Purification , Characterization and Antioxidant Potential of a Novel Lectin from Pterocarpus soyauxii Taub Seeds

The study investigated the presence of lectin in the seeds of Pterocarpus soyauxii, purified the lectin and determined its physicochemical properties as well as its antioxidant potential. Purification of P. soyauxii seeds lectin (PSSL) was accomplished by ammonium sulphate precipitation and gel filtration on Sephadex G-100, hemagglutination assay was by serial dilution and DPPH radical scavenging, metal chelating and lipid peroxidation inhibition assays were employed to evaluate its antioxidant potential. PSSL agglutinated erythrocytes from human blood group ABO, rabbit and rat. Trypsinization enhanced agglutination which was inhibited by galactose, lactose and arabinose. The lectin activity was independent of metal ion, thermal stable up to 60 °C and at pH range of 6 to 8. Gel filtration estimated molecular weight was 65 kDa and subunit molecular weight was 35 kDa. PSSL showed significant antioxidant activity. The lectin was able to scavenge DPPH radical, chelate iron metal and inhibit lipid peroxidation with IC50 of 0.14 ± 1.33 mg/ml, 3.40 ± 0.37 mg/ml and 0.50 ± 0.08 mg/ml respectively. The results indicate that PSSL might be applied as natural antioxidant for the development of drug and food products for the benefit of human health.


Introduction
Globally research towards the development of drug from natural sources for various ailments that is ravaging the world is increasing.Many plants have been examined for their pharmacological, nutraceutical and therapeutic potentials and different bioactive agents have also been isolated from them.Among biologically active agents is lectin, which is a protein or glycoprotein.It has the ability to bind reversibly and specifically to carbohydrate on the surface of the cells thereby precipitating or agglutinating them (Lam and Ng, 2011).Lectins are well distributed in nature, found in animals, plants and microorganism (Sharon and Lis, 2004;Hamid et al., 2013).Lectins have been found to be valuable tools in scientific research especially in the area of biocontroling, bone marrow transplantation, cancer diagnosis, HIV/AIDS and others (Inbar and Chet, 1997;Bouwman et al., 2006;Lam and Ng, 2011;Huskens and Schols, 2012;Yau et al., 2015).In addition, lectins have been shown to possess various biological activities like anticoagulant, antitumor, antibacterial, antiproliferative, anti-inflammatory, analgesic, insecticidal and antiviral (Singh et al., 1999;Lam and Ng, 2011;Kumar et al., 2012;Hamid et al., 2013;Santos et al., 2014;Dan et al., 2016;Lagarda-Diaz et al., 2017;Hendrickson and Zherdev, 2018).They are considered strong candidates for therapeutic usage because they are macromolecules with noticeable resistance to adverse conditions like pH, temperature variations and isotonicity, with no major changes to their biological functions (Coffey et al., 1993).Also, the characteristic property of lectins to recognise other molecules in a distinct way makes it relevant in various research and applications (Santos et al., 2014).
Recently, lectin research received a boost when it was reported to have the ability to scavenge free radicals (Pan and Ng, 2015;e Lacerda et al., 2017;Olodude et al, 2017) which provided them the potential to protect the human body against damage caused by free radicals and reactive oxygen species released during oxidative stress.The development of several chronic diseases such as cancer, aging, arthritis, cardiovascular diseases, diabetes and neurodegenerative disorder is linked to oxidative stress (Anitha and Sathisha, 2017).Treatment of these disorders needs supply of external antioxidant to improve the internal

Collection and identification of seeds
The fruits of Pterocarpus soyauxii were collected from the Botanical Garden, Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria.The plant and fruit were identified at IFE Herbarium in the same department.
Preparation of the crude extracts of P. soyauxii seeds Pterocarpus soyauxii seeds were removed from the fruits and air-dried.The seeds were dehulled and milled.The milled samples were defatted with petroleum ether and gentle stirring.The defatting process was further repeated for complete removal of the oily constituents.The defatted samples were homogenized in 25 mM phosphate buffered saline (PBS, pH 7.2) containing 100 mM NaCl.The mixtures were stirred overnight using a magnetic stirrer.The homogenates were centrifuged at 10000 rpm for 20 minutes using cold centrifuge and the collected supernatant termed crude lectin extract was frozen and stored.

Glutaraldehyde fixation of erythrocytes
Human blood groups A, B, O was obtained from healthy donors after informed consent.Rabbit and rat erythrocytes were obtained from healthy rabbit and rat purchased from Teaching and Research Farm, Obafemi Awolowo University, Ile Ife.
The animal and human red blood cells (RBC) were fixed with glutaraldehyde according to the method of Pattanapanyasat et al. (2010).Blood samples were collected into heparinized bottles and centrifuged at 3000 rpm for 15 min.The erythrocytes were collected and washed five times with PBS, pH 7.2.A 50% glutaraldehyde was diluted to 1% (v/v) with PBS and chilled at 4 °C.The chilled glutaraldehyde-PBS solution was used to dilute the red blood cells to 2% (v/v).The suspension of cells and glutaraldehyde was incubated for 1 h at 4 °C with occasional mixing.The fixed cells were collected by centrifugation at 3000 rpm for 5 min and washed five times with PBS.The cells were then suspended in PBS, containing 0.02% (w/v) sodium azide, to a final concentration of 2% (v/v) and stored at 4 °C until further use.

Trypsinization of red blood cells
Trypsinization was carried out using the method of Occena et al. (2007).Blood samples of blood groups A, B and O were collected in heparinized bottles and centrifuged at 3000 rpm for 15 min.The erythrocytes were collected and washed three times with PBS and 2% suspension was prepared in PBS for each of the blood groups.For trypsinization process, 1% trypsin was mixed with 2% red blood cells suspension in PBS in a ratio of 1:1 (v/v) and was incubated at 37 °C for 1 h.After incubation, the trypsinized cells were washed with PBS three times, diluted and resuspended in PBS (2% v/v), and stored until further use.

Hemagglutination assay
The crude lectin extract of P. soyauxii seeds was tested for the presence of lectin by hemagglutination assay performed by the method of Odekanyin and Kuku (2014).
defence system which may be overwhelmed by the excessive exposure to oxidative stress (Anitha and Sathisha, 2017).Therefore, there is need for development of more natural antioxidant from potential food sources which can reduce or thwart oxidative stress and its harmful effects.This possibly may replace the synthetic antioxidants which are high-priced and have adverse side effects.
Among the plant family which had their lectins well investigated for various biological properties is Leguminosae.Legume seeds have been the major focus of lectin-related research.Leguminous lectins are the most studied group of lectins and hundreds of these proteins have been isolated and extensively investigated in relation to their chemical, physicochemical, structural and biological properties (Sun et al., 2011).The studies of leguminous lectins are focused mainly on the subfamily Papilionoideae but yet some tribes of this family are rarely investigated.Few lectins from Dalbergieae tribe have been isolated, characterized and includes Lonchocarpus capassa (Joubert et al., 1986), Vatairea macrocarpa (Calvate et al., 1998), Pterocarpus angolensis (Echemendia-Blanco et al., 2009), Platypodium elegans (Benevides et al., 2012), Platymiscium floribundum (Pereira-Junior et al., 2012), Vatairea guianensis (Silva et al., 2012a), Centrolobium tomentosum (Almeida et al., 2016), and Centrolobium microchate (de Vasconcelos et al., 2015).Pterocarpus soyauxii is a species of Pterocarpus genus belonging to the Dalbergieae tribe and family Leguminosae.It is native to central and tropical West Africa countries like Nigeria, Congo-Kinshasa and Angola.The common English name is African padauk/coralwood while it is called Osun pupa by the Yoruba tribe of Western Nigeria.The leaves are edible and contain large amounts of vitamin C; the fresh leaves are used as vegetables and as livestock feed for goats, they are also recommended for consistent use for diabetics (Uzodimma, 2013).They are used in herbal medicine to treat skin parasites and fungal infections (Gill, 1992).The leaves have also been reported to be capable of normalising the hematological abnormalities associated with pathophysiology of diabetes mellitus (Saliu et al., 2012).The ethanolic leaf extract of Pterocarpus soyauxii has been reported to have antimicrobial activity.At high concentration, it significantly inhibited the growth of Escherichia coli, Salmonella typhi, Shigella flexneri, Staphylococcus aureus, Klebsiella pneumoniae and Candida albicans (Osuagwu and Akomas, 2013).Tchamadeu et al. (2011) reported that the aqueous stem bark extract of Pterocarpus soyauxii had very low toxicity in oral acute high dose administration and no toxicity in oral sub-chronic low dose administration in mice and thus concluded that the plant could be considered safe for oral medication.With enormous information available on the usage of this plant for the possible treatment of various disorders, no information is available at the time of complying this report on the health benefit of the seeds of Pterocarpus soyauxii.
The present study was therefore designed to isolate protein with hemagglutinating activity from Pterocarpus soyauxii seeds, purify the lectin, determine its physicochemical properties and evaluate the antioxidant potential of the purified lectin.
Hemagglutination assay was carried out in 96-well Ushaped microtiter plates.PBS (100 μl) was delivered sequentially into wells arranged in rows of 12 wells.The crude extract (100 μl) was added into the first well to obtain a 1:2 dilutions.A serial dilution was then performed by transferring 100 μl of the diluted sample in a particular well into the next well containing 100 μl PBS.This process was carried out for two complete rows of 12 wells each until the last 24 th well.Aliquots (50 μl) of the 2% glutaraldehydetreated erythrocytes suspension of the animal and human blood samples were added separately to each well and the wells were left for 2 h for agglutination to take place.The hemagglutination titre of the lectin is defined as reciprocal of the highest dilution of the extract exhibiting visible hemagglutination -this will be equivalent of one hemagglutination unit (HU).Blood group specificities of the lectins were determined as described above with different blood groups.

Sugar specificity test
The sugar specificities of the lectins were investigated by comparing sugars on the basis of minimum concentration required to inhibit the agglutination of erythrocyte by lectins as described by Kuku and Eretan, (2004).This was done in two steps.In the first step, lectin was successively diluted until the end-point dilution causing hemagglutination was obtained.0.2 M of sugar solution was added to each well at 50 μl per well, while the control well contained PBS instead of sugar solution.Fifty microlitres of erythrocyte suspension was added to each well, and the hemagglutination titre of the lectin was determined.Inhibitory sugars caused a reduction in the titre of the lectin activity shown by PBS-control experiment.The sugars tested are: maltose, D-(+)-mannose, lactose, L-(+)arabinose, sorbose, D-(+)-glucose, galactose, mannitol, Nacetyl-D-glucosamine, mannosamine, 2-deoxy-D-glucose, dulcitol, xylose, methyl α-D--glucopyranoside and D-(+)glucosamine HCl.In the second step, the minimum concentration of each inhibitory sugar required to inhibit lectin-specific hemagglutination of blood cells by 50% was determined (Kuku and Eretan, 2004).Two-fold successive dilutions of sugar samples were prepared in PBS (0.2 M initial concentration).Lectins were successively diluted in the microtiter plates until the 12 th well on each row.Fifty microlitres of the successively-diluted sugar solutions were added to each well containing the successively-diluted lectins while the control well contained PBS instead of sugar solution.Fifty microlitres of erythrocyte suspension was added to each well, and the hemagglutination titre of the lectin was determined.

Purification of P. soyauxii lectin Ammonium sulphate precipitation
The crude lectin extract of the P. soyauxii seeds was subjected to 70% ammonium sulphate precipitation.The ammonium sulphate equivalent to 70% precipitation was slowly added to the crude extracts.The mixture was gently stirred to dissolve completely the salt and centrifuged after 24 h at 3500 rpm for 15 min to obtain the precipitate.The precipitate was dialysed thoroughly against several changes of PBS to remove the salt and undissolved materials from the dialysates.

Gel-filtration on Sephadex G-100
The dialysate of ammonium sulphate precipitate of P. soyauxii crude lectin extract was applied on Sephadex G-100 column (2.5 х 40 cm) previously equilibrated with PBS, pH 7.2.The protein was eluted with the same buffer at a flow rate of 15 ml/hr and 5 ml fractions were collected.The fractions were monitored for protein by measuring the absorbance at 280 nm and assayed for hemagglutinating activity.

Determination of protein concentration
Protein concentration of the crude extract, dialysate and other fractions were determined by the method of Lowry et al. (1951) using Bovine Serum Albumin (BSA) as standard protein.The absorbance at 280 nm was also used to monitor protein elution in the chromatographic fractions.

Physicochemical characterization of purified lectin Molecular weight determination
Native molecular weight of the purified lectin was determined by carried out gel filtration on Sephadex G-100 column (1.5 x 100 cm) which had been calibrated with molecular weight markers.Standard protein (5 ml) was applied separately and ran using 10 mM phosphate buffer, pH 7.2 as eluants at a flow rate of 12 ml/h.Fraction of 5 ml were collected and the elution of the protein was monitored at 280 nm.The void volume of the column was determined using Blue dextran which elution's was monitored at 620 nm.The molecular weight markers used were Bovine serum albumin (Mwt 66,000), Ovalbumin (Mwt 45,000), Pepsin (Mwt 35,000), Chymotrypsin (Mwt 25,000); and Lysozyme (Mwt 14,000).5 mg/ml of each of the standard protein marker were prepared.The purified P. soyauxii seed lectin was also subjected to Sodium Dodecyl Sulphate-Polyacylamide gel electrophoresis (SDS-PAGE) for subunit molecular weight determination.The procedure described by Weber and Osborn (1975) was used along with lysozyme (Mwt 14,000), trypsin inhibitor (Mwt 20,000), carbonic anhydrase (Mwt 29,000), ovalbumin (Mwt 45,000), bovine serum albumin (Mwt 66,000), galactosidase (Mwt 118,000) and myosin (Mwt 212,000) as standard protein markers.

Effect of temperature on hemagglutinating activity
The effect of temperature on hemagglutinating activity of P. soyauxii seeds lectin and thermal stability of the lectin were done as described by Sampaio et al. (1998).The purified lectin was incubated in a water bath for 1 h at different temperatures (30 °C -100 °C).At each temperature, aliquots of the lectin were taken at 15 min interval for 60 min, rapidly cooled on ice and assayed for hemagglutinating activity.The hemagglutinating activity of the untreated samples at room temperature represented 100% hemagglutinating activity.

Effect of pH on hemagglutinating activity
The effect of pH on the hemagglutinating activity of the lectin was performed by incubating aliquots of the lectin solutions for 1 h with buffers of different pH values ranging from pH 3-13.The buffers used were 0.2 M citrate buffer (pH 3-5), 0.2 M Tris-HCl buffer (pH 6-8), and 0.2 M glycine-NaOH buffer (pH 9-13).The hemagglutination assays were carried out.Lectin incubated in PBS pH 7.2 was used as the control.

Effect of EDTA and divalent cations on hemagglutinating activity
The effect of ethylenediaminetetraacetic acid and divalent cations on P. soyauxii seeds lectin were carried out as described by Wang et al. (1996).The purified lectins were dialysed against 10 mM EDTA for 24 h and the hemagglutinating activity of the demetallized lectins was determined.The treated lectins were incubated with 50 μl of each of the following cations: CaCl2, BaCl2, MnCl2, SnCl2, and HgCl2 at 10 mM for 2 hrs and the hemagglutinating activity of each of the samples were determined.

Antioxidant assays 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
The hydrogen or radical scavenging properties of P. soyauxii seeds lectin was determined by the stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl) method as described by Brand-Williams et al. (1995).DPPH (0.3 mM, 1 ml) was added in methanol to 1 ml of varying concentrations of the lectin/standard (ascorbic acid).The mixture was vortexed and incubated in the dark for 30 min and the absorbance was measured at 517 nm against a DPPH negative control containing only 1 ml of the methanol instead of the lectin.
The percentage inhibition of the DPPH scavenging activity was calculated using the equation below: DPPH % inhibition = [1-(Abs sample/ Abs control)] ×100 Where: Abs sample = Absorbance of the lectins Abs control = Absorbance of the control at 517 nm Sample concentration providing 50% inhibition (IC50) was calculated from the graph by plotting inhibition percentage against sample concentration.

Lipid peroxidation inhibition assay
The assay of lipid peroxidation activity was carried out according to the thiobarbituric acid reaction method of Kimura et al. (1981) and Hattori et al. (1993) with BHT as standard.Ten percent (10%) liver homogenate (0.25 ml) was added to 150 mM Tris-HCl buffer (pH 7.2) (0.1 ml), 1% (w/v) ascorbic acid (0.05 ml), 0.07 M FeSO4 (0.05 ml) and varying concentrations of the purified lectin of P. soyauxii seeds.The reaction mixtures were incubated at 37 °C for 1 h.After incubation, 0.5 ml of 0.1 N HCl, 0.2 ml of 9.8% SDS, 0.9 ml of distilled water and 2.0 ml of 0.67% TBA were added sequentially.The reaction mixtures were heated in boiling water at 100 °C for 30 min., cooled and 2.0 ml of butan-1-ol was added and later centrifuged at 3000 rpm for 10 min.The supernatant was collected and measured at 532 nm against reagent blank.
The percentage inhibition was estimated using the expression: % Inhibition = (Abs control-Abs test /Abs test) ×100 where Abscontrol = MDA produced by fenton reaction in the absence of extract (control); Abstest = MDA produced by fenton reaction in the presence of extract.

Metal chelating activity assay
The metal chelating activity assay was carried out according to the method of Singh and Rajini (2004) with some modifications, using EDTA as standard.Solutions of 2 mM FeCl2•4H2O and 5 mM ferrozine were diluted 20 times.Briefly, an aliquot (1 ml) of varying concentrations of purified lectin of P. soyauxii seeds was mixed with 1 ml FeCl2•4H2O.After 5 min incubation, the reaction was initiated by the addition of ferrozine (1 ml).The mixture was shaken vigorously and after a further 10 min incubation period, the absorbance of the solution was measured spectrophotometrically at 562 nm.The percentage inhibition of ferrozine-Fe 2+ complex formation was calculated by using the formula: Chelating effect % = (Abs control -Abs sample/ Abs control) × 100; where Acontrol = absorbance of control sample (the control contains FeCl2 and ferrozine, complex formation molecules); Asample = absorbance of a tested samples.

Results and Discussion
Pterocarpus soyauxii seeds lectin (PSSL) was purified to homogeneity by combination of ammonium sulphate precipitation and gel filtration on Sephadex G-100.The crude protein extract of petroleum ether-defatted P. soyauxii seed powder was subjected to 70% ammonium sulphate precipitation and the precipitate obtained was thoroughly dialyzed.Dialysate was then layered on the Sephadex G-100 column.Three distinct protein peaks (GF-I, GF-II, GF-III) were obtained after the column chromatography.Hemagglutination assay carried out on the pooled fractions of each peak revealed that only the first peak agglutinated erythrocyte (Fig. 1).The same procedure was employed by Galbraith and Goldstein (1970) and e Lacerda et al. (2017).Galbraith and Goldstein (1970) used Sephadex G-200 while e Lacerda et al. (2017) used Sephadex G-100.E Lacerda et al. (2017) obtained three distinct protein peaks and only the first peak exhibited hemagglutinating activity.This is similar to the result of the present study.The methodology established for PSSL purification is efficient because a homogenous molecule was purified by 31 folds with about 13% recovery.The soluble protein concentration and specific activity of the crude lectin extract were 11.6 mg/ml and 176.6 HU/mg proteins respectively and for the purified lectin the value was 1.51 mg/ml and 5427.8HU/mg proteins respectively (Table 1).
Phosphate Buffer Saline (pH 7.2) extract of petroleum ether-defatted P. soyauxii seeds powder showed relatively high hemagglutinating activity against trypsin-treated and untreated erythrocyte of all human blood group and rabbit (Table 2).Only untreated rat erythrocyte was agglutinated.No blood group specificity was detected because all human blood erythrocyte was agglutinated to a similar degree.There was only slight preference for rabbit erythrocyte.Treatment of erythrocyte with trypsin improved the hemagglutination.Non-blood group specificity of P. soyauxii seeds lectin may be due to the presence of multiple binding sites where it can recognize all the determinants of each blood type (Saha et al., 2014).Agglutination of native and enzyme-treated rabbit erythrocyte was observed with Dioclea lasiocarpa seed lectin (do Nascimento et al., 2012).The same observation was reported by Silva et al. (2012a), Santiago et al. (2014) and e Lacerda et al. (2015) for lectins isolated from Vatairea guianensis, Canavalia oxyphylla and Mucuna pruriens respectively.Lectin present in the seed of Mucuna sloanei agglutinated both bromelain-treated and untreated human and rabbit erythrocyte (Teixeira-Sá et al., 2009).Bashir et al. (2010) also reported that extent of hemagglutination was the same when erythrocytes of all human blood group were incubated with purified soy-bean (Glycine max) seed lectin.Apuleia leiocarpa seeds lectin also showed hemagglutinating activity toward all blood tested with preference for rabbit erythrocyte (Carvalho et al., 2015).Opposing our findings on hemagglutinating activity, Pereira-Junior et al. (2012) andde Vasconcelos et al. (2015) reported that no hemagglutinating activity was observed for human erythrocytes when incubated with Platymiscium floribundum and Centrolobium microchaete seeds lectin respectively.
Lectin specificity with certain molecules or cells has been described by Gupta et al. (2012) as dependent on both lectin structure and free sugars or sugar residues of polysaccharides, glycoprotein or glycolipids ligands.
Difference in lectin structures and functions may be linked to many different binding site molecular compositions that bind to all kinds of glycosylated ligands.This makes each type of lectin to have high specificity with particular sugars.The sugar specificity of PSSL was investigated by hemagglutination-inhibition assay using different carbohydrates.PSSL hemagglutinating activity was completely inhibited by galactose and galactose-containing sugars.Also, a pentose sugar, arabinose, completely inhibited the lectin activity.Lactose showed better inhibition of PSSL hemagglutinating activity for all human erythrocyte.The minimum inhibitory concentration of sugars that completely inhibited the lectin activity is shown in Table 3.This inhibitory effect on lectin activity gave support to grouping of PSSL as a member of galactosebinding lectin.This is in accordance with sugar specificity reports for majority of leguminous lectins (Konozy et al., 2002 and2003;Bashir et al., 2010;Sun et al., 2011;Silva et al., 2012a;Kuku et al., 2012;Mishra et al., 2016;Khurtsidze et al., 2017).Some other leguminous lectins that displayed different specificity especially for glucose and mannose or their derivatives have also been reported (do Nascimento et al., 2012;Silva et al., 2013;Osterne et al., 2014;Santiago et al., 2014;Ng et al., 2015;Pompeu et al., 2015).It was observed that none of the sugars that inhibited PSSL Gel Filtration (Sephadex G-100) 1.51 8196 5427.8 30.7 a Protein concentration (mg), b Hemagglutinating activity expressed in hemagglutination units which is reciprocal of the highest dilution of the extract exhibiting visible hemagglutination, c Specific activity is calculated as the ratio of Total activity and total protein.
Fig. 1.Purification of PSSL on Sephadex G-100 Column.Approximately 7 ml of dialysate of 70% ammonium sulphate precipitate was layered on the column (2.5 х 40 cm) packed with Sephadex G-100 that was equilibrated with 25 mM Phosphate buffer pH 7.2 containing 100 mM NaCl.The PSSL was eluted with the same buffer at a flow rate of 12 ml/hr and fractions of 5 ml were collected, monitor for protein by measuring the absorbance at 280 nm and hemagglutinating activity of all the fractions was also determined.(Pooled fractions, •••••Hemagglutinating Activity, ■■■■ -OD 280, GF -Protein Peaks) hemagglutinating activity using human erythrocyte was able to inhibit the activity against rabbit erythrocyte completely.The presence of carbohydrate moiety on the rabbit erythrocyte surface that have higher affinity for the carbohydrate binding site of the lectin may possibly be preventing the sugars from binding to the lectin thereby stopping their inhibitory effects.There is little or no arabinose-specific lectin in the leguminous lectin family.Arabinose binding lectins are commonly isolated from microorganisms, mushrooms and also marine organisms (Engel et al., 1992;Wang and Ng, 2005).Recently, arabinose was reported to cause partial inhibition of hemagglutinating activity of Apuleia leiocarpa seeds lectin (Carvalho et al., 2015).Purified lectins from the Dalbergiae tribe have been reported to be composed of diverse isoforms (Calvete et al., 1998;Silva et al., 2012a;Pereira-Junior et al., 2012;de Vasconcelos et al., 2015).Two isolectins were identified in C. microchaete seeds lectin and diverge in lectin chain lengths (de Vasconcelos et al., 2015).There is high possibility of isolectins presence in P. soyauxii seeds lectin that diverge in their sugar specificity and likely other properties.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of purified PSSL in the presence and absence of reducing agent displayed an electrophoretic profile having a single band corresponding  to apparent molecular mass of 35 kDa.The native molecular weight of PSSL as determined by gel filtration chromatography was 64.6 kDa.The result revealed that PSSL is an homodimeric protein.The result was similar to what was obtained for P. coccineus seeds lectin which has 66 kDa as its native molecular weight and 33 kDa as the subunit mass (Pan and Ng, 2015).So also, it is comparable to two species of Mucuna lectins.Mucuna pruriens and M. sloanei gave a native molecular weight of 60 kDa and 65.6 kDa respectively.
Pterocarpus soyauxii seeds lectin (PSSL) was observed to possess high thermal stability, maintaining its full hemagglutinating activity even after incubation up to 60 °C for 1 h.However, activity was drastically reduced at 70 °C and completely lost at 80 °C and higher temperature (Fig. 2a).This implies that the lectins undergo conformational changes under extreme temperatures resulting in the loss of activity.The loss of activity of the lectins with increased temperature is due to destabilisation of sporadic weak interactions of tertiary structure responsible for native conformation of lectin (Singh and Saxena, 2013).These data suggest that PSSL is comparable to other leguminous lectins that exhibited high thermostability.Lectin from E. speciosa was also discovered to be stable up until 70 °C, but totally lost its activity at 80 °C (Konozy et al., 2003).Lectin isolated from P. floribundum seeds by Pereira-Junior et al.
(2012) maintained its full lectin activity at 60 °C for 1 h, but the activity decreases as the temperature increases.Canavalia oxyphylla seed lectin was likewise reported to retain full activity until 60 °C, lost considerable activity at 70 °C and no activity was observed at 90 °C (Santiago et al., 2014).Lectin purified from Phaseolus coccineus (Pan and Ng, 2015), Chenopodium quinoa (Pompeu et al., 2015), Apuleia leiocarpa (Carvalho et al., 2015) and Canavalia virosa (Shanmugavel et al., 2016) are all thermostable up to 60 °C for at least 30 min.High thermostability possessed by these lectins may be advantageous, as stable bioactive substance is more efficient during all phases of their processing and on the other hand this is considered as antinutritional factors that cause many adverse phenomena in animals if ingested (e Lacerda et al., 2017).
Several studies have revealed that stability under highly acidic and basic conditions is a common property of lectins (Pompeu et al., 2015).Lectins are resistant to harsh condition like drastic change in pH.Examination of the effect of pH on P. soyauxii seed lectin showed that the lectin was pH sensitive.Hemagglutinating activity was optimum at pH 6-8, relatively insensitive to acidic conditions where it maintained 50% maximal activity between pH 3-5 but was distinctly affected by basic pH with the hemagglutinating activity decreasing gradually by 50% for every unit rise in pH until pH 13 (Fig. 2b).This result is in agreement with were employed.This includes DPPH radical scavenging, metal chelating and lipid peroxidation assays.All methods showed that PSSL possessed antioxidant activity in a dosedependent manner and exhibited an IC50 of 0.14 ± 1.33 mg/ml, 3.40 ± 0.37 mg/ml and 0.50 ± 0.08 mg/ml for these methods respectively (Figs. 3 A, B and C).Apart from peptides, obtainable by enzymatic hydrolysis, chemical hydrolysis or bacterial fermentation, that have been established to possess strong antioxidant ability, some proteins from various sources have also been reported to have significant ability to ameliorate the harmful effect of free radicals and reactive oxygen species produced during oxidative stress.Previous investigations, especially on the leguminous seeds, have confirmed that protein from these seeds have antioxidant activity (Carrasco-Castilla et al., 2012;Petchiammal and Hopper, 2014;Pan and Ng, 2015;e Lacerda et al., 2017).Pan and Ng (2015) reported for the first time the ability of leguminous seed lectin to exert antioxidant activity though the antioxidant activity of lectins from other sources such as Moringa oleifera seeds (Santos et al., 2005), Pleurotus florida (Bera et al., 2011), and endophytic fungi (Sadananda et al., 2014) have been published.Our findings support other studies that reported antioxidant activity for lectins from the Leguminaceae family seeds.
the reported pH stability for lectin from Erythrina indica leaves lectin that exhibited pH stability at pH 6.0 -pH 8.0 (Konozy et al., 2002), A. hypogea seed which maintained its maximum activity at pH 5.0 to pH 8.0 (Sun et al., 2011), Dioclea lasiocarpa seeds lectin optimum pH was between 6.0 and 8.0 (do Nascimento et al., 2012) and C. virosa seeds lectin have highest activity at pH 4.0 -pH 9.0 (Shanmugavel et al., 2016).
The hemagglutinating activity of PSSL was not affected after prolong dialysis against EDTA.Also, addition of various divalent ions like Ca 2+ , Mn 2+ or Mg 2+ to the dialyzed lectin did not alter the activity.Though, it has been reported that leguminous lectins require divalent cations like Ca 2+ , Mn 2+ or Mg 2+ for their full hemagglutination (Echemendia-Blanco et al., 2009, Sun et al., 2011, do Nascimento et al., 2012, de Vasconcelos et al., 2015, e Lacerda et al, 2017) there are some exception.The current data proposes that, in contrast to other leguminous lectins, PSSL does not need metal ions for it to be fully active or that the metal ions are most likely bound tightly to the inner part of the molecule, shielding it from the EDTA effect.This is comparable to the report of Pereira-Junior et al. (2012), Silva et al. (2012a, b), Santiago et al. (2014), Pompeu et al. (2015) and Khurtsidze et al. (2017).
In determining the antioxidant capability of PSSL, three different in vitro methods for evaluating antioxidant activity

118Fig. 2 .
Fig. 2. Effect of temperature and pH on the hemagglutinating activity of PSSL

Table 1 .
Summary of purification of lectin from the seeds of Pterocarpus soyauxii

Table 2 .
Hemagglutinating activity of PBS extract of P. Soyauxii seeds against human and animal erythrocytes

Table 3 .
Inhibition of hemagglutinating activity of PSSL by various carbohydrates in the presence of different erythrocytes