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5 6 4 2 Gray's Inn Road, London WClX 8LU and P. J. HYLANDS. Chelsea Department of Pharmacy, King's College London,. Chelsea Campus, Manresa Road, ...
PURIFICATION AND PARTIAL CHARACTERIZATION OF A HEMAGGLUTININ FROM SEEDS OF JATROPHA CURCAS L. M . CAN0 ASSELEIH1, R. A . PLUMBLEY2 Overseas Development Natural Resources Institute 5 6 4 2 Gray’s Inn Road, London WClX 8LU

and P. J. HYLANDS Chelsea Department of Pharmacy, King’s College London, Chelsea Campus, Manresa Road, London SW3 6LX Received for Publication January 8, 1988. Accepted for Publication June 25, 198R

ABSTRACT An extract from Jatropha curcas seeds, purijied by geljltration on Sephadex G-75 and Sephacryl S-200, yielded an active hemagglutinin of high purity as assessed by electrophoresis and isoelectric focussing. The hemagglutinin had a molecular weight of around 660,000 and a p l value of5.75. The molecule was composed of two different subunits of molecular weights 23,450 and 11,500. Amino acid analysis suggested that the molecule lacked ‘/r cystine but contained a high proportion of acidic and basic amino acids. Agglutination of trypsinized erythrocytes, groups A , B and 0, took place over the range pH 4-10, and was prevented by D-galactose, D-galactosamine and N-acetyl-D-galactosamine . The hemagglutinin has only a weak binding capacity for D-galactose. Its activity was stable up to 60°C; at 80°C activity was lost in 50 min.

INTRODUCTION Jatrupha curcas L. belongs to the family Euphorbiacea and is a wild or semicultivated bush widely distributed in central and South America, Africa, India and S.E. Asia. The plant has many uses as an ornamental or in medicine and I

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Present address: Instituto Nacional de Investigaciones Sobre Recursos Biciticos, Apartado Postal 63, 91000 Xalpa, Veracruz, Mexico Correspondence to: R. A. PLUMBLEY Overseas Development and Natural Resources Institute 56-62 Gray’s Inn Road London WClX RLU

Journal of Food Biochemistry 13 (1989) 1-20. All Rights Reserved. 0 Copyright 1989 by Food & Nutrition Press, Inc.. Trumhull, Connrt.ti~~ut

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L. M. C A N 0 ASSELEIH, R. A. PLUMBLEY and P. J . HYLANDS

the oil has been used extensively in the manufacture of soap (Cano Asseleih 1986). The seeds have been reported as a powerful purgative (Aponte 1978) and are regarded as toxic. They are not commonly used for food but in Veracruz State, Mexico, where it is known as “piiioncillo”, they are dried, decorticated and roasted before being eaten salted or ground to a paste for use in sweets or the local dishes mole or tamales (Hernandez 1978). The kernels are reported to contain 24.8% protein and 48.2% oil and the plant is cultivated in South Veracruz where the seeds are fed to pigs without any apparent adverse effects (Cano Asseleih 1986). Studies on seeds from Sudan showed them to be highly toxic to mice (Adam 1974), calves (Ahmed and Adam 1979) and sheep and goats (Adam and Magzoub 1975) when whole ground seed was included in the diet. Toxic effects have been associated with a protein fraction and with the oil, although feeding trials of the oil from Mexican seeds showed no signs of toxicity towards rats (Panigrahi et al. 1984). The crude protein preparation, curcin, used by Stirpe et al. (1976) induced toxic symptoms in mice if administered by means of intraperitoneal injection. Curcin, associated with toxicity, has been shown to have an inhibitory effect on protein synthesis in vitro that is comparable with that exhibited by ricin and abrin but is, however, 1000 times less toxic (Stirpe et af. 1976). Like ricin, curcin is a lectin which has the ability to bind to carbohydrates and bring about hemaglutination of blood cells (Jaffe 1969). Ricin has been shown to consist of two different polypeptide chains which serve as a toxic “effectomer” and a nontoxic “haptomer” or carrier moiety (Jaffe 1980). The latter serves to anchor the toxin to the cell surface and it has been reported that binding probably involves a galactose-containing receptor site (Olsnes and Pihl 1973). Curcin does show differences from ricin, however, in that activity is not increased in the presence of 2-mercaptoethanol and it does not agglutinate Ehrlich ascites cells (Stirpe et al. 1976). This paper describes the purification of a hemagglutinin from Jutrophu curcus and examines some of its properties.

MATERIALS AND METHODS Extractability of the Hemagglutinin from J. curcas Seeds were supplied from Papantla, Veracruz, Mexico. They were harvested when mature and sun dried for four days prior to shipment. Finely ground decorticated seeds were extracted under conditions of varying pH (3-10) and sodium chloride concentration (0-. 15 M in 0.25 M steps) according to the method used by Dench (1982). Extracts were assayed for protein and hemagglutinin activity by the methods of Lowry et al. (195 I ) and Marquardt and Gordon (1974), respectively.

HEMAGGLUTININ FROM JATROPHA CURCAS

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Preparation of J. curcas Seed Extract Finely ground decorticated seeds (200 g) were suspended in 2 L of 0.5 M NaCl adjusted to pH 5.0 and stirred continuously for 1 h at 4°C. The suspension was filtered and the thin oily surface layer removed before the suspension was incubated in a water bath at 70°C for 30 min. The precipitate formed was removed by filtration and the supernatant was cooled to 4°C before dialysis against three changes of 0.5 M NaCl, pH 5.0 prior to freeze drying.

Purification of the Hemagglutinin The freeze dried extract was redissolved in distilled water and purified by gel filtration. Aliquots (20 mL) containing 8.7 mg mL-' protein were applied to a Sephadex G-75 (Pharmacia) column (35 cm x 4 cm) previously equilibrated with 0.5 M NaCI. Elution was with the same solution at a flow rate of 300 mL h- I . Fractions containing hemagghtinin activity were pooled, freeze dried, redissolved in distilled water and aliquots (2.5 mL) containing 24 mg mL- protein were applied to a Sephacryl S-200 (Pharmacia) column (50 cm X 3.2 cm) previously equilibrated with 0.1 M Tris-HC1 buffer, pH 7.0, containing 0.4 M NaCl. Elution was by the same buffer at a flow rate of 52 mL h-I and 6.0 mL fractions were collected. Active fractions were treated as before and aliquots (2.5 mL) containing 0.9 mg mL-' protein were rechromatographed on Sephacryl S-200. All purification steps were carried out at 4°C. Purification was also attempted using the method of Vretblad (1976). The crude hemagglutinin extract was applied to an N-acetyl-D-galactosamineSepharose column (15 cm X 0.9 cm) at a flow rate of 20 mL h-l until hemagglutinin could be detected in the eluent. The column was then washed extensively with 0.5 M NaCl, pH 5.0, before a 5.0 mL pulse of saline containing 40 mg mL-I of D-galactosamine or N-acetyl-D-galactosamine was applied followed by elution with saline. The elutant was monitored at 280 nm and the fractions showing positive readings were collected. The process was carried out at 4°C.

Purity Purity was determined after the rechromatography stage on Sephacryl S-200 by polyacrylamide disc gel electrophoresis on 10 cm polyacrylamide rods at pH 8.8 according to the discontinuous buffer system described by Laemmli (1970). Aliquots (14.6 p,g protein per gel) were applied and run for 4 h at 4°C with a current of 3 mA per gel. Gels were fixed for 1 h in 3% sulfosalicylic acid and stained overnight with 0.115% Coomassie Brilliant Blue (3-250 in 8% acetic acid dissolved in 25% methanol. Destaining was by diffusion of excess stain into repeated changes of 8% acetic acid in 25% methanol.

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L. M. C A N 0 ASSELEIH, R . A . PLUMBLEY and P. J. HYLANDS

A further test for purity was by isoelectrofocusing on agarose slab gels (1 I .5 cm x 11 .O cm X 0.2 cm) using the method recommended by Pharmacia (1980). Aliquots containing 5 or 10 pg protein were applied to the gels which were then run at a voltage of 1500 V and a power of 15 W for 1.5 h at 4°C. Gels were then fixed in 5% sulfosalicylic acid in 10% trichloroacetic acid for 1 h before being stained with 0.2% Coomassie Brilliant Blue G-250 in methanol: acetic acid: water (3: 1 :6) overnight. Gels were destained in repeated changes of methanol: acetic acid: water. The system was run with broad range (pH 3-10) and narrow range (pH 5-8) Phannalyte carriers. The isoelectric point (PI) for the hemagglutinin was determined by comparison with marker proteins of known PI. In both cases the hemagglutinin was detected by slicing unstained gels and assaying the leachate for hemagglutination activity after 2 h soaking in saline.

Molecular Weight Determination Molecular weight determination for the hemagglutinin was by the gel filtration method of Andrews (1964) using Sephacryl S-200, as described, or by gel electrophoresis using the discontinuous buffer system of Laemmli ( 1970). The electrophoresis was as previously described but both gel and buffer were modified by the addition of 0.1% sodium dodecyl sulfate (SDS). In addition, thin layer electrophoresis (17 cm X 17 cm X 0.5 cm) was done using 12% rather than 8% polyacrylamide and 0.1% SDS run at 150 V for 5.5 h. Samples contained 250 Fg protein mL-' in 0.0625 M Tris/HCI buffer, pH 6.8, containing 10% glycerol, O.oc)l% bromophenol blue, 1 mM EDTA, 1 M 2-mercaptoethanol and 2% SDS. Immediately after mixing, the samples were heated at 100°C for 5 min prior to application. Standard proteins of known molecular weight were used as comparisons: thyroglobulin (669,000), catalase (232,000), aldolase (158,000), bovine serum albumin (67,000), ovalburnin (43,000) and cytochrome C (12,270) were used in gel filtration and thyroglobulin (669,000), ferritin (440,000), catalase (232,000), lactate dehydrogenase (140,000) and bovine serum albumin (67,000) (high molecular weight standards) or phosphorylase B (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000) and a-lactalbumin ( 14,400) (low molecular weight standards) were used in gel electrophoresis.

Amino Acid Composition A sample of the freeze-dried hemagglutinin obtained after re-chromatography on Sephacryl S-200 was hydrolyzed under nonoxidative acid conditions as described by Moore and Stein ( 1963). The amino acid composition of the hydrolyzate was determined by ion-exchange chromatography on a Beckman I2 I MB auto amino acid analyzer. A standard amino acid mixture was used for comparison and norleucine was used as an internal standard.

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Inhibition by Sugars, Effect of pH and Heat Stability Crude seed extract (20 mL) prepared by homogenizing finely ground seed in 0.8% saline (1 g/20 mL), was made up to 140 mM with respect to different sugars shown in Table 1 and allowed to stand for 30 min at 21°C. Further 20 mL samples were adjusted to obtain a pH range of 3-10 or heated for 5 h at 40" to 90°C in 10°C steps before being assayed for hemagglutinin activity. In the latter case, assessment was every 10 min. The experiment was repeated after Sephacryl S-200 gel filtration with the same result.

Assays for Hemagglutinin, Protein and Total Nitrogen Human erythrocytes of groups A, B and 0 were first trypsinized according to the method of Marquardt and Gordon (1974) before being mixed with hemagglutinin in Microtiter wells according to the procedure described by Vretblad (1976). Activity was based on time for agglutination ( + ) > 20 min, (2 +) > 10 min or ( 3 + ) < 10 min. Protein was assessed by absorbance at 280 nm or by the method of Lowry et at. (1951). Total nitrogen was assessed by microKjeldahl as described by Dench (1982) followed by colorimetric determination of ammonia in the digest as described by Weatherburn (1967).

TABLE 1. HEMAGGLUTINATION OF TRYPSINIZED HUMAN ERYTHROCYTES, IN THE PRESENCE OF SUGARS, BY THE HEMAGGLUTININ EXTRACTED FROM JATROPHA CURCAS SEEDS

Agglutination of trypsinized Sugar Added (20 mPJ)

human erythrocytes

D-glucose

D-glucosamine N-acetyl-g-glucosamine D-galactose D-galactosamine N-acetyl-g-galactosamine L-fucose D-rnannose N-acetylneuraminic acid

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A

B

0

+ + +

+ +

+ + +

-

+ + +

+ -

+ + +

-

+ + +

L. M. C A N 0 ASSELEIH, R. A. PLUMBLEY and P. J. HYLANDS

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RESULTS AND DISCUSSION Extractability of the Hemagglutinin Extraction of the finely ground meal from decorticated seeds of J . curcas showed that hemagglutinin activity was only detectable in extracts containing 0.25 M or 0.5 M sodium chloride at a pH of 4-6 (Fig. 1). Maximum extractability was obtained with 0.5 M sodium chloride at pH 5.0. High molarity salt extracts contained inactive hemagglutinin which became active when the salt concentration was reduced by dialysis against 0.5 M sodium chloride at pH 5.0 prior to the assay. Extracts obtained under conditions above pH 6.0 showed no activity even when dialyzed. This implies that acid conditions are required for the extraction of the hemagglutinin. Active extracts remained active, however, if the pH was raised to pH 10.0. Extracts obtained using optimum conditions (0.5 M sodium choloride at pH 5.0) contained around 30% protein (Fig. 1).

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8

I a

6

10

20

30

LO

50

60

% Protein extrortobility

FIG. 1. EFFECTS OF SODlUM CHLORIDE CONCENTRATION AND pH ON THE EXTRACTABILITY OF THE HEMAGGLUTININ FROM JATROPHA CURCAS SEEDS Percentage protein is based on the value of 57% in the seed-meal; analysis was by micro-Kjeldahl. Sodium chloride concentration (-o-), pH (-m-). Hemagglutination ( + ), ( + 2) or (3 + ) according to agglutination time of >20 min >10 min or z

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0

L l L

0

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P

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0 Ln

C

E

-

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10

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Fractions ( 6 m l )

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15

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20

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FIG. 3. GEL FILTRATION ON SEPHACRYL S-200 OF A HEMAGGLUTININ SAMPLE OBTAINED FROM SEPHADEX (3-75 GEL FILTRATION Elution was at 52 mL h-' using Tris/HCI buffer, pH 7.0, containing 0.4 M NaCI. Protein was assayed by the method of Lowry et al. (1951) and hemagglutination is represented as ( + ) or (3 +) according to agglutination time of >20 min or 20 min or