127
PLANT SEED LECTINS. A POSSIBLE MARKER FOR
1
CHEMOTAXONOMY OF THE GENUS Canavalia
2
3
RENATO DE AZEVEDO MOREIRA , ENILENE DE FRANÇA CORDEIRO ,
2
4
BENILDO SOUSA CAVADA , EDSON PAULA NUNES , AFRÂNIO GOMES
5
2
FERNANDES and JOSÉ TADEU ABREU OLIVEIRA .
Departamento de Bioquímica e Biologia Molecular, Centro de Ciências,
Universidade Federal do Ceará, CP 6020, Fortaleza, Ceará, 60451-970, Brazil
ABSTRACT- Canavalia bicarinata, Canavalia bonariensis,
Canavalia brasiliensis, Canavalia dictyota, Canavalia
ensiformis, Canavalia gladiata, Canavalia maritima and
Canavalia plagyosperma seeds were investigated with respect
to phenologic aspects (size, mass, hilum length) and with
respect to their chemical composition. Thus, the total nitrogen
content of the seed flours were determined, and the flours were
submitted to extraction at different pH values. The seed
extracts were investigated with respect to their behavior in
affinity chromatography on Sephadex G-50 column,
polyacrylamide gel electrophoresis in the presence of SDS and
2-mercaptoethanol (SDS-PAGE) and Ouchterlony double
immunodiffusion. Although all the species investigated have
shown the presence of lectins, their contents and extractibility
at different pH show some differences. By affinity
chromatography, it was noted that although every extracted
lectin has been retained in the same column, their content in
the seeds studied varied somehow. When the SDS-PAGE
protein bands were compared, it was found very close
qualitative similarities although important quantitative
differences were observed. On the other hand, when the seed
extracts were allowed to diffuse against IgG anti Canavalia
brasiliensis lectin, it was obtained total immunological identity
among all the seeds. Preliminary phenograms prepared with
the data obtained are shown.
Additional index terms: seeds.
LECTINAS VEGETAIS. UM MARCADOR
PARA A QUIMIOTAXONOMIA DO
GENERO Canavalia
RESUMO- Sementes de Canavalia bicarinata, Canavalia
bonariensis, Canavalia brasiliensis, Canavalia dictyota,
Canavalia ensiformis, Canavalia gladiata, Canavalia maritima
e Canavalia plagiosperma foram estudadas quanto aos
aspectos fenológicos (tamanho, massa e comprimento do hilo)
e quanto à sua composição química. Assim, farinhas de
sementes tiveram o teor de nitrogênio total determinado e
1Recebido em 02/04/1993 e aceito em 19/10/1993. Research supported by CAPES, CEDCT, CNPQ, FBB and IFS.
2Prof. Adjunto, Dr., Depto de Bioquímica e Biologia Molecular.
3Bolsista IC CNPQ, Depto de Bioquímica e Biologia Molecular.
4Prof. Assistente, Agrônomo, Depto. de Biologia.
5Prof. Visitante, Agrônomo, Depto. de Biologia, Centro de Ciências, Universidade Federal do Ceará, CP 6020, Fortaleza, Ceará,
60451-970, Brazil
foram submetidas à extração a diferentes valores de pH. Os
extratos obtidos foram investigados com respeito ao seu
comportamento por cromatografia de afinidade em coluna de
Sephadex G-50, eletroforese em gel de poliacrilamida em
presença de SDS e imunodifusão dupla de Ouchterlony.
Embora as sementes de todas as espécies estudadas tenham
mostrado a presença de lectinas, estas diferem não só no seu
teor total como na extratibilidade a diferentes valores de pH.
Por cromatografia de afinidade em Sephadex G-50 nota-se,
embora todas as lectinas extraidas tenham ficado retidas na
matriz, seus teores nas sementes variam. Quando as bandas
protéicas obtidas por SDS-PAGE são comparadas, há uma
semelhança qualitativa bastante acentuada embora com
importantes diferenças quantitativas. Por outro lado, quando
os extratos totais foram deixados difundir contra IgG anti lectina
de Canavalia brasiliensis, foi obtida identidade imunológica das
lectinas em todas as amostras. Fenogramas preliminares
preparados com os dados obtidos são apresentados.
Termos adicionais para indexação: sementes.
INTRODUCTION
The plants belonging to the genus Canavalia are
widely distributed in the tropical regions and are
important for animal nutrition due to their high protein
content. Despite this fact, very little has been done
about its taxonomy and ecology. The genus has been
arranjed (Aymard & Cuello, 1991) in the family
Leguminosae, Sub-Family Papilionoideae, Tribe
Phaseolae, Sub-tribe Diocleinae. The tribe Phaseoleae
comprises eight sub-tribes (Cajaninae, Phaseolinae,
Clitoriinae, Ophrestiinae, Kennediinae, Erythrininae,
Diocleinae and Glycininae) and the sub-tribe Diocleinae
is divided in 13 genera (Canavalia, Cleobulia,
Pachyrhizus, Collaea, Camptosema, Dioclea, Cratylia,
Galactia,
Cymbosena,
Herpiza,
Calopogonium,
Macropsichanthus and Luzonia) from which only the last
two are not present in the neotropics. The genus
Canavalia has been divided in four sub-genera,
Catadonia, Wenderothia, Canavalia, and Maunaloa
(Sauer, 1964; Lackey, 1977; Lackey, 1981; Aymard &
Cuello, 1991). Other authors (Hutchinson, 1964;
Heywood, 1971; Harborne, Boulter & Turner, 1971)
arrange the genus Canavalia in a different sub-tribe
(Diocleae, B. & H.f.) of the tribe Phaseoleae or even in
a separate tribe (Diocleae Hutch.)
R. Bras. Fisiol. Veg., 127-132, 1993.
128
TABLE 1- Numerical data for cluster analysis.
C.bic.
C.bon
C.bra.
C.dic.
Species1
C.ens.
C.gla
12.2
7.2
26.00
0.22
17.0
13.3
52.17
0.62
19.7
13.8
17.24
1.51
16.2
12.4
16.81
1.52
20.8
13.4
16.4
1.93
38.2
19.8
26.31
4.73
9.9
12.9
32.41
0.96
19.1
14.7
19.16
1.45
35.07
0.59
7.58
0.89
9.00
1.51
7.32
5.52
9.58
4.98
9.58
7.91
9.00
5.20
32.40
0.37
8.30
0.26
8.58
1.26
7.58
3.83
5.58
3.47
9.58
3.32
7.00
1.90
34.34
1.17
8.80
0.79
7.58
1.78
7.58
6.51
8.77
3.64
9.58
9.51
7.17
18.50
32.39
1.53
8.00
0.85
7.58
2.49
7.00
4.25
9.58
4.44
9.58
8.63
6.00
11.60
36.42
1.40
6.60
0.98
8.80
2.56
7.58
6.78
8.80
5.63
9.00
8.66
5.81
12.60
36.40
0.97
8.00
0.43
9.00
2.86
8.58
4.72
8.80
5.09
8.17
9.97
8.17
23.80
39.15
1.47
9.60
0.95
7.58
2.32
7.58
2.11
8.00
4.60
9.00
5.75
8.17
15.10
35.53
1.18
7.80
1.22
8.81
2.62
6.58
4.83
7.90
3.85
9.58
9.44
8.17
14.20
Data
Seed parameters
Longitudinal lenght (mm)
Transversal Length (mm)
Hylum (%)
Mass (g)
Bichemichal parameters
2
Total protein
pH 2.6 0.1 M NaCl3
pH 4.0 0.1 M NaCl3
pH 4.0 0.2 M NaCl3
pH 6.0 0.1 M NaCl3
pH 8.0 0.1 M NaCl3
pH 10.0 0.1 M NaCl
% Peak III4
C.mar.
C.pla.
1C. bicarinata (C.bin.), C. bonariensis (C.bon.), C. brasiliensis (C.bra.), C. dictyota (C.dic.), C. ensiformis (C.ensi.), C. gladiata (C.gla.), C.
maritma (C.mar.), C. plagiosperna (C.pla.).
2Obtained by multiplying the total nitrogen per 6.25 and given as mg per g of flour
3 The numbers given for each extraction correspond to extracted protein (mg per g of flour, upper) and hemagglutinating titer (lower).
4Obtained as percent of total protein from the Sephadex G-50 affinity chromatography (P I + PIII).
The use of proteins, metabolically important for the
plant, as taxonomic marker is well stablished. Thus, the
presence of seed lectins in tribes, genera and species
of the same family and the high homology found
between lectins from sources taxonomically related
makes them a suitable molecular tool for
chemotaxonomy and phyllogenetic investigations (Toms
& Western, 1971).
Lectins are carbohydrate-binding proteins of
non-immune origin, widely distributed in nature, that
interact with sugar containing substances, and are
capable of specific recognition and reversible binding to
carbohydrate, without altering covalent structures of any
glycosyl ligands (Moreira et al., 1991; Pusztai, 1991).
The lectins present in seeds of the sub-tribe
Diocleinae are glucose (mannose)-specific and, thus,
can be isolated by affinity chromatography on Sephadex
G-50 column (Moreira et al., 1983; Moreira & Cavada,
1984; Grangeiro et al., 1990; Cavada et al., 1990;
Oliveira et al., 1991; Vasconcelos et al., 1991).
The most rational classification of the lectins is based
on their carbohydrate-binding specificity (Goldstein &
Hayes, 1978). However, even within a particular group,
the
lectins
show
some
differences
in
carbohydrate-binding and physicochemical properties.
These differences are less pronounced when the lectins
are extracted from botanically related plants (Moreira &
Oliveira, 1983 a, 1983 b), reflecting evolutionary
relationships among species or tribes from the same
family. Thus, very close similarities have been found in
the amino acid sequences of the lectins present in
seeds of Dioclea grandiflora, Dioclea lehmanni,
Canavalia maritima and Canavalia ensiformis, all
belonging to the sub-tribe Diocleinae (Moreira et al.,
1983; Richardson et al., 1984, Ainouz et al., 1987; Perez
et al., 1991). Striking structural similarities were also
found among lectins of other members of this sub-tribe,
such as Canavalia ensiformis, Canavalia maritima,
Dioclea grandiflora, Dioclea selerocarpa and Cratylia
floribunda (Moreira et al., 1985; Oliveira et al., 1990).
The lectins from seeds of Viceae species have been
considered phylogenetic markers in this tribe (Rouge et
al., 1987).
In this paper, a comparative study has been made
between the whole proteins and the lectins in particular,
from seeds of eight species belonging to three
subgenera of the genus Canavalia, as a tentative of their
use as marker for chemotaxonomy of this genus.
MATERIAL AND METHODS
R. Bras. Fisiol. Veg., 127-132, 1993.
129
V
U
V
FIGURE 1- Influence of the pH on the solubility of seed proteins
of Canavalia species: ( ) C. bicarinata, ( ) C. bonariensis,
( ) C. brasiliensis, ( ) C. dictyota, („) C. ensiformis, () C.
gladiata, C. maritima, (z) C. plagiosperma.
FIGURE 2-. Influence of the pH on the solubility of the
hemagglutinating activity of Canavalia species: ( ) C.
bicarinata, (∆) C. bonariensis, ( ) C. brasiliensis ( ), C.
dictyota, ( ) C. ensiformis, ( ) C. gladiata, () C. maritima,
(z) C. plagiosperma.
Seeds. The seeds (sub-genus Wenderothia: Canavalia
bicarinata, sub-genus Catadonia: Canavalia bonariensis, and sub-genus Canavalia: Canavalia brasiliensis,
C. dictyota, C. ensiformis, C. gladiata, C. maritima, C.
plagiosperma) used were collected in the State of Ceará
and State of Rio Grande do Sul or obtained from germoplasm bank of CENARGEN (Centro Nacional de Recurses Genéticos - Empresa Brasileira de Pesquisas
Agripecuária, Brasília).
unit (HU) was defined as the reciprocal of the highest
dilution still giving a visible agglutination (Moreira & Perrone, 1977). The hemagglutination titer was defined as
log2 HU and the minimum dose as the minimum amount
of protein still promoting a visible agglutination.
S
T
Erythrocytes. Rabbit blood was obtained by puncture of
the marginal ear’s vein of healthy animals.
Freund’s complete adjuvant was a product of B.D.H.
Acrylamide and methylene bisacrylamide were products
of Sigma Chemical Co. and Sephadex G-50 was from
Pharmacia, Uppsala.
Protein determination. The method described by Bradford (1976) was used with bovine serum albumin (BSA)
as standard. Readings at 280 nm were also employed
to determine protein content of the column eluates.
Agglutination assay. This was done in small glass tubes
where to a series of 1:2 dilutions of the crude extract
or purified lectins in 0.15 M NaCl containing 5 mM
CaCl2, 5 mM MnCl2 (0.25 ml) were added 0.25 ml of a
2% suspension of erythrocytes. The degree of agglutination was monitored visually after the tubes had been
left at 37 oC for 30 min and subsequently left at room
temperature for further 30 min. The hemagglutination
V
„
S
…
Protein extraction. The dehulled seeds were ground in
a coffee grinder (40 mesh) and the protein extracts were
prepared by treating the seed meal (1 g) with 20 ml of
0.1 M Glycine-HCl pH 2.6, 0.1 M Na-Acetate pH 4.0,
0.2 M Na-Acetate pH 4.0, 0.1 M Tris-HCI pH 6.0, 0.1
M Na-Borate pH 8.0 and 0.1 M Na-Borate pH 10.0, all
containing 0.1 M NaCl. The suspensions were left at
room temperature for 3 h and spun at 10,000 x g for
20 min, at 7 oC. The clear supernatant were used for
the various analyses.
Affinity chromatography. This was done as described
previously (Moreira & Cavada, 1984). The extracts obtained with borate pH 8.0 buffer were applied to a
Sephadex G-50 column (36 cm x 1.0 cm) equilibrated
with the same buffer, containing 5 mM CaCl2 and 5
mM MnCl2. After removing unbound material, the lectin
was desorbed from the column with 0.1 M glucose
added to the equilibrium solution. The eluates were collected in 3.6 ml fractions and analyzed for hemagglutinating activity and protein content.
IgG preparation. Antibody specific for the lectin from
Canavalia brasiliensis was raised in rabbits. Immuno-
R. Bras. Fisiol. Veg., 127-132, 1993.
130
FIGURE 3- Affinity chromatography of the seed proteins of Canavalia species. The seed flour was extracted (1:10 w:v) in 0.1 M
Na-borate, 0.15 M NaCl, pH 8.0 buffer (1 ml) and applied (1 ml) to a Sephadex G-50 (36 cm long; 10 cm diameter; 29 ml column
volume) column, equilibrated with the same buffer, in which it was dissolved 5 mM CaCl2 and 5 mM MnCl2. Elution flow rate was
22 ml h-1 and fractions of 2.0 ml were collected and read at 280 nm. Elutions were performed with the equilibrium buffer and
changed to 0.1 M glucose, dissolved in the same buffer, at fraction 25 (arrow). The total activity emerged with the glucose eluted
peak. 1- C. bicarinata, 2- C. bonariensis, 3- C. brasiliensis, 4- C. dictyota, 5- C. ensiformis, 6- C. gladiata, 7- C. maritima, 8- C.
plagiosperma.
specific IgG were obtained by fractionation of immunosera with ammonium sulfate followed by DEAE
Sephadex A-50 chromatography according to Harboe
and Ingild (1973).
13 mA for 4 h. The protein bands were visualized by
staining with Coomassie Brilliant Blue R-250.
Immunodiffusion. Ouchterlony radial double immunodiffusion were carried out according to Clausen (1969),
using 1% agarose gels prepared with 0.15 M NaCl,
containing 0.2 % sodium azide. The seed crude extracts
(20 µg) were applied in 8 wells in a circular distribution,
around a central well containing the IgG anti Canavalia
brasiliensis lectin (20 µg). The samples were allowed to
diffuse for 48 h, at room temperature, and the precipitated arcs examined by coloring the 0.15 M NaCl
washed gel plates with 0.005 % Coomassie Brilliant
Blue R-250.
Seed characteristics. The seed mass and size, and the
hylum lenght were determined and is presented in table
1.
Sodium Dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE). This was carried out according to
Laemmli (1970) in vertical 2 mm gel slabs consisting of
a stacking gel mix of 4% total acrylamide gel concentration, pH 6.8 and a main running gel mix containing
17.5% acrylamide concentration. Samples were dissolved in 0.01 M sodium phosphate, pH 7.0, 2% SDS
buffer with 1% 2-mercaptoethanol and incubated at 100
o
C for 15 min. A few crystals of sucrose were dissolved
in the samples which were then applied to the gel. The
electrophoresis was conducted at a constant current of
RESULTS
Nitrogen content. The total protein content (nitrogen
basis) of the seeds of the eight Canavalia species were
determined (Table 1), and it was found close values,
varying from 32.40 ± 3.50 to 39.15 ± 3.25 (n=12).
Solubility. The influence of pH on the solubility of the
proteins and hemagglutinating activity of Canavalia
were examined (Table 1, Fig. 1 and 2). It was found
that although the curves obtained for the solubility of
proteins behaved quite similarly (with some exceptions
as C. bonariensis and C. maritima), the curves for the
hemagglutinating activity varied significantly. It was also
found the maximum hemagglutinating activity at pH 8.0
(with the exception of C. gladiata).
Affinity chromatography. When the pH 8.0 extracts were
applied to a Sephadex G-50 affinity column (Fig. 3), it
was found typical chromatograms for the Diocleinae
proteins (Moreira et al, 1983; Moreira & Cavada, 1984;
Oliveira et al, 1991). Important differences were found
when the lectin peaks (PIII) were compared to the high
R. Bras. Fisiol. Veg., 127-132, 1993.
131
FIGURE 4- PAGE-SDS of the seed proteins of Canavalia
species. 1, 11 - Molecular mass marker (P- Phosphorylase B,
97.4 KDa; B- BSA, 66 KDa; C- carbonic anhydrase, 29 KDa;
L- Lysozyme, 14.3 KDa; 2- Dioclea grandiflora lectin; 3- C.
bicarinata; 4- C. bonariensis; 5- C. brasiliensis; 6- C. dictyota;
7- C. ensiformis; 8- C. gladiata; 9- C. maritima; 10- C.
plagiosperma.
molecular mass non retained protein peaks (PI), mainly
for C. bicarinata and C. bonariensis (Table 1).
SDS-PAGE. When the seed flours were extracted in
Na-phosphate buffer containing SDS and 2-mercaptoethanol and electrophoresed in polyacrylamide gel in
the presence of SDS and 2-mercaptoethanol (Fig. 4),
only small differences were observed in the distribution
of protein bands.
Ouchterlony immunodiffusion. Total identity was found
among the lectins from the Canavalia lectins investigated, when the pH 8.0 extrats were allowed to diffuse
against IgG anti C. brasiliensis lectin (data not shown).
Cluster analysis. The quantitative data obtained for the
eight Canavalia species were submitted to statistical
cluster analyses (Oliveira, 1991). The similarities found
were utilized for the construction of the tentative phenogram of Fig. 5.
DISCUSSION
The cluster analysis showed no apparent correlation
between the seed morphological characteristics (size,
mass and hylus lenght) and the species taxonomic
identification. The same result was obtained when the
protein contents (total and extrated protein) were used
for the analysis. On the other hand, when the lectin
parameters (hemagglutinating activity and the lectin
affinity peak) were analysed, alone or grouped with the
protein parameters, a statistical correlation was found,
with the 6 seeds belonging to the sub-genera Canavalia
grouped together, but separated from the other two
species which belongs to the sub-genera Catadonia and
Wenderothia. A less significant correlation was found
when the above parameters were analysed together
with the seed morphological characteristics.
FIGURE 5- Phenetic relationships of the Canavalia species.
1- C. dictyota, 2- C. ensiformis, 3- C. plagiosperma, 4- C.
brasiliensis, 5- C. gladiata, 6- C. maritima, 7- C. bicarinata, 8C. bonariensis.
In the phenogram prepared (Fig. 5), with the
quantitative data obtained, it can be clearly seen that, as
should be predicted by the systematic relationships, the
species belonging to the sub- genus Canavalia can be
grouped separated from the other two species
belonging to the sub-genera Catadonia, and
Wenderothia. In the case of Canavalia dictyota, not
clearly located in the sub-genus Canavalia or Maunaloa
(Aymard & Cuello, 1991), from the results of cluster
analysis, we can suggest its classification as Canavalia
(as suggested by Sauer, 1964).
When, on the other hand, the (qualitative) results
obtained from SDS-PAGE were compared, it was found
only small differences among the eight species, with a
very high similarity when the lectin bands were
compared. The same was found when the pH 8.0 crude
extracts were submitted to Ouchterlony double
immunodiffusion, with IgG raised against the Canavalia
brasiliensis lectin. Every extract had an arc recognized
by the IgG and, moreover, these arcs showed complete
immunological identity (data not shown).
Thus, from the results obtained, we suggest that the
lectins can be used as chemotaxonomic marker within
the genus Canavalia. More investigation have to be
done with the seeds of other Diocleinae genera, before
its general use as a marker for the tribe.
REFERENCES
AINOUZ, I. L.; MOREIRA, R. A.; CAMPOS, F. A. P.,
RICHARDSON, M.; BEGBIE, R.; STEWART, J. C.;
WATT, W. B. & PUSZTAI, A. The isolation and amino acid
sequence of the beta and gama subunits of the lectin
from seeds of Dioclea grandiflora. Phytochemistry,
26:1435-1440, 1987.
AYMARD, G.C. & CUELLO, N.A. Catalogo y adiciones a las
especies neotropicales del genero Canavalia (Leguminosae- Papilionoideae-Diocleinae). Seminario-Taller de
Trabajo sobre Canavalia (Canavalia ensiformis (L) DC),
Maracay, Venezuela, 1991.
R. Bras. Fisiol. Veg., 127-132, 1993.
132
BRADFORD, M. M. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Analytical Biochemistry , 72:248-254, 1976.
CAVADA, B.S.; VIEIRA, C.C.; SILVA, L.M.A.; OLIVEIRA,
J.T.A. & MOREIRA, R.A. Comportamento da lectina de
sementes de Canavalia brasiliensis, Mart. durante a
germinação em presença da luz. Acta Botanica
Brasilica , 4(2):13-20, 1990.
CLAUSEN, J. Immunochemical techniques for identification
and estimation of macromolecules. In: T. S. Work & E.
Work (Eds) Laboratory Techniques in Biochemistry
and Molecular Biology. North-Holland Publishing Co.,
Amsterdam-London, 1969.
GRANGEIRO, T.B., OLIVEIRA, J.T.A., MOREIRA, R.A. &
CAVADA, B.S. Atividade hemaglutinante em sementes
de Dioclea restrata Benth. Acta Botanica Brasilica ,
4(2):61-68, 1990.
HARBOE, N. & INGILD, A. Immunization, isolation of immunoglobulins, estimation of antibody titre. In: N.H.Axelsen,L. Kroll & B. Week (Eds) A manual of Quantitative
Immunoelectrophoresis, Methods and Applications ,
Blakwell Scientific Publications, London, 1973.
HARBORNE, J.B., BOULTER, D. & TURNER, B.L. Chemotaxonomy of the Leguminosae. Academic Press, London-New York, 1971.
HEYWOOD, V.H. The Leguminosae - A Systematic Purview
In: Harborne, J. B., Boulter, D. & Turner, D. L. (Eds.)
Chemotaxonony of the Leguminosae , Academic
Press, London New York, 1971.
HUTCHINSON, J. The Family of Flowering Plants. v. 1.,
Clarenden Press, Oxford, 1964, quoted by Heywood,
1971.
LACKEY, J.A. A synopsis of Phaseolae (Leg. Pap.). Thesis Ph.D. 290 p. Iowa State University, 1977, quoted
by Aymard & Cuello, 1991.
LACKEY, J.A. Phaseoleae. In: Pohlhill, R.M. & Raven, P.H.
(Eds.) Advances in Legume Systematics. Part 1.
Royal Bot. Gard. Kew, 301-327, 1981, quoted by Aymard
& Cuello, 1991.
LAEMMLI, U. K. Cleavage of structural proteins during the
assembly of the bacteriophage T4. Nature , 227:68O685, 1970.
MOREIRA, R. A., AINOUZ, I. L., OLIVEIRA, J. T. A. &
CAVADA, B. S. Plant lectins, chemical and biological
aspects. Memórias do Instituto Oswaldo Cruz , 86:211218, 1991.
MOREIRA, R.A., BARROS, A.C.H., OLIVEIRA, J.T.A. &
RICHARDSON, M. Comparative studies of lectins from
seeds of the tribe Diocleae. Arquivos de Biologia e
Tecnologia , 28(1), 1985.
MOREIRA, R. A., BARROS, A. C. H., STEWART, 0. &
PUSZTAI, A. Isolation and characterization of a lectin
from the seeds of Dioclea grandiflora Mart. Planta ,
158:63-69, 1983.
MOREIRA, R.A. & CAVADA, B.S. Lectin from Canavalia
brasiliensis (Mart.). Isolation, characterization and beha vior during germ ination. Biologia Plantarum ,
26(2):113-120, 1984
MOREIRA, R.A., CAVADA, B.S., OLIVEIRA, J.T.A. & AINOUZ, I.L. Plant lectins. In: Oliveira-Filho & Sgarbieri
Eds. Proceedings of the First Brazilian Congress on
Proteins , UNICAMP, Campinas, SP. Editora UNICAMP,
1991, 639 p.
MOREIRA, R.A. & OLIVEIRA, J.T.A. Comparative studies
of seed proteins of the genus Artocarpus with respect to
lectins. Biologia Plantarum , 25:336-342, 1983 a.
MOREIRA, R.A. & OLIVEIRA, J.T.A. Lectins from the genus
Artocarpus. Biologia Plantarum , 25:343-348, 1983 b.
MOREIRA, R. A. & PERRONE, J. C. Purification and partial
characterization of a lectin from Phaseolus vulgaris.
Plant Physiology , 59:783-787, 1977.
OLIVEIRA, J.G.B. Cluster analysis computer program, personal comunication, 1991.
OLIVEIRA, J.T.A.; CAVADA, B.S.; MARTINS, J.L.;
JARENKOW, J.A.; VASCONCELOS, I.M. & MOREIRA,
R.A. Lectinas de sementes como marcadores taxonômic o s d a t r ib o Diocleae. Acta Botanica Brasilica ,
4(2):169-173, 1990.
OLIVEIRA, J.T.A., CAVADA, B.S. & MOREIRA, R.A. Isolation and partial characterization of a lectin from Cratylia
floribunda seeds. Revista Brasileira de Botânica,
14:61-66, 1991.
PEREZ, G.; PEREZ, C.; CAVADA, B. S.; MOREIRA, R. A.
& RICHARDSON, M. Comparison of the amino acid
sequence the lectins from seed Dioclea lehmanni and
Canavalia maritima. Phytochemistry, 30:2619-2621,
1991.
PUSZTAI, A. Plant Lectins , Cambridge University Press,
Cambridge, 1991, 263 p.
RICHARDSON, M.; CAMPOS, F. A. P.; MOREIRA, R. A.;
AINOUZ, I.L BEGBIE, R.; WATT, W. B. & PUSZTAI, A.
The complete amino acid sequence of the major alphasubunit of the lectin from the seed Dioclea grandiflora
Mart. European Journal of Biochemistry, 114:101-111,
1984.
ROUGE, P.; RICHARDSON, M.; RANFAUSG, P.; YARWOOD, A.; & CAVADA, B.S. Single and two-chain legume lectins as phylogenetic marker speciation. Biochemical Systematics and Ecology, 15:341-348,
1987.
SAUER, J. Revision of Canavalia. Brittonia , 16:106-181,
1964.
TOMS, G.C. & WESTERN, A. Phytohaemagglutinins. In:
Harborne, J. B., Boulter, D. & Turner, B. L. (Eds.) Chemotaxonony of the Leguminosae, Academic Press, London, 1971.
VASCONCELOS, I.M., CAVADA, B.S., MOREIRA, R.A. &
OLIVEIRA, J.T.A. Purification and partial characterization of a lectin from the seed of Diaclea guianensis.
Journal of Food Biochemistry, 15:137-154, 1991.
R. Bras. Fisiol. Veg., 127-132, 1993.