Molecular characterization and tandem mass spectrometry of the lectin extracted from the seeds of Dioclea sclerocarpa Ducke.
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).
Laboratório de Moléculas Biologicamente Ativas (Biomol-Lab), Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Av. Humberto Monte s/n, Bloco 907, Lab. 1075, Campus do Pici, Fortaleza-CE, 60440-970, Brazil.
Jorge Luis Almeida
JL
Correia
Antônia Sâmia Fernandes
AS
do Nascimento
João Batista
JB
Cajazeiras
Ana Cláudia Silva
AC
Gondim
Ronniery Ilario
RI
Pereira
Bruno Lopes
BL
de Sousa
André Luiz Coelho
AL
da Silva
Wanius
W
Garcia
Edson Holanda
EH
Teixeira
Kyria Santiago
KS
do Nascimento
Bruno Anderson Matias
BA
da Rocha
Celso Shiniti
CS
Nagano
Alexandre Holanda
AH
Sampaio
Benildo Sousa
BS
Cavada
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D052598:Dioclea / Q000033:anatomy & histology / Q000737:chemistry*; D006384:Hemagglutination / Q000187:drug effects; D037102:Lectins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D011817:Rabbits; D012639:Seeds / Q000737:chemistry*; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry / Q000379:methods*
D037102:Lectins