Purification and characterisation of a lectin from the red marine alga Pterocladiella capillacea (S.G. Gmel.) Santel. & Hommers.

Abstract

-(Purification and characterisation of a lectin from the red marine alga Pterocladiella capillacea (S.G. Gmel.) Santel. & Hommers.). A lectin present in the marine red alga Pterocladiella capillacea was purified and characterised by extraction of soluble proteins (crude extract) in 20 mM Tris-HCl buffer, pH 7.5. Among the analysed erythrocytes (human blood group A, B and O and the animals ox, goat, chicken and rabbit) the lectin agglutinated specifically rabbit erythrocytes. The hemagglutinating activity assay showed that the lectin was not dependent on divalent cations and was shown to be inhibited by the glycoproteins avidin and mucin. The purification procedure was conduced by precipitation of the crude extract with 80% saturation ammonium sulfate (F0/80) followed by affinity chromatography on guar-gum column. The lectin of P. capillacea was purified 14.5 fold and had a recovery of 27.4% of the original total specific activity present in the crude extract. The absence of carbohydrate suggested that the lectin is not a glycoprotein. The molecular mass of P. capillacea lectin, determined by gel filtration, was 5.8 kDa. SDS-PAGE in the presence of β-mercaptoethanol gave one band, indicating that the native lectin is a monomeric protein. The activation energy of denaturation process (∆G') was calculated to be 106.87 kJ . mol -1 at 70 o C.RESUMO -(Purificação e caracterização de uma lectina da alga marinha vermelha Pterocladiella capillacea (S.G. Gmel.) Santel. & Hommers.). Uma lectina presente na alga marinha vermelha Pterocladiella capillacea foi purificada e caracterizada pela extração de proteínas solúveis (extrato bruto) em tampão Tris-HCl 20 mM, pH 7,5. Entre os eritrócitos testados (grupos sangüíneos humanos A, B e O e os animais boi, cabra, galinha e coelho) a lectina aglutinou especificamente eritrócitos de coelho. O ensaio de atividade hemaglutinante evidenciou que a lectina não era dependente de cátions divalentes e inibida pelas glicoproteínas avidina e mucina. O procedimento de purificação foi conduzido por precipitação protéica do extrato bruto com sulfato de amônio até 80% de saturação (F 0/80), seguido por cromatografia de afinidade em coluna de goma de Guar. A lectina de P. capillacea foi purificada 14,5 vezes e teve uma recuperação de 27,4% da atividade específica total original presente no extrato bruto. A ausência de carboidrato sugeriu que a lectina não é uma glicoproteína. A massa molecular da lectina de P. capillacea, determinada por filtração em gel, foi de 5,8 kDa. PAGE-SDS em presença de β-mercaptoetanol mostou uma única banda protéica, indicando que a lectina de P. capillacea é uma proteína monomérica. A energia de ativação do processo de desnaturação (∆G') foi calculada em 106,87 kJ . mol -1 a 70 o C.

Purification and partial characterisation of a lectin from the red marine alga Vidalia obtusiloba C. Agardh

“…The energy of activation of denaturation process (∆G') was estimated to be 25.4 Kcal.Mol -1 , which is similar to the values found for the lectin isolated from Pterocladiella capillacea Santel & Hommers (Oliveira et al 2002).…”

“…The lectin was also inhibited by D-galactose and others derivatives (α-lactose and D-galactosamine), and was strongly inhibited by the glycoprotein porcine stomach mucin (62.5 µg.mL -1 ), a glycoprotein with a terminal GalNAc residue, and fucose and galactose as internal residues (Slomiany & Mayer 1972). Similar observations of inhibition by mucin on , Enantiocladia duperreyi (C. Agardh) Falkenberg (Benevides et al 1998), Ptilota serrata Kützing and Pterocladiella capillacea Santel & Hommers (Oliveira et al 2002). The hemagglutinating activity of Vidalia obtusiloba lectin was completely abolished when 5 mM EDTA was added to the reaction medium.…”

“…The lectin of the red marine alga Vidalia obtusiloba was purified by extraction of soluble proteins in 20 mM Tris-HCl buffer, pH 7.5, containing 0.005 M CaCl 2 (TB) following the procedures as described by Oliveira et al (2002). The crude extract was previously submitted to the treatment with 60% saturated ammonium sulphate and the protein fraction 0%-60% obtained was subjected to ion exchange chromatography on a column of DEAE-cellulose.…”

“…The blood specificity from the Vidalia obtusiloba lectin was determined by use of erythrocytes from different animals (rabbit, chicken, ox, goat) and humans from the ABO system, native and treated with the enzymes trypsin, bromelain, papain and subtilisin, according to Oliveira et al (2002).…”

“…They occur mainly in monomeric forms and have a high proportion of acidic amino acids, with isoeletric points from 4 to 6 (Shiomi et al 1981. Most algal lectins have been isolated from red algae, such as Bryothamnion seaforthii (Turner) Kützing and B. triquetrum (Gmelim) Howe (Ainouz et al 1995), Solieria filiformis (Kützing) Gabrielson (Benevides et al 1996), Enantiocladia duperreyi (C. Agardh) Falkenberg (Benevides et al 1998) and Pterocladiella capillacea Santel & Hommers (Oliveira et al 2002). The lectins isolated from red marine algae Bryothamnion triquetrum (Gmelim) Howe and Hypnea japonica Tanaka were the first lectins to have their primary structure elucidated by Calvete et al (2000) and Hori et al (2000), respectively.…”