Anais da Academia Brasileira de Ciências (2017) 89(3 Suppl.

): 2113-2117
(Annals of the Brazilian Academy of Sciences)
Printed version ISSN 0001-3765 / Online version ISSN 1678-2690
http://dx.doi.org/10.1590/0001-3765201720160150
www.scielo.br/aabc | www.fb.com/aabcjournal

Detection, purification and characterization of a lectin

from freshwater green algae Spirogyra spp.

ANTÔNIA S. DE OLIVEIRA1, CLÁUDIA F. LÓSSIO1, ANNE J. RANGEL2, MARIA G.Q.

MARTINS1, FERNANDO E.P. DO NASCIMENTO1, MARIA L.L. DE ANDRADE 3, BENILDO
S. CAVADA1, SÍRLEIS R. LACERDA2 and KYRIA S. DO NASCIMENTO1

¹Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará,

Rua José Aurélio Câmara, s/n, Pici, 60440-970 Fortaleza, CE, Brazil
²Departamento de Química Biológica, Universidade Regional do Cariri, Rua Cel.
Antônio Luis, 1161, Pimenta, 63100-000 Crato, CE, Brazil
³Colegiado de Medicina, Universidade Federal do Vale do São Francisco, Av. José
de Sá Maniçoba, s/n, Centro, 56304-917 Petrolina, PE, Brazil

Manuscript received on March 17, 2016; accepted for publication on June 17, 2016

ABSTRACT
Freshwater algae are rich sources of structurally biologically active metabolites, such as fatty acids,
steroids, carotenoids and polysaccharides. Among these metabolites, lectins stand out. Lectins are proteins
or glycoproteins of non-immune origin which bind to carbohydrates or glycoconjugates, without changing
ligand structure. Many studies have reported on the use of Spirogyra spp. as effective bioindicators of heavy
metals; however, reports on Spirogyra molecular bioprospecting are quite limited. Therefore, this study
aimed to detect, isolate, purify and characterize a lectin present in the freshwater green algae Spirogyra.
Presence of the lectin protein in the extract was detected by hemagglutination assays. Subsequently,
the protein extract was subjected to a sugar inhibition assay to identify the lectin-specific carbohydrate.
Following this, the extract was applied to a guar gum column to afford the pure lectin. The lectin was
inhibited by N-acetyl-glucosamine and N-acetyl-beta-D-mannose, but more strongly by D-galactose. The
apparent molecular mass of the purified lectin was evaluated by Polyacrylamide gel electrophoresis in the
presence of sodium dodecyl sulfate (SDS-PAGE). Electrophoretic analysis revealed a single protein band
with an apparent molecular mass of 56 kDa. Thus, it could be concluded that a lectin was purified from
Spirogyra spp.
Key words: algae, characterization, lectin, purification.

INTRODUCTION fixation, organic carbon uptake and oxygen release.

Their by-products are important tools with high
Together with their symbiotic partners, algae form a
economic value, such as polysaccharides, lipids,
diverse group of organisms that play a fundamental proteins and pigments (Cardozo et al. 2007).
role in biogeochemical cycles, food chains, nitrogen In particular, lectins are proteins of non-
Correspondence to: Benildo Sousa Cavada immune origin which bind specifically and
E-mail: [email protected] reversibly to carbohydrates (Peumans and Van

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2114 ANTÔNIA S. DE OLIVEIRA et al.

Damme 1995). As such, lectins have been used to a large system (Lee and Chang 2011, Kumar and
as tools to identify aberrant glycans expressed Oommen 2012, Mane and Bhosle 2012).
by neoplastic cells and verify the interactions Apart from Kumar et al. (2011) who
between pathogens and carbohydrates of host cells characterized the fatty acid profile of Spirogyra and
to determine the etiology of microbial diseases Kang et al. (2015) who isolated gallic acid with
(Bennett and Roberts 2005, Teixeira et al. 2012). vasorelaxant and antihypertensive effects, reports
The ability of lectins to decipher glycocodes on Spirogyra molecular bioprospecting are quite
makes them molecules with high biotechnological limited.
potential. Moreover, lectins isolated from species Thus, to broaden our understanding of the
of Cyanobacteria, such as Nostoc ellipsosporum, phytochemical potential of Spirogyra spp., the
Microcystis viridis, M. aeruginosa, Oscillatoria present study aimed to detect, isolate, purify and
agardhii, and Scytonema varium, as well the red characterize the lectin extracted from this algae.
alga Griffithsia sp., possess antiviral properties and Thus contributing to molecular research on
thus can be a potent candidate for the prevention of Spirogyra spp. and enriching the characterization
HIV transmission (Li et al. 2008). Lectins isolated of biomolecules produced by species of this genus.
from Griffithsia and the Cyanobacteria Scytonema
MATERIALS AND METHODS
varium also showed antiviral activity against the
hepatitis C virus (HCV) (Takebe et al. 2013). Such MATERIAL

antiviral activity is attributed to binding between

Algal biomass of Spirogyra spp. was collected at
lectins and high-mannose glycans present in the
Rosário Dam (6°46’7.53 “S and 38°57’9.79”W),
glycoproteins of viral membranes, thus blocking Lavras da Mangabeira, Ceará, Brazil. Rabbit
the entry of viruses into target cells. erythrocytes were obtained from the Federal
Spirogyra (Charophyta) is a filamentous type University of Ceará (UFC), and human blood was
of green algae belonging to the Zygnemataceae obtained from donors at the Hematology Center
family usually found in freshwater environments. of UFC. Reagents were purchased from Sigma-
These algae are characterized by helical, or AldrichTM, Bio-Rad and GE HealthcareTM.
spherical, arrangement of the chloroplasts and
HEMAGGLUTINATION AND INHIBITION ASSAY
filamentous green masses surrounded by mucilage
stems. These algae are frequently found in Assay of hemagglutinating activity was
relatively clean eutrophic, well-oxygenated water, accomplished through serial dilution of the
especially in acidic medium (Guiry and Guiry protein sample in 0.1 M Tris-HCl pH 7.6 buffer
2015, Franceschini et al. 2010). containing 0.15 M NaCl, followed by addition of
Moreover, they are indicative of environmental 2% erythrocytes treated or untreated enzymatically.
ecological state (Hainz et al. 2009), because The result was assigned as H.U./mL, being defined
lectins are related to high levels of pollution as the reciprocal of the highest dilution capable
with a high concentration of nutrients and heavy of agglutinating erythrocytes. To determine the
metals (Naskar et al. 2009). These characteristics lectin-specific sugar, hemagglutinating activity
have prompted many studies reporting the use of was inhibited using various sugars (D-mannose,
Spirogyra spp. as effective bioindicators of heavy D-glucose, N-acetyl- ɑ-D-glucosamine, ɑ-lactose,
metal accumulation, such as copper, chromium D-galactose, methyl-ɑ-D-galactopyranoside,
and zinc, which may be used in the future scaled ß-lactose, N-acetyl-ß-D-mannose and mannitol).

An Acad Bras Cienc (2017) 89 (3 Suppl.)

 DETEC, PURIF AND OF A LEC FROM FRESH GREEN alg Spirogyra spp. 2115

Each sugar at a concentration of 0.1 M was MOLECULAR MASS DETERMINATION

submitted to serial dilution with 0.1 M Tris-HCl

Purity and apparent molecular weight were
buffer pH 7.6 with 0.15 M NaCl. Subsequently, a
evaluated by polyacrylamide gel electrophoresis
solution containing 4 H.U. of the lectin was added
in the presence of dodecyl sodium sulfate (SDS-
to each sugar dilution, followed by incubation at 37 PAGE) performed on a 12% gel according to
°C for 1 hour. Then, native rabbit erythrocytes were Laemmli (1970). Samples were solubilized in
added to the sugars incubated with the lectin. The sample buffer (80 mM Tris-HCl pH 6.8, 10%
results were determined as the minimum inhibitory glycerol, 0.02% bromophenol blue and 2% SDS)
concentration (MIC) of sugar capable of inhibiting to a final concentration of 4 mg/mL. The proteins
lectin hemagglutinating activity (Verbet 1995). were stained with a 0.12% Coomassie brilliant
Inhibition of hemagglutination activity by sugars blue R-250 solution. Excess dye was removed by
was conducted with the protein extract and the washing the gel in hot distilled water.
isolated lectin.
RESULTS
LECTIN PURIFICATION
Based on quantification of total protein extract, it
Spirogyra specimens were washed with distilled was observed that Spirogyra spp. has low protein
water and macerated in liquid N2 to obtain a fine content (0.153 mg/mL). The lectin (SpyL) detected
powder. This powder was then subjected to protein in the protein extracts preferentially agglutinated
extraction at a 1:3 (w/v) ratio in 0.1 M Tris-HCl native rabbit erythrocytes (16 HU/ ml).
pH 7.6 buffer containing 0.15 M NaCl and 0.01 Carbohydrate binding specificity was
M phenylmethanesulfonylfluoride (PMSF) under determined by inhibition of hemagglutinating
constant stirring for 4 hours. Subsequently, the activity. The lectin was inhibited by N-acetyl-
extract was centrifuged at 9.000 g for 30 minutes glucosamine (MIC 0.025 M) and N-acetyl-β-D-
at 4 °C (Eppendorf Centrifuge 5810R) and the mannose (MIC 0.0125 M), but more strongly by
D-galactose (MIC 0.00625 M) (Table I).
supernatant applied to a column (6 mL bed volume)
The Spirogyra spp. lectin (SpyL) was purified
of guar gum (GE Healthcare, USA) and equilibrated
from the total extract using a guar gum column
with the extraction buffer. After 12 hours of gel
chromatographic step. The lectin was eluted with
contact, the proteins not bound to the matrix were
galactose 0.1 M in NaCl 0.15 M, and its activity
eluted with extraction buffer (1 mL/min), and the
was observed after removal of sugar by dialysis in
lectin was eluted with a solution of 0.1 M galactose
0.1 M sodium acetate buffer pH 4.0 with 0.15 M
with 0.15 NaCl. Fractions of 1 mL were collected
NaCl, followed by dialysis in distilled water. The
and monitored at 280 nm (Ultrospec 2100 pro UV/ hemagglutinating activity of the peak was detected
Vis spectrophotometer, Amersham Biosciences). using native rabbit erythrocytes and resulted in
The protein fractions for each peak were dialyzed activity of 32 H.U./mL.
exhaustively against distilled water and lyophilized. The apparent molecular mass of the lectin SpyL
The total extract and the chromatographic fractions was determined by SDS-PAGE which showed a
were subjected to soluble protein dosage (Bradford single band with an apparent molecular mass of 56
1976) and hemagglutination activity. kDa (Figure 1).

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2116 ANTÔNIA S. DE OLIVEIRA et al.

TABLE I and biomedical research development, for instance,

Inhibitory effect of sugars on the hemagglutinating assay
the lectins of the microalgaes Nostoc ellipsosporum
of Spirogyra spp. total extract.
(11 kDa), Scytonema varium (9,7 kDa), Microcystis
Minimum Inhibitory
Carbohydrate
Concentration (MIC) viridis (13 kDa) and Oscillatoria agardhii (13
D-mannose NI kDa), all mannose specific, are capable of binding
D-glucose NI the glycoprotein gp120 of virus HIV (Boyd et al.
N-acetyl-ɑ-D-glucosamine 0.025 1997, Bokesch et al. 2003, Yamaguchi et al. 1999,
ɑ-lactose NI Sato et al. 2007, Li et al. 2008).
D-galactose 0.00625
Among the lectins of microalgae studied, a
ß-lactose NI
lectin from cyanobacterium Microcystis aeruginosa
N-acetyl-ß-D-mannose 0.0125
presented the highest physicochemical similarity to
Methyl-ɑ-D-galactopyranoside NI
Spirogyra spp., due to its high molecular weight
Mannitol NI
and galactose inhibited hemagglutinating activity
NI: not inhibited.
(Yamaguchi et al. 1998). More studies, however,
are needed to further evaluate the biotechnological
potential of galactose specific microalgae lectins.
This paper described the detection and
purification of a galactose-specific lectin present
in green microalgae Spirogyra spp. collected from
Rosario Dam in Lavras da Mangabeira-CE, Brazil.
Properties of Spirogyra lectin are similar to those
of lectins isolated from other green algae, such as
high molecular weight and specificity for galactose.

ACKNOWLEDGMENTS
Figure 1 - a. Chromatographic profile of Spirogyra spp. total
extract applied in guar gum column; PI eluted with 0.1 M Tris- This work was supported by Conselho Nacional de
HCl pH 7.6 with 0.15 M NaCl; PII eluted with 0.1 M galactose
Desenvolvimento Científico e Tecnológico (CNPq)
with 0.15M NaCl. b. 12% Polyacrylamide gel electrophoresis
in the presence of sodium dodecyl sulfate (SDS-PAGE). Well and Coordenação de Aperfeiçoamento de Pessoal
1: Molecular markers (phosphorylase b, 97 kDa; bovine serum de Nível Superior (CAPES). B. S. Cavada is senior
albumin, 66 kDa; ovalbumin, 45 kD; trypsin inhibitor 20.1 kDa
investigator of CNPq.
and α-lactalbumin 14.4 kDa). Well 2: Total extract. Well 3:
Guar gum PII.
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