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Molecular Characterization and Tandem Mass Spectrometry of the Lectin Extracted from the Seeds of Dioclea sclerocarpa Ducke

2011; Multidisciplinary Digital Publishing Institute; Volume: 16; Issue: 11 Linguagem: Inglês

10.3390/molecules16119077

1433-1373

Jorge Luís Almeida Correia, Antônia Sâmia Fernandes do Nascimento, João Batista Cajazeiras, Ana C. S. Gondim, Ronniery Ilário Pereira, Bruno Lopes de Sousa, André Luiz Coelho da Silva, Wanius García, Edson Holanda Teixeira, Kyria Santiago Nascimento, B.A.M. Rocha, Celso Shiniti Nagano, Alexandre Holanda Sampaio, Benildo Sousa Cavada,

Transgenic Plants and Applications

Lectin from the seeds of Dioclea sclerocarpa (DSL) was purified in a single step by affinity chromatography on a Sephadex G-50 column. The primary sequence, as determined by tandem mass spectrometry, revealed a protein with 237 amino acids and 81% of identity with ConA. DSL has a molecular mass of 25,606 Da. The β and γ chains weigh 12,873 Da and 12,752 Da, respectively. DSL hemagglutinated rabbit erythrocytes (both native and treated with proteolytic enzymes), showing stability even after one hour of exposure to a specific pH range. The hemagglutinating activity of DSL was optimal between pH 6.0 and 8.0, but was inhibited after incubation with D-galactose and D-glucose. The pure protein possesses a molecular mass of 25 kDa by SDS-PAGE and 25,606 Da by mass spectrometry. The secondary structure content was estimated using the software SELCON3. The results indicate that b-sheet secondary structures are predominant in DSL (approximately 42.3% antiparallel b-sheet and 6.7% parallel b-sheet). In addition to the b-sheet, the predicted secondary structure of DSL features 4.1% a-helices, 15.8% turns and 31.3% other contributions. Upon thermal denaturation, evaluated by measuring changes in ellipticity at 218 nm induced by a temperature increase from 20 °C to 98 °C, DSL displayed cooperative sigmoidal behavior with transition midpoint at 84 °C and permitted the observation of two-state model (native and denatured).