicated the presence of lectins only able to agglutinate erythrocytes of type A. This was inhibited only by lectin-carbohydrate N-acetyl-D-galactosamine at a concentration of 25 mM. The molecular exclusion was enough to isolate the lectin, where the peak I obtained from chromatography was active against type A red blood cells (PLUN). The estimated weight by EGPA indicated that it has about 128 kDa. The final purification yield was 80%, and this purified protein about 28 times. Thermostability and pH indicated variations in resistance to 80°C and pH between 2 and 11. Only the 8 M urea was able to inhibit the lectin. The 250 mM EDTA was inhibitory effect on hemagglutination activity was recovered in the presence of manganese ions. PLUN is a glycoprotein, with approximately 2% of carbohydrates in its composition. The lectin has no anti-hemolytic effect for no human blood type. PLUN has antioxidant action with AAEAA (μMAA/g) of 418.20, 326 and 82.9 for total antioxidant activity, ABTS radical capture and capture of DPPH radical, respectively. The IC50 (mg/ml) stood at 1.22, 0.21 and 4.39 for the tests cited above, respectively. The lectin has antitumor activity against melanoma derived cells at doses of 100 and 50 mg/ml, reducing up to 83% tumor cells, and gastroprotective action in a concentration of 1000 mg / kg, reducing up to 63% of the injured area stomach.