Plant Physiol. (1991) 97, 856-8620032-0889/91/97/0856/07/$01 .00/0

Received for publication January 29, 1991Accepted May 9,1991

Purification and Characterization of Two Major Lectins fromAraucaria brasiliensis syn. Araucaria angustifolia

Seeds (Pinhao)1

Pradip K. Datta, Maria 0. D. C. R. Figueroa, and Franco M. Lajolo*Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta 700 032, India (P.K.D.) and Faculdade de

Ciencias Farmaceuticas - USP, Depto. de Alimentos e Nutriqao Experimental, Cx.Postal 66355, CEP 05389,Sao Paulo, SP, Brasil (M.O.D.C.R.F., F.M.L.)

ABSTRACT

Two major lectins (lectin I and lectin 11) were purified to homo-geneity from the seeds of Araucaria brasiliensis (Gymnosper-mae). The purity of the lectins was confirmed by polyacrylamidegel electrophoresis, isoelectric focusing, and high performanceliquid chromatography. They are glycoproteins in nature contain-ing 6.3 and 2.9%, respectively, of neutral sugar and have absorp-tion coefficients of 3.8 and 4.7, respectively, at 280 nanometers.The molecular weights of both lectins obtained by gel filtrationon Sephacryl S-400 were equal: 200,000. After dissociation bysodium dodecyl sulfate-polyacrylamide gel electrophoresis, mo-lecular weights were 20,000 and 34,000, respectively, for lectin Iand lectin 11, suggesting they are decameric and hexameric innature. The amino acid composition of both lectins showed littledifference, but both had high amounts of acidic amino acids andlacked methionine in their molecule. The carbohydrate bindingspecificity of lectins was directed towards mannose, glucose,and their oligomers. High inhibitory activity was also found withthyroglobulin. The erythroagglutinating activity of the lectins wasenhanced in the presence of high-molecular-weight substancesboth at 37 and 40C. Divalent cations do not appear to be essentialfor activity. They maintained their agglutinating activity over abroad but different range of pH: 5.5 to 7.5 and 6.5 to 7.5, respec-tively. Both lectins agglutinated erythrocytes of human ABO bloodtypes equally well.

Lectins are a special group of sugar-binding proteins ofnonimmune origin able to agglutinate cells and/or precipitateglucoconjugates (l1). They have been identified in naturefrom microorganisms to animals (10) and display a widevariety of unique and interesting biological properties (18).Several hypotheses concerning their biological functions havebeen reported, but this remains an unsettled question.

"Pinhao" is the seed of the tree "Pinheiro de Parana"; it isconsumed as food. This tree is a Gymnospermae that growsin the south of Brazil and in the northern region of Argentina.It belongs to the Coniferae group, which forms, nowadays,the biggest living group of Gymnospermae (21). No infor-mation was found in the literature on the presence and

' The research was developed and funded within the Depto. deAlimentos e Nutriaio Experimental of Sao Paulo University whileDr. P. K. Datta was a postdoctoral fellow ofthe program CNPq-USP.

description of lectins in Gymnospermae. Here, we describethe isolation, purification, and some of the properties of thepinhao lectins.

MATERIALS AND METHODS

Pinhao, Araucaria brasiliensis, was purchased from a localsupermarket. Normal typed human erythrocytes were ob-tained from the University Medical College Blood Bank (SaoPaulo, Brazil). HPLC grade methanol was from E. Merck(FRG). Bio-Gel P- 150 was from Bio-Rad Laboratories (Rich-mond, CA). Phenyl-Sepharose CL-4B, DEAE-Sephadex,Pharmalite (pH 3.0 to 10.0), and Sephacryl S-400 were allfrom Pharmacia Fine Chemicals (Uppsala, Sweden). Thefollowing chemicals were all obtained from Sigma ChemicalCo. (St. Louis, MO): thyroglobulin, pepsin, arabinogalactan,polyvinylpyrrolidone, myosin from rabbit muscle (Mr,205,000), ,3-galactosidase from Escherichia coli (Mr, 116,000),phosphorylase b from rabbit muscle (Mr, 97,400), BSA (Mr,66,000), albumin from egg (Mr, 45,000), carbonic anhydrasefrom bovine erythrocyte (Mr, 29,000), I3-lactoglobulin (Mr,18,400), lysozyme (Mr, 14,300), D-xylose, a-D-fucose, D-man-nitol, D-galactose, D-fructose, D-mannose, raffinose, lactose,D-glucosamine HCl, L-rhamnose, D-glucose, para-nitro-phenyl-a-D-glucoside, para-nitrophenyl-fl-D-glucoside, para-nitrophenyl-a-D-mannoside, para-nitrophenyl-N-acetyl f3-D-galactosaminide, para-nitrophenyl-N-acetyl-,-D-glucosamin-ide, para-nitrophenyl-a-D-galactoside, para-nitrophenylmal-toside, para-nitrophenyl-fl-D-fucoside and L-arabinose. Allother chemicals were of analytical grade.

Purifications of Lectins

Step I. Dehusked pinhao (100 g) was soaked in 1000 mLof PBS (pH 7.2) (8 mM phosphate buffer containing 0.15 MNaCl) for 12 h at 4°C and was then homogenized. Thehomogenate was filtered through cheesecloth. The superna-tant solution (crude extract) was collected after centrifugationfor 30 min at 10,000g. The purification was continued from30 mL of the crude extract, containing 82.2 mg of proteins(Table I). This extract was dialyzed overnight against Mc-Ilvaine buffer (pH 4.8) in 0.2 M NaCl; protein precipitatedduring dialysis was centrifuged at 25,000g for 20 min. Theprecipitate was dissolved in 0.1 M phosphate buffer (pH 7.2)containing 0.20% sodium azide in 0.75 M NaCl at 25°C. This

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CHARACTERIZATION OF LECTINS FROM ARAUCARIA BRASILIENSIS SEEDS (PINHAO)

solution was dialyzed and centrifuged as described above. Theprecipitate obtained on centrifugation was called GF- 12. Thesupernatant obtained at this stage was pooled with the super-natant obtained in the previous step. The combined super-natants were dialyzed against deionized water with severalchanges for 24 h and centrifuged at 25,000g for 20 min. Theprecipitate was called GF-2. The supernatant at this stage stillcontained protein and was called albumin protein. The GF- 1and GF-2 preparations were dissolved in 0.2 M NaCl in 0.15M phosphate buffer ofpH 7.5.

Step II. Gel filtration of the albumin protein fraction wascarried out on a Bio-Gel P-150 (87 x 2.5 cm) column. Thecolumn was washed and equilibrated with PBS. The albuminprotein fraction was concentrated and dialyzed against PBSand applied to the Bio-Gel P-150 column. Protein was elutedfrom the column with PBS at a flow rate of 12 mL/h, 3 mL/tube, and fractions showing agglutinating activity were pooledand concentrated.

Step III. The hydrophobic separation of the most activefraction on the gel filtration column was done on a Phenyl-Sepharose CL-4B (56 x 1.5 cm) column. The column waswashed and equilibrated with 8 mm phosphate buffer (pH7.2). The minor peak from gel filtration column was dialyzedagainst the same buffer, and, after being loaded into thehydrophobic column, protein fractions were eluted with aNaCl gradient (0 to 1.0 M) at a flow rate of 10 mL/h, 2 mL/tube. The proteins eluted at 0.16 M salt concentration pos-sessed erythroagglutinating activity. The active fractions werepooled and concentrated.

Step IV. The concentrated fraction was then applied to aDEAE-Sephadex column. Before application of the protein,the column was washed and equilibrated with 8 mm phosphatebuffer (pH 7.2). The protein was dialyzed against the samebuffer added to the column and was washed with 20 mL ofthe same buffer. Afterward, a linear gradient of NaCl (0 to0.5 M) was started at a flow rate of 20 mL/h, 1 mL/tube. Thepeaks showing hemagglutinating activity were pooled sepa-rately and concentrated.

Step V. HPLC on an LKB (Bromma, Sweden) 2150 HPLCpump system was used for further analysis of the lectins. Theindividual peaks obtained from the DEAE-Sephadex columnwere dialyzed against PBS, and a portion containing 50 ,ug ofprotein was applied to the HPLC column (Ultropac TSK G-3000 SW) previously equilibrated with PBS. The columneffluent, at a flow rate of 0.5 mL/min, was monitored at 280nm.

Electrophoresis

PAGE of the purified lectins was performed by the Davismethod (6). The molecular mass of the lectins treated withSDS in the presence of 2-mercaptoethanol was determined(14) by using standard Mr markers.

Isoelectric Focusing

Isoelectric focusing was carried out in a rod gel (12 x 0.6cm) containing 4% ampholine (pH 3.0 to 10.0) and 10%polyacrylamide (12). After a run for 6 h at 300 V, the gel was

2Abbreviation: GF, globulin fraction.

cut into slices, the slices were immersed in 1 mL of water,and the pH of the extract was determined. Lectin activity ineach slice was then measured.

Protein Estimation

The protein was determined by the method of Lowry et al.(19). Eluates from the columns were also monitored spectro-photometrically at 280 nm.

Hemagglutination Assay

Hemagglutination was assayed in V-microtiter plates (4) byserially diluting a 50-ML sample into 50 ML of PBS. Added toeach well was 50 ,L of 2% rabbit erythrocyte suspension inthe same buffer, and hemagglutination was determined aftera 1-h incubation at 37C as the reciprocal of the greatestdilution that gave visible aggregation. Effects of saccharides,glycoprotein, arabinogalactan, and polyvinylpyrrolidone onhemagglutination were examined by preincubation of thelectin with the test substance at 37°C for 30 min, followed byincubation at 37C for 1 h after addition of erythrocytesuspension.

Agglutinating activity of these high-molecular-weight poly-mers in the absence of lectins was determined by the additionof 50 ,L of PBS, 50 ,L of polymer solution (1 mg/mL), and50 ,L of 2% rabbit erythrocyte suspension to each well andby the incubation of the plates for 1 h at 37TC.

Molecular Mass Determination

The molecular weights of lectin I and lectin II were deter-mined (2) by gel filtration on a Sephacryl S-400 column (70x 1 cm; 1 mL/tube; flow rate, 10 mL/h) calibrated withmyosin (Mr, 205,000), ,B-galactosidase (Mr, 116,000), phos-phorylase b (Mr, 97,400), BSA (Mr, 66,000), ovalbumin (Mr,45,000), and carbonic anhydrase (Mr, 29,000).

Divalent Cation Requirement

The purified lectins were dialyzed against PBS (pH 7.2)containing 10 mM EDTA. Excess EDTA was removed byextensive dialysis against the same buffer. Hemagglutinatingactivity was assayed on the dialysis residue alone and in thepresence of CaCl2, MnCl2, and MgCl2 at concentrations vary-ing from 10 to 40 mM.

Carbohydrate Analysis of the Agglutinating Proteins

The neutral sugar content of the purified lectins was esti-mated by the method of Dubois et al. (7) with D-glucose as astandard.

Effect of pH and Temperature

The following were used for pH studies: acetate buffers, pH4.0 to 6.5; phosphate buffers, pH 6.5, 7.2, and pH 7.5 to 8.0;borate buffers, pH 8.5 to 9.5; glycine buffers, pH 9.5 to 10.5.All buffers contained 0.15 M NaCl, and pH intervals of 0.5units each.The effect of temperature on hemagglutination activity of

lectins was determined by incubating the lectin samples for

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Plant Physiol. Vol. 97, 1991

30 min at different temperatures from 25 to 80°C at 10°Cincrements.

Amino Acid Analysis

The amino acid analysis of purified lectin I and lectin IIwas performed by the method of Spackman et al. (22) on a

Beckman 121 MB automatic amino acid analyzer (BeckmanInstrument Co., Fullerton, CA) equipped with a HewlettPackard Integrater (Model HP3396A) after the hydrolysis ofthe samples in 6 M HCI for 24 h at 1 10°C. Cysteine andmethionine were analyzed after performic acid oxidation andHCI hydrolysis. Tryptophan was calculated by the method ofEdelhoch (8).

RESULTS AND DISCUSSION

The purification steps are depicted in Table I. The seedcoat had no agglutinating activity; maxim lectin activity wasrecovered from the albumin fraction. This fraction by chro-matography on Bio-Gel P-150 yielded one minor and one

major peak (Fig. 1). The minor peak showed maximum lectinactivity and was purified further through a Phenyl SepharoseCL-4B column. This gave only one major peak with highlectin activity, but it was found heterogeneous on electropho-resis. For further purification, this fraction was applied toDEAE-Sephadex column. With a 0 to 0.5 M NaCl gradient,we obtained 10 peaks, 6 of them having hemagglutinatingactivity. Among them, the last two peaks presented 90% oftotal activity. They were called lectin I and lectin II and were

purified by TSK G-3000 SW HPLC (Fig. 2). Lectin I andlectin II eluted as single symmetrical peaks and were foundto be homogeneous with 1 17.9-fold and 76.7-fold purification,respectively, and with high specific activities (Table I). Theminimum amount of purified lectin that gives positive agglu-

150

Tube N!:

c

4-

cm

EI

Figure 1. Elution profile of A. brasiliensis seed lectins on Bio-Gel P-150 column. Flow rate, 12 mL/h; 3 mL/tube; solid line, absorbanceat 280 nm.

tination was 0.0028 ,ug/mL for lectin I and 0.0043 ,g/mL forlectin II. In our laboratory under similar conditions, forPhaseolus vulgaris, soybean, etc., we obtain values from 1 to7 ,ug/mL approximately.

Generally, seeds contain one lectin, even though there are

some reports on the presence of more than one (3, 13, 15,17). It is possible that plants contain families of lectin genes

that may have altered patterns of expression in a tissueor in different tissues of the same plant. Common lectingene products may also differ during posttranslationalmodification (9).

Table 1. Purification Scheme of Lectins from A. brasiliensis Seeds

FractionTotal Specific Total Purifi- Rcvr

Fraction Protein Activity Activity cation Rve

mg prlmg of titer x 10-3 -fold %

Crude extracta 82.2 29.9 2457.8 1 100GF-1 20.0 0.6 12.0 0.0 0.5GF-2 14.5 2.6 37.7 0.1 1.5Albumin proteins 23.6 97.2 2294.0 3.3 93.3

Bio-Gel P-150Peak (minor) 3.5 420.1 1470.4 14.1 59.8Peak (major) 17.6 12.8 225.3 0.4 9.2

Phenyl-Sepharose 1.4 963.8 1349.3 32.2 54.9DEAE-SephadexPeak I 0.1 25.8 2.6 0.9 0.1Peak II 0.1 6.8 0.7 0.2 0.0Peak III 0.3 157.5 47.3 5.3 1.9Peak IV 0.2 63.2 12.6 2.1 0.5Peak V 0.2 2978.9 595.8 99.6 24.2Peak VI 0.3 2048.0 614.4 68.5 25.0

HPLCPeak V (lectin 1) 0.2 3524.6 704.9 117.9 28.7Peak VI (lectin 11) 0.2 2292.7 458.5 76.7 18.7a 30 mL.

858 DATTA ET AL.

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CHARACTERIZATION OF LECTINS FROM ARAUCARIA BRASILIENSIS SEEDS (PINHAO)

0,02

0,01

I

humuumlihiiulfhfhmulin

a

I._

b

0,03

0,02

0,01

0 5 10 15 20

min0 10 15 25 30

min

Figure 2. HPLC assay of protein from peak V (a) and peak VI (b) obtained from DEAE-Sephadex column. The sample protein (50 ,g in 100 /IL)was applied to the column. The column effluent was monitored at A280 = 0.1 absorbance unit full scale. Inset: PAGE from lectin I (a) and lectin 11(b), 30 Mg of protein in each gel tube, 5 mA/gel (7.5%).

The homogeneity of the purified lectins, lectin I and lectinII, were confirmed by several criteria. On PAGE at pH 8.3(Fig. 2) and SDS-PAGE (Fig. 3), they gave a single proteinband. The Mr values of lectins I and II by SDS-PAGE werecalculated to be 20,000 + 1,000 and 34,000 ± 1,000, respec-tively (Table II). The homogeneity of the purified lectinswas also confirmed by isoelectric focusing. Both showed asingle band at pH values of about 7.3 and 7.5, respectively(Table II).Gel filtration on Sephacryl S-400 column oflectin I resulted

in one major and four minor peaks; the Mr of the major peakwas 200,000 + 1,000 and the minor peaks were 79,000 ±1,000; 59,500 + 1,000; 39,000 ± 1,000 and 20,000 + 1,000,respectively. This suggests that lectin I exists as a decamer inthe native stage with aggregation of one single monomer.However, lectin II, on Sephacryl S-400 gel filtration, showedtwo peaks with Mr values of 200,000 + 1,000 and 68,000 ±1,000, respectively, which indicates that it probably exists asa hexamer formed by the association of three dimers (TableII). Minor polypeptide fragments may result from differentassociation of subunits.

14.3 Kd -

a b

Figure 3. SDS-PAGE in presence of 2-mercaptoethanol of lectin I (a)and lectin 11 (b) at pH 8.3. Agglutinating protein (50 Mg) was appliedin each gel tube. A current of 3 mA/gel (10%) was applied for 8 h at1OC.

Table II. Characteristics of Lectin I and Lectin /1

Properties Lectin Lectin II

Native M, 200,000 ± 1,000 200,000 ± 1,000Subunit M, 20,000 ± 1,000 34,000 ± 1,000Possible structure Decamer HexamerIsoelectric point -7.3 -7.5Neutral sugar (%) 6.3 2.9Al%,' cmat 280 nm 3.8 4.7

974 Kd -

66.0 Kd -

45.0 Kd-

29.0 Kd_-

184 Kd-

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Plant Physiol. Vol. 97, 1991

Table ll. Effect of High-Molecular-Weight Substances and DivalentCations on Specific Activity of Lectin I and Lectin 11

Specific Activity x 10-3Conditions

Lectin Lectin 11

370C 3,524 2,292370C + Arabinogalactan 14,124 9,204370C + PVP 3,524 36,81740C 3,524 2,29240C + Arabinogalactan 14,124 9,20440C + PVP 70,620 73,635370C + EDTA 3,524 2,292370C + 40 mM MgCI2 70,620 9,204370C + 40 mm CaC12 3,524 4,602370C + 40 mM MnCI2 3,524 2,292

Measurements of the molecular weights of lectins by gelfiltration may be subject to some uncertainty owing to inter-actions in the lectin matrix, but, in most cases, good estimateswere possible by this technique.

Lectins I and II are glycoproteins in nature, containing 6.3and 2.9% neutral sugars (Table II).The absorption coefficients A'%, cm of lectins I and II were

3.8 and 4.7, respectively (Table II).In the presence of high-molecular-weight substances, both

lectins showed different behavior at 37°C (Table III). Specificactivity of lectin I increased in the presence of arabinogalac-tan, but it was not affected by PVP, whereas specific activityof lectin II was increased by the presence of both substances.In the absence of lectin, these substances did not presentagglutinating activity.At low temperature, interesting results were obtained. At

4°C, the activity of neither lectin changed but, at this temper-

Table IV. Hapten Inhibition Study of Purified A. brasiliensis LectinsMinimum ConcentrationRequired to Inhibit the

Sugarsa Hemagglutinating Activity

Lectin I Lectin 11

mM

D-Mannose 3.12 1.56D-Glucose 3.12 3.12D-Glucosamine 12.50 6.25p-nitrophenyl 1.56 0.78a-D-glucOsidep-nitrophenyl 0.78 1.56O-D-glucosidep-nitrophenyl 0.78 0.39a-D-mannosidep-nitrophenyl N-acetyl 25.0 12.50fl-D-glucosaminideThyroglobulin 0.156b 0.312bPepsin 50.Qb 50.Ob

aD-Xylose, D-fucose, mannitol, D-galactose, D-fructose, L-arabi-nose, raffinose, lactose, L-rhamnose, p-nitrophenyl-a-D-galactoside,p-nitrophenyl-N-acetyl-l-D-galactosaminide, p-nitrophenylmaltoside,and para-nitrophenyl-h-D-fucoside were not inhibitors up to 100mM. b Concentration in mg/mL.

Table V. Amino Acid Compositions of A. brasiliensis LectinsLectin Lectin II

Amino acid Residue Neaest Residue Nearestper 100 integer per 100 inerresidue residue

Asp 7.82 8.0 8.90 9.0Thr 5.18 5.0 7.88 8.0Ser 5.93 6.0 4.19 4.0Glu 10.71 11.0 10.15 10.0Pro 2.97 3.0 2.80 3.0Gly 15.15 15.0 17.76 18.0Ala 4.79 5.0 4.32 4.0Val 5.23 5.0 8.21 8.0Met 0 0 0 0Cysa 8.25 8.0 6.08 6.0lie 4.76 5.0 5.98 6.0Leu 3.78 4.0 3.75 4.0Tyr 2.76 3.0 3.68 4.0Phe 2.29 2.0 3.00 3.0Lys 9.69 10.0 5.24 5.0His 6.34 6.0 3.06 3.0Arg 3.00 3.0 3.08 3.0Trpb 1.34 1.0 2.49 3.0

a Cystein was calculated as cysteic acid. bTryptophan wascalculated by the method of Edelhoch (1967).

ature and in the presence of arabinogalactan or PVP, specificactivity of both lectin I and lectin II increased. This indicatesthat in the presence of high-molecular-weight substances,there are conformational changes of the molecules that helpincrease their activities. It was reported earlier that lectinsmay increase their specific activity in the presence of high-molecular-weight substances because of the enhancement of,3-conformation (5).

Metal ions do not appear to be an essential requirement forthe actions of either lectins, because the activity of each lectinwas not inhibited by EDTA, but their specific activity in-creased from normal when high concentrations of metal ionslike 40 mM MgCl2 and 40 mr CaCl2 were added to the system(Table III). Ca2' and Mn24 content of some lectins increases,as does agglutinating activity (20). We do not know the metalcontent of native lectins. It is possible that the metal ionshave different avidities for certain reactive groups on theprotein (1), which explains the minimal action of Ca2" onlyon lectin II. At lower concentrations (from 10 to 30 mM),there was no significant change of activity.The carbohydrate-binding specificity was determined by

the hapten inhibition study in Crook's microtiter plates, andthe results are shown in Table IV. It is evident that, in thecase of lectin I, p-nitrophenyl-a-D-mannoside and p-nitro-phenyl f3-D-glucoside are the most potent inhibitors, whereas,for lectin II, p-nitrophenyl-a-D-mannoside is the most effi-cient. Other sugars tested did not inhibit hemagglutination,even at higher concentrations (Table IV). Agglutination oferythrocytes caused by the crude extract was inhibited by N-acetyl-D-glucosamine (12.6 mM) but not by maltose or sucrose(up to 100 mM). The glycoprotein thyroglobulin was the mostpotent inhibitor among the saccharides tested for both lectins.Pepsin had no inhibitory activity.

DATTA ET AL.860

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CHARACTERIZATION OF LECTINS FROM ARAUCARIA BRASILIENSIS SEEDS (PINHAO) 861

A Both lectin I and lectin II agglutinated erythrocytes ofhuman ABO blood types equally well.

* A AA. brasiliensis lectins are interesting substances because oftheir high specific activities and their structural features.

In this article, we have described some properties of theselectins, which have two main characteristics that have not

* been found in plant lectins before: extremely high agglutin-* * ating activity and their presence in Gymnospermae.

The high agglutinating activity observed in lectins I and IImay be explained by the numerous subunities presented by

0 *° them (decameric and hexameric, respectively), creating a highvalence number and, thus, a high possibility of binding.

0 Hundreds of lectins have been recognized in plants, some0 of them already have been purified and some are being

commercialized. However, the presence of lectins in Gym-^0 5.0 6.0 70 8.0 9.0 10.0 11.0 nospermae had not been described before. A. brasiliensis

p H lectins could be used for taxonomic and phylogenetic studiesand, what is more, for evolutionary correlation studies (16).

B

a I A

.-

0.-_

.4_

C

0

4-

.n

I

1024

256

64

16

*A 2048,

A

.

.

.L-

512

C

0

X 1284-

32cUE

8

2

0

4.0 5.0 6.0 7.0

pH

ao 9.0 10.0 11.0

Figure 4. pH dependence activity profile of A. brasiliensis lectin I (A)and lectin 11 (B). P, Acetate buffer; A, phosphate buffer; 0, boratebuffer; 0, glycine buffer. For experimental details, see the text.

The amino acid compositions of lectins are presented inTable V. Analysis revealed high amounts of acidic aminoacids, glycine, cysteine, and lysine for lectin I. In lectin II,

acidic amino acids, threonine, glycine, and valine were higherin content when compared with other amino acids. Thecontent of aromatic amino acids was higher in lectin II thanin lectin I. Methionine was absent in both lectins.

Purified lectin I and lectin II exhibited maximal specificactivities between pH 5.5 to 7.5 and pH 6.5 to 7.5, respectively(Fig. 4). No significant difference was observed with differentbuffers at overlapping pH values in both the cases.The thermal stability of the lectins is shown in Figure 5,

where the change of specific activities is depicted. Lectin IIwas more temperature labile than lectin I. With increasingtemperature, both lectins lost their activities, which essentiallycease at 80C.

2048

a-

'U

5128

01

En

X 32E

IU8

2

0

0

.

0

!O 30 40 50 60 70 80

Temp (-C)

B

0

0

0

20 30 40 50 60 70 80

Temp ('C)

Figure 5. Temperature dependence activity profile of A. brasiliensislectin I (A) and lectin 11 (B). For experimental details, see the text.

1024

,, 256._-

c 640.2_

c

, 16cm

4I

40961

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Plant Physiol. Vol. 97, 1991

ACKNOWLEDGMENT

We thank Miss C. R. Denani for the careful and efficient typing ofthe manuscript.

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