EXTRACTION OF LECTIN FROM NATURAL RAW MATERIALS AND STUDY OF ITS PHYSICOCHEMICAL PROPERTIES
Keywords:
Lectin, Phaseolus vulgaris, protein purification, Affi-gel blue gel, FPLC (fast protein liquid chromatography), ion-exchange chromatography, gel filtration, subunits, N-terminal structure, hemagglutination activity, pH and temperature stability, MCF-7 cells, breast cancer, HIV-1 reverse transcriptase, inhibition, mitogenic response, thymidine incorporation, macrophages, nitric oxide production, chemical modification, tryptophan, and activity.Abstract
Lectin was extracted from Phaseolus vulgaris seeds and purified using Affi-gel blue gel, fast protein liquid chromatography (FPLC) – ion-exchange chromatography, and FPLC-gel filtration methods. The lectin consists of two 30 kDa subunits, and its N-terminal structure was found to be similar to other Phaseolus lectins. The hemagglutination activity of the lectin remained stable within a pH range of 1-14 and a temperature range of 0-80°C. The lectin strongly inhibited the proliferation of MCF-7 (breast cancer) cells with an IC50 of 1.3 μM and suppressed HIV-1 reverse transcriptase activity with an IC50 of 7.6 μM. The lectin also exhibited activity by increasing [3H-methyl]-thymidine incorporation in mouse splenocytes in response to a mitogenic stimulus. However, it did not stimulate nitric oxide production in murine peritoneal macrophages. Chemical modification results indicated that tryptophan is crucial for the hemagglutination activity of the lectin.
References
Peumans W.J. and Van Damme E.J.M., Lectins as plant defense proteins, Plant Physiology 1995; 109:347-350
Barondes S.H. Bifunctional properties of lectins: lectins redefined, Trends Biochem Sci 1988; 13:480-2.
Sumner J.B., Gralën N., Eriksson-Quensel I.B., "The molecular weights of canavalin, concanavalin A and Concanavalin B", The Journal of Biological Chemistry 2002; 125: 45–48.
Goldstein I.J., Poretz R.D., "Isolation, physicochemical characterization, and carbohydrate-binding specificity of lectins", The Lectins Properties, Functions and Applications in Biology and Medicine 1986; 25: 233–247.
Van Parijs J., Willem F., Broekaert W.F., Peumans W.J., Hevein: an antifungal protein from rubber-tree latex, Planta 1991; 183: 258-264.
Van Damme E.J.M., Willy J.P., Annick B., Pierre R., Plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biological roles, Crit. Rev. Plant Science 1998; 17: 575–692.
Ofek I. and Sharon N., Adhesins as lectins: specificity and role in infection, Curr Top Microbiol Immunol 1990; 151, 91–113.
Ofek I. and Doyle R.J., Common themes in bacterial adhesion, In: Bacterial Adhesion to Cells and Tissues 1994; 35: 513-562.
Ofek I. and Doyle R.J., Recent developments in bacterial adhesion to animal cells. In: Bacterial Adhesion to Cells and Tissues 1994; 33: 321-512.
Etzler M.E., Distribution and Function of Plant Lectins. In: The lectins Properties Functions and Applications in Biology and Medicine, Liener, Academic Press 1986; 3: 371-435.
Van Damme E.J.M., Willy J.P., Annick B., Pierre R., Plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biological roles, Crit. Rev. Plant Science 1998; 17: 575-692.
Vijayan M. and Chandra N., Lectins, Curr. Opin. Struct. Biol 1999; 9: 707- 714.
Rahbe Y., Sauvion N., Febvay G., Peumans W.J., Gatehouse A.M.R., Toxicity of lectins and processing of ingested proteins in the pea aphid Acyrthosiphon pisum, Entomol Exp Appl 1995; 76: 143-155
Hilder V.A., Powell K.S., Gatehouse A.M.R., Gatehouse J.A., Gatè-house L.N., Shi Y., Hamilton W.D.O., Merryweather A., Newell C., Timans J.C., Peumans W.J., Van Damme E.J.M., Boulter D., Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids Transgenic Res. 1995; 4: 18-25.
Collinge D. B., Kragh K. M., Mikkelsen J. D., Nielsen K. K., Rasmussen U., Vad K., Plant chitinases, The Plant Journal 1993; 3, 31–40.
Kieliszewski M.J., O'Neill M., Leykam J., Orlando R., Tandem mass spectrometry and structural elucidation of glycopeptides from a hydroxyproline-rich plant cell wall glycoprotein indicate that contiguous hydroxyproline residues are the major sites of hydroxyproline-O-arabinosylation, J Biol Chem 1995; 270: 2541–2549.
Laemmli U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 1970; 227: 680–685.
Favre J., A simple and rapid method for the determination of the molecular weights of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, J Biol Chem 1975; 250: 7453–7455.
Collins P.W., Ng T.B., Tam J.P., A simple chemical method for the synthesis of bifunctional heterocrosslinkers, J Biol Chem 1991; 266: 14106–14109.
Wong C.K., Ng T.B., Anti-inflammatory and immunomodulating properties of Cinnamomum zeylanicum, Biochem Pharmacol 1999; 58: 1791–1798.
