Purification and physicochemical characterization of a cotyledonary lectin from Luetzelburgia auriculata

• Published in: Phytochemistry (Oxford) Vol. 61; no. 3; pp. 301 - 310
• Main Authors: Oliveira, José T.A, Melo, Vânia M.M, Câmara, Maria F.L, Vasconcelos, Ilka M, Beltramini, Leila M, Machado, Olga L.T, Gomes, Valdirene M, Pereira, Silvano P, Fernandes, Cléberson F, Nunes, Edson P, Capistrano, Gina G.G, Monteiro-Moreira, Ana C.O
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 01.10.2002; Elsevier
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbohydrates - analysis; Characterization; Chromatography, Ion Exchange; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Fabaceae - chemistry; Fundamental and applied biological sciences. Psychology; Glycoproteins; Hemagglutination; Hot Temperature; Hydrogen-Ion Concentration; Isoelectric Focusing; Lectin; Lectins - chemistry; Lectins - isolation & purification; Lectins - metabolism; Leguminoseae; Luetzelburgia auriculata; Molecular Sequence Data; Molecular Weight; Proteins; Purification; Sequence Analysis, Protein; Lectin; NBT; Purification; Luetzelburgia auriculata; IEF; EDTA; LAA; PVDF; Characterization; BSA; BCIP; Leguminoseae; PHT; Hemagglutination; Cotyledon; Leguminosae; Affinity chromatography; Dicotyledones; Angiospermae; Spermatophyta; Biological activity
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/S0031-9422(02)00239-X

A lectin was purified from the cotyledons of Luetzelburgia auriculata (Fr. All) Ducke by affinity chromatography on agarose-N-acetyl-d-galactosamine. The lectin is a potent agglutinin for rabbit erythrocytes, reacts with human red cells, but is inactive against cow, sheep, and goat erythrocytes. Hemagglutination of rabbit erythrocytes was inhibited by either 0.39 mM N-acetyl-neuraminic acid or N-acetyl-d-galactosamin, 12.5 mM d-lactose or d-melibiose, 50 mM d-galactose or raffinose. Its hemagglutinating activity was lost at 80 °C, 5 min, and the activation energy required for denaturation was 104.75 kJ mol−1. Chromatography on Sephadex G-100, at pH 7.6, showed that at this hydrogenic ionic concentration the native lectin was a homotetramer (123.5 kDa). By denaturing SDS-PAGE, LAA seemed to be composed of a mixture of 29 and 15 kDa polypeptide subunits. At acidic and basic pHs it assumed different conformations, as demonstrated by exclusion chromatography on Superdex 200 HR 10/30. The N-terminal sequence of the 29 kDa band was SEVVSFSFTKFNPNQKDII and the 15 kDa band contained a mixture of SEVVSFSFTKFNPNQKDII and KFNQIVAVEEDTDXESQPQ sequences, indicating that these bands may represent full-length and its endogenous fragments, respectively. The lectin is a glycoprotein having 3.2% neutral carbohydrate, with a pI of 5.8, containing high levels of Asp+Asn and Glu+Gln and hydroxy amino acids, and low amount or absence of sulfur amino acids. Its absorption spectrum showed a maximum at 280 nm and a ε1%1cm of 5.2. Its CD spectrum was characterized by minima near 228 nm, maxima near 196 nm and a negative to positive crossover at 210 nm. The secondary structure content was 6% α-helix, 8% parallel β-sheet, 38% antiparallel β-sheet, 17% β-turn, 31% unordered and others contribution, and 1% RMS (root mean square). In the fluorescence spectroscopy, excitation of the lectin solution at 280 nm gave an emission spectrum in the 285–445 nm range. The wavelength maximum emission was in 334.5 nm, typical for tryptophan residues buried inside the protein. A lectin from the cotyledons of Luetzelburgia auriculata is purified and characterized physicochemically.