LECTINS FROM THE SUB-TRIBE

DIOCLEINAE (LEGUMINOSAE- PHASEOLAE): A CHEMOTAXONOMIC

TOOL

Ana Cristina Oliveira Monteiro-Moreira1, Walbert Edson Muniz-Filho3and Ana Cecília Goes Horta2, Renato Azevedo Moreira*

1 Curso de Ciências Farmacêuticas, CCS, Universidade de Fortaleza 2Laboratório de Lectinas e Glicoconjugados, Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Caixa Postal 6020, 60451-970, Fortaleza (CE), Brasil. Fax: (55 85) 288-9789, email [email protected] 3Departamento de Bioquímica, Universidade Federal do Maranhão, São Luis (MA), Brasil *Corresponding author.

Abstract

Seeds of fourteen species from the genera and ( Leguminosae - Papilionoideae - Phaseolae - Diocleinae) were investigated with respect to morphologic (size, mass, hylum length) and chemical characteristics. Thus, The total nitrogen content and amino acid composition of the seed flour were determined and these were submitted to extraction at different pH values. The seed extracts were investigated with respect to their content of protein and hemagglutinating activity, fractionated by affinity chromatography and the lectin content evaluated. The isolated lectins were investigated with respect to their amino acid composition, quaternary structure, thermal stability and immunochemical characteristics. The numerical data obtained were submitted to cluster analysis and a phenogram constructed.

Key Word Index - Diocleinae, Canavalia, Dioclea, lectin, chemotaxonomy

Introduction Ophrestiinae and Phaseolinae (Aymard & Cuello, The Leguminosae is one of the three 1991). The sub-tribe Diocleinae, sometimes classi- largest family of flowering , exceeded fied as a separate tribe (Hutchinson, only by Compositae and Orchidaceae, and is 1964) is composed of tropical plants and comprises the most economically important family from 13 genera Calapogonium, , Cana- the Dicotyledonae, with many species (beans valia, , Collea, , , and ) cultivated as source of food and feed- Dioclea, , Herpiza, Luzonia Macropsy- ing. They are also botanically full of interest canthus, and Pachyrhizus. The genera Canavalia since their more than 17.000 species constitute and Dioclea are largely spread in the tropics and almost 10% of the flowering plants, being virtu- important sources of animal feeding due to their ally ubiquitous and are present in all the envi- high protein content. The Canavalia is di- ronments (except the epiphyte and maritime) in vided in 4 sub-genus: Canavalia, Catadonia, Mau- almost all the continents, except from Artartic naloa and Wenderothia, and the genus Dioclea is (Lackey, 1977; Lackey, 1981). divided in 3 sub-genus: Dioclea, Pachilobium and Three sub-families Caesalpinioideae, Platylobium (Sauer, 1964; Maxwell, 1969; Hey- Mimosoideae and Papilionoideae, are recog- wood, 1971; Aymard & Cuello, 1991). nised (although sometimes as separate families, Numerical taxonomy is understood as the Caesalpiniaceae, Mimosaceae and Papiliona- grouping, by numerical methods, of taxonomic ceae). From the family Leguminosae, the Papil- units on the basis of their character states (Sneath ionoideae is the largest (with two thirds of the & Sokal, 1973). The term includes the drawing of genera and species). is divided in 30 tribes and phyllogenetic inferences from the data by statisti- can be found mainly in the tropics and temper- cal methods. These methods require the use of ate regions. The tribe Phaseolae is sub-divided numerical characters or the conversion of informa- in 8 sub-tribes, Cajaninae, Clitoriinae, Dio- tion about taxonomic entities into numerical quan- cleinae, Erythrininae, Glycininae, Kenediinae, tities.

VI Reunião Regional da SBBq – Nordeste. 1 Biochemical data have been used for gladiata, C. maritima, C. plagiosperma) and Dio- numerical taxonomy mainly when they deal clea (D. altissima, D. guianensis, D. rostrata, D. with physiologically key substances. seed violacea, D. virgata), sub-family Diocleinae, were proteins play such role, as they are responsible collected in the State of Ceará and one of them (D. for the plant metabolism, defence and nutrient lehmanii) was kindly supplied by Dr. Gerardo reserve. Lectins (carbohydrate binding proteins, Perez (Universidad Nacional de Bogota, Colmbia). capable of cell recognition and showing high Voucher specimens of the studied samples are homology) are believed to play some of these deposited in the Herbário Prisco Beserra of the roles and varied very little during evolution. Universidade Federal do Ceará. Thus the presence, in related taxa, of seed lec- Erythrocytes. Rabbit blood was obtained by punc- tins makes them a suitable molecular tool for ture of the marginal earl's vein of healthy animals. chemotaxonomic and phyllogenetic investiga- Freund’s complete adjuvant was a product of tions (Toms & Western, 1971; Moreira et al, B.D.H. Acrylamide and methylene bisacrylamide 1991; Lis & Sharon, 1998; Peumans & Van were product of Sigma Chemical Co., Sephadex Damme, 1998). G-50, Superose 12 HR and MW markers were The lectins present in of the tribe from Pharmacia, Uppsala, Sweden. Diocleinae are glucose(mannose)-specific and, The morphologic characteristics were determined thus, can be isolated by affinity chromatography with 100 seeds. The hylum (%) was determined as a on Sephadex G-50 column (Moreira et al, 1983; relation between the hylum length and the total seed Moreira & Cavada, 1984; Oliveira et al, 1991; circumference. Vasconcelos et al, 1991; Moreira et al, 1996; Total nitrogen was determined by the method of Moreira et al, 1997; Monteiro et al, 1998). The Baethgen & Allen (1969), and protein concentra- lectins are classified in the basis of their carbo- tion was obtained by the method of Bradford hydrate specificity (Goldstein & Hayes, 1978). (1976) using bovine serum albumin as standard. Although minor differences have been found, Readings at 280 nm were also employed to deter- even within a particular group, these are less mine protein content of the column eluates. noticeable when the lectins are extracted from Hemagglutinating activity was determined by the botanically related plants (Moreira & Oliveira, method described by Moreira & Perrone (1977), in 1983 a; Moreira & Oliveira, 1983 b), reflecting small glass tubes where to a series of 1:2 dilutions the evolutionary relationships. Thus, very close of the crude extract or purified lectins in 0.15 M similarities have been found in the amino acid NaCl containing 5 mM CaCl2 and 5 mM MnCl2 sequences of the seed lectins from Dioclea (250 µl) were added the same volume of a 2% grandiflora, Dioclea lehmanii, Canavalia ensi- suspension of erythrocytes. The degree of agglutina- formis, Canavalia gladiata and Canavalia mari- tion was monitored visually after the tubes have been tima, all belonging to the sub-tribe Diocleinae left at 37 oC for 30 min and subsequently left at room (Perez et al, 1991). temperature for further 30 min. The hemagglutina- In this paper, a comparative study was tion unit (HU) was defined as the reciprocal of the done using the whole proteins and the lectins in highest dilution still giving a visible agglutination. particular, from the seeds of eight species be- The hemagglutination titre was defined as log2 HU longing to three sub-genera of the genus Cana- and the minimum dose as the minimum amount of valia (sub-genus Wenderothia: Canavalia bi- protein still promoting visible agglutination. carinata; sub-genus Catadonia: Canavalia Protein extraction. The dehulled seeds were ground bonariensis and sub-genus Canavalia: Cana- in a coffee grinder (60 mesh) and the protein extracts valia brasiliensis, C. dictyota, C. ensiformis, C. were prepared by treating the seed meal (1 g) with 20 gladiata, C, maritima and C. plagiosperma) and ml of 0.1 M Glycine-HCl pH 2.6; 0.1 M Na-Acetate six species belonging to the three sub-genera of pH 4.0; 0.2 M Na-Acetate pH 4.0; 0.1 M; Tris-HCl the genus Dioclea. (sub-genus Dioclea: Dioclea pH 6.0; 0.1 M Na-Borate pH 8.0 and 0.1 M Na- guianensis, D. lehmanii, D. virgata; sub-genus Borate pH 10.0 buffers, all containing 0.1 M NaCl. Pachylobium: D. altissima, D. violacea; and The suspensions were left at room temperature for 3 sub-genus Platylobium: Dioclea rostrata). h and spun at 10,000 x g for 20 min. at 7 oC. The clear supernatants were used for the various analyses. Material and Methods Affinity chromatography. This was done as described previously (Moreira et al. 1983). The The species studied, belonging to the genera extracts obtained with 0.1 M borate pH 8,0 buffer Canavalia (C. bicarinata, C. bonariensis, C. containing 0.15 M NaCl were applied to a Sephadex brasiliensis, C. dictyota, C. ensiformis, C. G-50 column (40 cm x 1.6 cm) equilibrated with the

VI Reunião Regional da SBBq – Nordeste. 2 same buffer, containing 5 mM CaCl2 and 5 mM determined using the expression of Arrhenius MnCl2. After removing unbound material, the (Dawes, 1972). lectin was desorbed from the column with 0.1 M Amino acid analyses were performed after hy- glucose added to the equilibrium solution. The drolysis of the seed material (flour and lectins) eluates were collected in 3.0 ml fractions and samples in sealed glass tubes under N2, for 24 hr, analysed for hemagglutinating activity and protein with 6 N HCl at 110 oC. After hydrolysis HCl was content. removed by evaporation and the residue was ana- Gel filtration. The purified lectins were submitted lysed in a Pharmacia-LKB Biochrom 20 system. to gel filtration on a Superose 12 HR column, in For Cluster analysis, a basic matrix was con- the absence and presence of 0.1 M glucose, in 0.1 structed with the numerical data using the proce- M Glycine-HCl pH 2.6, 0.1 M Na-Acetate pH dures of Oliveira (1991). The degrees of similarity 4.0, 0.1 M Na-Acetate pH 6.0, 0.1 M Tris-HCl pH found were utilised for the construction of the 8.0 buffers all containing 0.15 M NaCl, 5 mM tentative phenogram. In the case of SDS/PAGE, CaCl2 and 5 mM MnCl2. the gel slabs were scanned and the area of each Antibodies specific for the lectins from Canavalia band was calculated in relation to total area. The brasiliensis and Dioclea altissima were raised in gel filtration results were presented was relative rabbits. Immunospecific IgG was obtained by area of each peak. fractionation of immunosera with ammonium sulphate followed by DEAE-Sephadex A-50 Results and Discussion chromatography according to Harboe and Ingild (1973). The seed morphologic characteristics (size, Ouchterlony radial double immunodiffusion were hylum length % and mass) were determined (Table carried out according to Clausen (1969), using 1% 1). These values were quite different (varying from agarose gels prepared with 0.15 M NaCl, 0.29 cm to 3.82 cm and from 0.26 cm to 2.21 cm for containing 0.1 M Glucose and 0.2 % sodium longitudinal and transversal lengths, respectively, azide . The seed crude extracts (20 µg) were from 0.11g to 12.12g for the mass and from 16.44 % applied in wells in a circular distribution, around a to 62.00 % for the hylum) . Quite different values central well containing the IgG anti Canavalia were also obtained when the seed total protein brasiliensis, or Dioclea altissima lectins (20 ug). content (nitrogen base) were determined (Table 1) The samples were allowed to diffuse for 48 h, at and it was found values varying from 214.0 ± 23.1 to room temperature, and the precipitated arcs in the 391.5 ± 30.6 (n=12). 0.15 M NaCl washed gel plates were stained with The influence of pH on the solubility of the 0.005% Coomassie Brilliant Blue R-250. proteins and hemagglutinating activity of Diocleinae Sodium dodecyl sulphate polyacrylamide gel was examined (Table 1). It was found that, although electrophoresis (SDS-PAGE) was carried out the data obtained for the solubility of proteins according to Laemmli (1970) in vertical 2 mm gel behaved somewhat similarly, the data for the slabs, consisting of a stacking gel mix of 4% total hemagglutinating activity varied significantly, with acrylamide gel concentration, pH 6.8 and a main the Dioclea showing higher activities. Moreover, running gel mix, containing 17.5 % acrylamide although the protein contents showed higher values concentration. Samples were dissolved in 0.01 M in alkaline pH, the hemagglutinating activity did not sodium phosphate, pH 7.0, 2% SDS buffer with show any pattern. -mercaptoethanol and incubated at 100 oC for 15 When the pH 8.0 extracts were applied to a min. Sucrose were dissolved (to 10%) in the Sephadex G-50 column, it was found typical samples which were then applied to the gel. The chromatograms for the Diocleinae proteins (Moreira electrophoresis was conducted at a constant et al., 1983; Moreira & Cavada, 1984; Oliveira et al., current of 13 mA for 4 h. The protein bands were 1991; Vasconcelos et al., 1991; Moreira et al., 1996; visualised by staining with Coomassie Brilliant Moreira et al, 1997; Monteiro et al, 1998). Important Blue R-250, and the gels scanned in a Laser differences were found when the lectin peaks (PIII) densitometer LKB 2222-020 Ultroscan XL with were compared to the high molecular mass non monochromatic Helium-Neon lamp at 633 nm. retained protein peaks (PI), with values varying from The thermostability of the hemagglutinating 1.90% to 32.19% (Table 1). activity was determined treating the crude extracts When the Diocleinae seed flours, extracted at different temperatures during different times in Na-borate pH 8.0 buffer were submitted to heat and the residual activity determined. The treatment at temperatures varying between 78 and 80 activation energy of the inactivation process was oC, and the inactivation curves obtained used to calculate the activation free energy of the process, it

VI Reunião Regional da SBBq – Nordeste. 3 was obtained values varying from 24.07 to 26.97 peak) were analysed, alone or grouped with the Kcal/mol. These results, showing a variation protein parameters, strong statistical correlation was somewhat significant, suggest differences in found at the sub-genus level, in spite of the structures that reflect in the stability (Table 1). immunological identity found between the lectins The amino acid composition of the from all the species investigated. When to these data Diocleinae seed flour and lectins were also was added the SDS/PAGE quantitative data and analysed and minor but significant differences amino acid composition, the correlation was more were found (Table 2). consistent and a phenogram could be drawn. From The Diocleinae seed flours were the plot, it can be seen that, as it should be predicted extracted in 0.1 M Na-phosphate buffer pH 7.5 by the classical systematic relationships, the species containing SDS and β-mercaptoethanol and belonging to each of the sub-genera can be grouped electrophoresed in polyacrylamide gel in the together and separated from the others. presence of SDS (Fig 1). Only small but From the results obtained using these significant differences were observed in the morphological and biochemical characters (151 in distribution of protein bands. The gel slabs were total), we suggest that the lectins can be used as scanned and the area obtained for each band (total selective taxonomic markers within both genera of 27) was compared and is presented in Table3). Canavalia and Dioclea and, moreover, as a tool for Total identity was found among the distinguishing species from species belonging to lectins from the Canavalia and Dioclea seed these genera. As an initial contribution for the investigated, when the pH 8.0 extracts were classification of the Diocleinae species, we can see allowed to diffuse against IgG anti Canavalia that in the case of Canavalia dictyota, not clearly brasiliensis or Dioclea altissima lectins (Fig. 2). located in the sub-genus Canavalia or Maunaloa When the quaternary structure of the (Aymard & Cuello, 1991), our results suggest its Diocleinae lectins were investigated by gel classification as Canavalia (as suggested by Sauer, filtration (Table 4) it was found that these lectins 1964). behaved as an equilibrium system among the More investigations have to be done with monomeric and dimeric forms, dependent of the seeds of other Diocleinae genera in order to use these pH of the medium. Thus, at pH 2.6, all the lectins characters as taxonomic markers for the whole sub- showed Mr corresponding to the monomeric form tribe. (Mn ≅ 26 kDa) and to the dimers (Mr ≅ 50 kDa) at pH 8.0, with intermediary values at pH 4.0 and REFERENCES 6.0, corresponding to mixtures of these two forms. When, on the other hand, the gel filtration Aymard, G.C. & Cuello, N.A. (1991). Catalogo y was done in the presence of the ligand glucose adiciones a las especies neotropicales del (Table 5) the transition from monomer to dimer genero Canavalia (Leguminosae- was favoured. The equilibrium between the two Papilionoideae-Diocleinae). Seminario- forms is a characteristic of each lectin and is due, Taller de Trabajo sobre Canavalia (Cana- ultimately, to the intrinsic interactions between valia ensiformis (L) DC), Maracay, Vene- the charged amino acid side chains. When this zuela. behaviour was compared, important differences Beathgen, W.E. & Alley, M.M. (1969) A manual were found among the Diocleinae lectins. colorimetric procedure for measuring am- The quantitative data obtained for the monium nitrogen in soil and plant Kjeldahl eight Canavalia and six Dioclea species were digests. Communications in Soil Science submitted to statistical cluster analysis, for the and Plant Analyses, 20(9-10): 961-969. construction of the tentative phenogram (Fig. 3). Bradford, M. M. (1976). A rapid and sensitive When the analysis were done using different sets method for the quantitation of microgram of data, in most cases showed no apparent quantities of protein utilising the principle correlation with the species taxonomic of protein-dye binding. Analytical Bio- identification. Thus, when the morphological seed chemistry, 72: 248-254. characteristics, the amino acid composition, the Clausen, J. (1969). Immunochemical techniques for SDS/PAGE and gel filtration were used identification and estimation of macro- separately, no correlation was found. Only when molecules. In T. S. Work & E. Work (Eds) the hemagglutinating activity and/or the lectin Laboratory Techniques in Biochemistry content were used, it was found good correlations. and Molecular Biology. North-Holland Thus, when the lectin parameters Publishing Co., Amsterdam-London. (hemagglutinating activity and the lectin affinity

VI Reunião Regional da SBBq – Nordeste. 4 Dawes, E.A. (1972) Quantitative Problems in dictyota seeds. Revista Brasileira de Fisio- Biochemistry. Edinburgh, Churchill logia Vegetal, 10(3): 167-172. Livingston, 470 p. Moreira, R. A., Ainouz, I. L., Oliveira, J. T. A. & Goldstein, I.J. & Hayes, C. E. (1978). The Cavada, B. S. (1991). Plant lectins, chemi- lectins: carbohydrate-binding proteins of cal and biological aspects. Memórias do plant and animals. Advances in Carbo- Instituto Oswaldo Cruz, 86: 211-218. hydrate Chemistry and Biochemistry, Moreira, R. A., Barros, A. C. H., Stewart, J. & Pusz- 35: 127-340. tai, A. (1983). Isolation and characterisa- Harboe, N. & Ingild, A. (1973) Immunization, tion of a lectin from the seed Dioclea isolation of immunoglobulins estimation grandiflora Mart. Planta, 158: 63-69. of antibody titre. In N.H.Axelsen, L. Moreira, R.A. & Cavada, B.S. (1984). Lectin from Kroll & B. Week (Eds) A Manual of Canavalia brasiliensis (Mart.). Isolation, Quantitative Immunoelectrophoresis, characterisation and behaviour during ger- Methods and Applications, Blakwell mination. Biologia Plantarum, 26(2), 113- Scientific Publications, London. 120, Harborne, J.B., Boulter, D. & Turner, B.L. Moreira, R.A., Cordeiro, E.F., Ramos, M.V., Gran- (1971) Chemotaxonomy of the Legumi- geiro, T.B., Martins, J.L., Oliveira, J.T.A. nosae. Academic Press London-New & Cavada, B.S. (1996). Purification and York. partial characterisation of a lectin from Heywood, V.H. (1971) The Leguminosae - A Dioclea violacea Benth seeds. Revista Bra- Systematic Purview IN Harborne, J. B., sileira de Fisiologia Vegetal, 8(1): 23-29. Boulter, D. & Turner, B. L. (Eds.) Moreira, R.A., Monteiro, A.C.O., Horta, A.C.G., Chemotaxonony of the Leguminosae, Oliveira, J.T.A. & Cavada, B.S. (1997). Academic Press, London - New York. Isolation and partial characterisation of Hutchinson, J. (1964) The Family of Flowering Dioclea altissima var. megacarpa seed Plants. Vol. 1., Clarenden Press, Ox- lectin. Phytochemistry, 46(1): 139-144. ford, 1964, quoted by Heywood (1971) Moreira, R.A. & Oliveira, J.T.A. (1983 a). Com- Lackey, J.A.(1977) A synopsis of Phaseolae parative studies of seed proteins of the ge- (Leg. Pap.). Thesis Ph.D. 290 p. Iowa nus Artocarpus with respect to lectins. Bio- State University, quoted by Aymard & logia Plantarum, 25: 336-342. Cuello (1991). Moreira, R.A. & Oliveira, J.T.A. (1983 b). Lectins Lackey, J.A. (1981) Phaseolae. In: Pohlhill, from the genus Artocarpus. Biologia Plan- R.M. & Raven, P.H. (Eds.) Advances in tarum, 25: 343-348. Legume Systematics. Part 1. Royal Bot. Moreira, R. A. & Perrone, J. C. (1977) Purification Gard. Kew, 301-327, 1981, quoted by and partial characterisation of a lectin from Aymard & Cuello (1991). Phaseolus vulgaris. Plant Physiology, 59: Laemmli, U. K. (1970). Cleavage of structural 783-787. proteins during the assembly of the bac- Oliveira, J.T.A., Cavada, B.S. & Moreira, R.A. teriophage T4. Nature, 227: 680-685. (1991). Isolation and partial characterisa- Liener, I.E., Sharon, N. & Goldstein, I.J. (1986). tion of a lectin from Cratylia floribunda The lectins: Properties, Functions, and seeds. Revista Brasileira de Botânica, 14: Applications in Biology and Medicine. 61-66. Academic Press, New York. Oliveira, J.G.B. (1991). Cluster analysis computer Lis, H. & Sharon, N. (1998). Lectins: Carbohy- program, personal communication. drate-specific proteins that mediate cel- Perez, G.; Perez, C.; Cavada, B. S.; Moreira, R. A. lular recognition. Chemical Reviews 98: & Richardson, M. (1991). Comparison of 637-674 the amino acid sequence of the lectins from Maxwell, R.H. (1969). The genus Dioclea (Fa- seed Dioclea lehmanni and Canavalia baceae) in the New World. PhD Thesis, maritima. Phytochemistry, 30: 2619-2621. Dept. of Botany, Southern Illinois Uni- Peumans W.J., & Van Damme, E.J.M. (1998). Plant versity. lectins: specific tools for the identification. Monteiro, A.C.O., Muniz-Filho, W.E., Horta, Isolation, and characterisation of O-linked A.C.G., Beltramini, L.M. & Moreira, glycans. Critical Reviews in Biochemistry R.A. (1998). Isolation and partial char- and Molecular Biology, 33(3): 209-258 acterisation of a lectin from Canavalia Sauer, J. (1964). Revision of Canavalia. Brittonia, 16: 106-181.

VI Reunião Regional da SBBq – Nordeste. 5 Sneath, P.H. & Sokal, R.R. (1973). Numerical Taxonomy. W.H. San Francisco, Free- man, 573 p. Toms, G.C. & Western, A. (1971) Phytohemag- glutinins. In J. B. Boulter & B. L. Turner (Eds) Chemotaxonomy of Legu- minosae. Academic Press, London. 1971. Vasconcelos, I.M., Cavada, B.S., Moreira, R.A. & Oliveira, J.T.A. (1991).Purification and partial characterisation of a lectin from the seed of . Journal of Food Biochemistry.15: 137- 154.

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Fig. 2. DOUBLE DIFFUSION PATTERNS OF Fig. 1. PAGE-SDS OF THE SEED PROTEINS THE LECTINS FROM SEEDS OF DIOCLEINAE OF DIOCLEINAE (CANAVALIA AND SPECIES, OBTAINED BY AFFINITY DIOCLEA) SPECIES. 1 and 16 - Molecular CHROMATOGRAPHY ON SEPHADEX G-50 weight marker (lectin from Dioclea altissima, 26, COLUMNS. The middle wells contained 50 µg of 14 and 9 kDa, respectively), 2. Canavalia immunoglobulins raised against Dioclea altissima bicarinata, 3 - Canavalia bonariensis, 4 - (A) or Canavalia brasiliensis (B), respectively. The Canavalia brasiliensis; 5 - Canavalia dictyota; 6 - contents of the peripheral wells were 20 µg of the Canavalia ensiformis, 7 - Canavalia gladiata, 8 - lectins from: (A) 1- Dioclea altissima, 2 - Dioclea Canavalia maritima, 9 - Canavalia plagiosperma, guianensis, 3 - Dioclea lehmanii, 4 - Canavalia 10 - Dioclea altissima; 11 - Dioclea guianensis, brasiliensis, 5 - Dioclea rostrata, 6 - Dioclea 12 - Dioclea lehmanii, 13 - Dioclea rostrata, 14 - violacea, 7 - Dioclea virgata and (B) 1 - Canavalia Dioclea violacea, 15 - Dioclea virgata. bicarinata, 2 - Canavalia bonariensis, 3 - Canavalia

brasiliensis, 4 - Canavalia dictyota, 5 - Canavalia

ensiformis, 6 - Canavalia gladiata, 7 - Canavalia

maritima, 8 - Canavalia plagiosperma.

VI Reunião Regional da SBBq – Nordeste. 7

Fig. 3. PHENETIC RELATIONSHIPS OF THE DIOCLEINAE (CANAVALIA AND DIOCLEA) SPECIES. 1 - Canavalia bicarinata, 2 - Canavalia bonariensis, 3 - Canavalia plagiosperma, 4 - Canavalia. dictyota, 5 - Canavalia brasiliensis, 6 - Canavalia ensiformis, 7 - Canavalia maritima, 8 - Canavalia gladiata, 9 - Dioclea rostrata, 10 - Dioclea violacea, 11 - Dioclea altissima, 12 - D. virgata, 13 - Dioclea lehmanii, 14 - Dioclea guianensis.

VI Reunião Regional da SBBq – Nordeste. 8 Table 1. Seed parameters and biochemical characters of Diocleinae seeds Species1 CanBic CanBon CanBra CanDic CanEns CanGla CanMar CanPla DioAlt DioGui DioLeh DioRos DioVio DioVir Seed parameters Longitudinal length (cm) 1.22 1.70 1.97 1.62 2.08 3.82 0.99 1.91 2.96 0.29 0.97 2.56 3.00 1.03 Transversal length (cm) 0.72 1.33 1.38 1.24 1.34 1.98 1.29 1.47 2.21 1.29 0.30 0.54 2.15 0.26 Hylum (%) 26.00 52.17 17.24 16.81 16.44 26.31 32.41 19.16 62.00 32.00 31.00 32.00 32.00 30.00 Mass 0.22 0.62 1.51 1.52 1.93 4.73 0.96 1.45 12.12 0.11 0.16 1.97 10.41 0.12 Biochemical Characters

Total protein (N2 x 6.25) 350.7 324.0 343.4 323.9 364.2 344.0 391.5 355.3 300.4 306.1 369.0 276.4 214.0 273.3 Soluble protein2 pH 2.6, 0.1 M 11.80 7.40 23.40 30.60 28.0 19.40 29.4 23.60 24.60 19.00 42.40 24.00 22.40 29.00 pH 4.0, 0.1 M 17.80 5.20 15.80 17.01 19.60 8.60 19.00 24.40 59.60 41.80 63.60 49.20 42.80 29.00 pH 4.0, 0.2 M 30.20 25.20 35.60 49.80 51.20 57.20 46.40 52.40 24.00 25.80 27.40 32.60 23.80 22.60 pH 6.0, 0.1 M 110.40 76.60 130.20 85.00 135.60 94.40 42.20 96.60 55.20 70.60 88.80 64.40 67.20 74.40 pH 8.0, 0.1 M 99.60 69.40 72.80 88.80 112.60 101.80 92.00 77.00 75.00 100.40 116.00 82.60 62.40 96.40 pH 10.0, 0.1 M 158.20 66.40 190.20 172.60 173.20 199.40 115.50 188.80 88.40 109.00 98.60 85.00 108.20 102.60 Hemagglutinating activity3 pH 2.6, 0.1 M 7.58 8.30 8.80 8.00 6.60 8.00 9.60 7.80 13.00 13.50 14.80 13.90 13.30 14.20 pH 4.0, 0.1 M 9.00 8.58 7.58 7.58 8.80 9.00 7.58 8.81 13.30 14.90 15.10 12.30 12.90 14.90 pH 4.0, 0.2 M 7.32 7.58 7.58 7.00 7.58 8.58 7.58 6.58 13.40 13.60 14.50 14.60 12.90 14.70 pH 6.0, 0.1 M 9.58 5.58 8.77 9.58 8.80 8.80 8.00 7.90 12.50 13.20 13.50 14.90 12.50 13.90 pH 8.0, 0.1 M 9.58 9.58 9.58 9.58 9.00 8.17 9.00 9.58 13.70 14.10 14.70 13.20 11.90 14.50 pH 10.0, 0.1 M 9.00 7.00 7.20 6.00 5.80 8.20 8.20 8.20 12.50 13.00 13.90 14.50 10.60 13.90 Lectin4 (%) 5.20 1.90 18.50 11.60 12.60 23.80 15.10 14.20 13.59 29.96 32.19 16.52 20.38 26.67 ∆G5 25.93 26.97 24.29 24.07 25.07 24.72 25.23 24.29 25.57 24.18 26.46 26.46 25.04 25.74

1 CanBic: Canavalia bicarinata; CanBon: C. bonariensis; CanBra: C. brasiliensis; CanDic: C. dictyota; CanEns: C. ensiformis; CanGla: C. gladiata; CanMar: C. maritima; CanPla: C. plagiosperma; DioAlt: Dioclea altissima; DioGui: D. guianensis; DioLeh: D. lehmanii; DioRos: D. rostrata; DioVio: D. violacea; DioVir: D. virgata. 2 3 4 5 (mg/g of flour); log2 UH; recovered lectin by affinity chromatography (%); Activation energy (Kcal/mol) of the process of lectin thermo inactivation.

VI Reunião Regional da SBBq – Nordeste. 9 Table 3. Protein bands (Rf) by PAGE/SDS electrophoresis. SDS/PAGE parame- Species1 ters2 CanBic CanBon CanBra CanDic CanEns CanGla CanMar CanPla DioAlt DioGui DioLeh DioRos DioVio DioVir Band 1 5.13 2.75 3.44 1.91 3.14 3.84 5.34 4.62 3.28 4.30 6.58 5.93 4.00 4.99 Band 2 7.44 4.24 4.76 5.93 8.78 6.89 6.83 6.71 4.64 9.41 10.75 8.26 7.46 9.62 Band 3 1.90 2.25 2.29 2.53 0.00 0.00 3.11 1.62 0.00 2.44 0.66 0.00 1.24 0.70 Band 4 2.01 0.71 2.42 1.69 1.43 2.88 1.43 2.00 2.45 0.87 1.11 2.72 3.01 0.00 Band 5 5.35 5.18 4.23 3.96 4.51 5.90 5.42 5.26 4.72 4.16 4.63 4.20 4.57 3.86 Band 6 1.06 0.81 1.67 2.51 0.00 0.00 0.52 0.60 0.94 0.00 0.67 0.53 1.52 0.77 Band 7 0.00 3.74 0.00 0.00 0.00 0.00 7.91 0.82 0.00 0.00 2.81 1.00 0.57 1.90 Band 8 0.00 1.18 3.60 3.87 1.09 0.00 4.90 3.00 3.59 5.43 2.53 0.00 3.23 0.00 Band 9 16.52 10.77 19.13 17.16 16.45 23.73 14.14 15.03 12.18 9.66 17.91 8.21 10.46 12.40 Band 10 4.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10.28 0.00 10.38 0.00 15.16 Band 11 0.98 0.00 5.19 6.01 5.94 2.08 5.40 5.33 5.74 4.82 3.50 2.08 12.20 3.86 Band 12 0.00 3.44 1.19 1.62 1.40 2.57 0.00 0.00 2.37 2.78 1.90 2.44 0.00 2.10 Band 13 4.69 1.49 0.00 0.00 0.00 0.00 0.00 1.20 2.04 0.54 1.15 1.24 0.00 1.10 Band 14 1.47 2.63 1.35 2.01 1.87 1.61 0.00 1.89 1.15 2.06 1.51 2.11 0.00 0.00 Band 15 0.00 2.14 0.00 0.00 0.00 0.00 0.00 0.00 1.81 2.85 5.72 0.00 0.00 5.14 Band 16 24.18 30.05 33.22 33.45 32.34 30.60 28.27 30.01 27.11 8.37 22.46 24.80 29.29 6.90 Band 17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 8.77 0.00 0.00 0.00 13.95 Band 18 1.85 4.68 0.00 0.00 0.00 2.85 0.00 0.00 1.77 1.34 0.00 0.00 0.00 0.00 Band 19 0.00 6.71 6.10 0.00 5.27 0.00 1.84 4.79 2.93 1.78 1.70 0.00 1.15 1.44 Band 20 7.50 0.00 0.00 6.15 2.25 3.19 3.24 3.90 2.00 3.99 3.59 0.00 1.71 2.55 Band 21 0.00 2.09 2.27 0.90 1.73 0.00 2.16 0.00 2.05 0.00 0.00 0.00 0.00 0.55 Band 22 1.01 0.00 0.00 0.00 0.00 1.40 0.00 0.00 4.25 2.91 1.54 0.00 2.11 2.74 Band 23 1.27 2.78 2.70 1.03 1.16 1.15 0.00 1.16 0.00 1.78 0.00 0.00 3.24 0.00 Band 24 1.05 2.14 0.00 1.03 1.17 1.55 0.00 1.57 0.00 0.61 1.92 0.00 0.00 2.02 Band 25 1.33 1.70 0.00 0.61 0.66 1.36 1.45 0.73 1.95 5.74 4.19 3.62 1.13 5.05 Band 26 3.04 3.67 3.65 3.64 5.30 3.80 2.98 2.74 2.41 0.00 0.00 0.00 0.00 0.00 Band 27 4.59 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

1 CanBic: Canavalia bicarinata; CanBon: C. bonariensis; CanBra: C. brasiliensis; CanDic: C. dictyota; CanEns: C. ensiformis; CanGla: C. gladiata; CanMar: C. maritima; CanPla: C. plagiosperma; DioAlt: Dioclea altissima; DioGui: D.

VI Reunião Regional da SBBq – Nordeste. 10 guianensis; DioLeh: D. lehmanii; DioRos: D. rostrata; DioVio: D. violacea; DioVir: D. virgata. 2 (%) of total stained area

Table 2. Amino acid composition of Diocleinae seed flour and lectins Amino acid Species1 composition2 CanBic Can- CanBra CanDic CanEns CanGla Can- CanPla CraFlo DioAlt DioGui DioLeh DioRos DioVio DioVir Bon Mar Seed flour ASX 16.22 15.43 14.47 13.32 14.78 14.35 14.38 13.45 11.30 13.42 15.54 15.40 15.68 14.10 15.19 THR 7.11 8.02 8.33 7.04 8.26 8.26 8.91 6.46 6.96 7.79 10.11 10.27 8.05 6.84 8.86 SER 7.05 11.12 13.60 7.57 13.48 13.04 12.59 8.10 13.04 14.34 16.93 13.15 11.86 10.68 12.24 GLX 5.68 5.91 5.26 6.58 5.22 4.78 5.02 7.43 5.65 5.64 4.61 5.25 6.78 7.26 5.06 GLY 9.21 10.02 7.02 8.00 6.52 7.83 8.24 9.34 13.48 7.48 9.37 7.52 4.66 5.98 8.02 ALA 7.64 7.82 6.14 9.09 6.52 7.39 8.09 8.76 10.43 7.79 9.16 7.31 5.93 5.56 8.44 VAL 8.22 5.71 7.46 7.36 7.39 7.39 7.08 7.84 7.39 6.97 3.41 6.83 5.08 5.56 6.33 ILE 7.64 6.21 6.58 6.79 6.52 6.52 5.20 6.33 6.09 6.05 3.30 6.54 5.51 6.41 5.91 LEU 8.40 7.31 7.89 8.33 7.83 7.83 7.29 7.87 6.96 7.79 6.92 8.67 8.90 8.55 7.59 TYR 3.16 3.01 3.07 3.40 7.04 3.04 3.02 3.06 3.91 3.07 3.09 3.12 4.24 3.85 2.95 PHE 4.46 4.71 4.82 5.22 4.78 4.78 5.00 4.55 3.48 4.82 4.58 4.67 6.36 7.26 5.06 HIS 1.95 1.60 2.63 3.06 2.61 2.61 2.57 2.94 1.30 1.74 1.06 1.40 2.12 2.56 1.69 LYS 5.42 5.31 5.26 5.96 5.22 4.78 5.45 6.28 3.91 5.23 3.30 2.39 5.51 5.98 4.22 ARG 3.21 3.01 3.07 2.91 3.04 2.61 2.66 2.74 3.04 3.07 3.30 3.28 4.66 5.13 2.95 PRO 4.64 4.81 4.39 5.37 4.78 4.78 4.50 4.85 3.04 4.82 5.32 4.19 4.66 4.27 5.49 Lectin ASX 12.74 12.31 11.52 13.24 11.68 11.48 11.64 10.24 12.91 12.37 11.96 12.96 12.35 12.83 11.95 THR 5.00 4.04 5.98 5.09 5.89 5.89 5.96 4.66 6.13 5.60 7.28 5.21 6.97 5.75 6.79 SER 4.55 3.77 7.19 4.91 7.15 7.39 7.44 4.49 7.37 6.85 9.51 4.83 8.54 6.76 7.18 GLX 12.37 14.51 14.45 12.57 11.98 14.61 11.30 15.10 11.07 13.07 11.13 11.29 10.24 13.68 9.89 GLY 8.88 8.50 7.83 8.02 7.82 7.87 7.92 8.52 7.84 7.63 8.32 8.14 8.27 7.66 7.76 ALA 7.20 7.67 7.40 7.24 7.33 7.37 7.28 7.73 7.36 6.52 7.75 7.21 7.37 6.47 7.55 ½ CYS 1.18 1.58 0.96 0.44 1.16 1.00 0.89 0.77 1.23 1.05 1.23 1.00 0.95 1.00 1.51 VAL 7.24 6.64 6.04 6.67 6.26 6.34 6.60 7.00 6.34 6.29 6.66 7.17 6.26 5.87 6.13 MET 0.68 0.51 0.21 0.43 0.22 0.22 0.18 0.70 0.47 0.26 0.20 0.49 0.19 0.24 0.35 ILE 3.23 3.02 5.51 3.02 4.87 5.14 4.78 3.19 4.99 4.67 5.06 3.25 4.91 4.47 5.26 LEU 8.83 8.77 8.41 9.17 8.53 8.34 8.15 9.97 8.02 9.30 8.53 9.24 8.12 9.31 7.85 TYR 3.05 3.10 2.49 3.14 2.56 2.56 2.92 3.38 2.62 2.43 2.57 3.05 2.63 2.38 2.58 PHE 4.38 4.11 4.79 4.39 4.51 4.63 4.51 4.66 4.69 4.21 2.53 4.61 4.34 4.05 5.00 HIS 3.02 2.62 3.29 3.05 3.00 3.07 3.10 3.21 2.78 3.12 4.83 3.19 2.89 2.70 3.18 LYS 7.15 9.16 6.51 7.03 6.54 6.80 7.75 7.25 6.65 7.13 2.92 8.85 5.83 4.84 6.95 ARG 5.13 4.87 4.96 6.06 4.86 4.61 4.23 3.36 4.20 4.11 3.80 3.85 4.66 4.66 3.87 PRO 5.27 4.79 5.48 5.39 5.26 4.45 5.20 5.60 5.08 5.20 5.49 5.42 5.30 5.11 5.41 TRP 0.12 0.06 1.39 0.17 0.29 0.23 0.16 0.17 0.24 0.16 0.26 0.26 0.19 0.23 0.28 1 CanBic: Canavalia bicarinata; CanBon: C. bonariensis; CanBra: C. brasiliensis; CanDic: C. dictyota; CanEns: C. ensiformis; CanGla: C. gladiata; CanMar: C. maritima; CanPla: C. plagiosperma; DioAlt: Dioclea altissima; DioGui: D. guianensis; DioLeh: D. lehmanii; DioRos: D. rostrata; DioVio: D. violacea; DioVir: D. virgata. 2 (mol %)

VI Reunião Regional da SBBq – Nordeste. 11 Table 4. Gel filtration of Diocleinae lectins Buffer2 Mr3 Species1 CanBic CanBon CanBra CanDic CanEns CanGla CanMar CanPla CraFlo DioAlt DioGui DioLeh DioRos DioVio DioVir 0.1 M Glycine- <23 0 0 0 0 0 0 0 0 100.00 0 0 0 0 0 0 HCl pH 2.3 23/28 100 100 100 100.00 100.00 100.00 100.00 100.00 0 100.00 72.90 100.00 100 100 100 28/33 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 33/38 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 38/43 0 0 0 0 0 0 0 0 0 0 27.1 0 0 0 0 43/48 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 48/53 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 53/58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 >58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.1 M Na-Acetate <23 0 0 0 0 0 0 0 0 0 0 2.23 0 0 0 0 pH 4.0 23/28 0 0 0 0 0 0 0 0 100.00 0 0 0 0 0 0 28/33 100 100 100.00 100.00 0 0 0 100.00 0 100.00 66.14 100.00 100 100 100 33/38 0 0 0 0 100.00 100.00 100.00 0 0 0 0 0 0 0 0 38/43 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 43/48 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 48/53 0 0 0 0 0 0 0 0 0 0 31.62 0 0 0 0 53/58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 >58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.1 M Na-Acetate <23 0 0 23.36 0 0 3.21 10.69 0 0 0 4.34 0 0 0 0 pH 6.0 23/28 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28/33 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0 33/38 0 100.00 0 100.00 100.00 0 89.31 100.00 0 0 0 0 100 0 100 38/43 100.00 0 0 0 0 0 0 0 0 0 0 100.00 0 0 0 43/48 0 0 76.64 0 0 0 0 0 100.00 0 0 0 0 0 0 48/53 0 0 0 0 0 96.79 0 0 0 100.00 95.66 0 0 0 0 53/58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 >58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.1 M Tris-HCl <23 0 46.3 34.61 0 43.05 58.20 0 0 0 17.11 54.36 0 0 44.83 0 pH 8.0 23/28 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28/33 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 33/38 0 53.7 0 0 56.95 0 0 0 0 0 0 0 0 0 0 38/43 0 0 0 100.00 0 0 0 0 0 0 0 0 0 0 0 43/48 100.00 0 65.39 0 0 41.80 100.00 100.00 100.00 0 0 100.00 0 55.17 0 48/53 0 0 0 0 0 0 0 0 0 75.94 45.64 0 100.00 0 100.00 53/58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 >58 0 0 0 0 0 0 0 0 0 6.95 0 0 0 0 0 1 CanBic: Canavalia bicarinata; CanBon: C. bonariensis; CanBra: C. brasiliensis; CanDic: C. dictyota; CanEns: C. ensiformis; CanGla: C. gladiata; CanMar: C. maritima; CanPla: C. plagiosperma; CraFlo: Cratylia floribunda: DioAlt: Dioclea altissima; DioGui: D. guianensis; DioLeh: D. lehmanii; DioRos: D. rostrata; DioVio: D. violacea; DioVir: D. virgata. 2 All buffers contains 0.15 M NaCl. 3 Apparent Molecular Mass

VI Reunião Regional da SBBq – Nordeste. 12 Table 5. Gel filtration of Diocleinae lectins, in the presence of 0.1 M Glucose Buffer2 Mr3 Species1 CanBic CanBon CanBra CanDic CanEns CanGla CanMar CanPla CraFlo DioAlt DioGui DioLeh DioRos DioVio DioVir 0.1 M Glycine- <23 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 HCl pH 2.3 23/28 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 78.91 100.00 100.00 100.00 100.00 + 0.1 M Glucose 28/33 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 33/38 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 38/43 0 0 0 0 0 0 0 0 0 0 21.09 0 0 0 0 43/48 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 48/53 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 53/58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 >58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.1 M Na-Acetate <23 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 pH 4.0 23/28 0 0 0 0 0 0 0 0 100.00 0 0 0 0 0 0 + 0.1 M Glucose 28/33 0 0 0 0 0 0 0 0 0 0 0 100.00 0 0 0 33/38 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 0 100.00 88.77 0 100.00 41.33 87.70 38/43 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 43/48 0 0 0 0 0 0 0 0 0 0 0 0 0 58.67 0 48/53 0 0 0 0 0 0 0 0 0 0 11.23 0 0 0 12.30 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 53/58 >58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.1 M Na-Acetate <23 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 pH 6.0 23/28 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0.1 M Glucose 28/33 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 33/38 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 38/43 0 0 100.00 100.00 100.00 0 0 0 0 0 0 0 0 0 0 43/48 0 0 0 0 0 0 100.00 100.00 100.00 100.00 100.00 0 100.00 100.00 0 48/53 0 100.00 0 0 0 100.00 0 0 0 0 0 100.00 0 0 100.0 53/58 100.00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 >58 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.1 M Tris-HCl <23 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 pH 8.0 23/28 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0.1 M Glucose 28/33 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 33/38 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 38/43 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 43/48 0 0 0 0 100.00 0 0 0 100.00 0 0 0 0 0 0 48/53 0 100.00 100.00 100.00 0 100.00 100.00 100.00 0 0 0 100.00 0 100.00 100.00 53/58 100.00 0 0 0 0 0 0 0 0 100.00 84.16 0 100.00 0 0 >58 0 0 0 0 0 0 0 0 0 0 15.84 0 0 0 0 1 CanBic: Canavalia bicarinata; CanBon: C. bonariensis; CanBra: C. brasiliensis; CanDic: C. dictyota; CanEns: C. ensiformis; CanGla: C. gladiata; CanMar: C. maritima; CanPla: C. plagiosperma; CraFlo: Cratylia floribunda: DioAlt: Dioclea altissima; DioGui: D. guianensis; DioLeh: D. lehmanii; DioRos: D. rostrata; DioVio: D. violacea; DioVir: D. virgata. 2 All buffers contains 0.15 M NaCl and 0.1 M Glucose. 3 Apparent Molecular Mass

VI Reunião Regional da SBBq – Nordeste. 13