Isolation of soybean agglutinin (SBA) from soy meal

gradient chromatography on Bio-Gel HTP was used to obtain the purified SBA as fraction C, and the hemagglutinating procedure was simplified. Purity of...
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Isolation of Soybean Agglutinin (SBA) from Soy Meal Prem D. Sattsangi', Saroj Sattsangi, and Herbert H. Grossman Pennsylvania State University, Fayette Campus, Uniontown, PA 15401 Modifications of previously reported procedures to purify soybean agglutinin (SBA) were used to isolate a lectin from soybean meal which showed 5000-6000 HUImg hemagglutinating activity, inhibited by very low concentrations of Nacetyl-D-galactosamine.A combination of stepwise and linear gradient chromatography on Bio-Gel HTP was used to obtain the purified SBA as fraction C, and the hemagglutinating procedure was simplified. Purity of the lectin was ascertained by polyacrylamide gel electrophoresis under standard alkaline conditions and by molecular weight determination by SDSPAGE. A decrease in the yield of crude SBA with the aging of the soybean meal was noticed. Soybean agglutinin (SBA) is a glycoprotein belonging to a group called lectin. Lectins are proteins that bind specifically with carbohydrates (1). They are mainly found in plants and agglutinate red blood cells, hence, the earlier name Phytohemagglutinin. Recently lectins have also been found in bacteria, invertebrates, and in humans (2). There are numerous excellent reviews available on lectins and their applications in biology and medicine (3-6). Lectins are used (a) to differentiate between blood group types, (b) to act as mitogenic agents in immunology, and (c) to serve as reagents to distinguish between normal and malignant cells. All these and a variety of other uses for lectins depend on their sugar specificity. The glycolipids and glycoproteins present on the cell wall have mono- and oligo-saccharide residues studding the cell surface. These sugar residues nre involwcl in intercellular communication, regnlation of cell crowth and differenti~ntion, and immune, response and malign&cy. Thus, lectins are used as reagents in these studies. There is added interest in SBA because of its possible role in the process of nitrogen fixation (7,8). We are interested in developing the use of fluorescence techniques to the study of lectin-substrate binding interactions. This necessitates a constant supply of chemically and biologically homogeneous SBA. Although commercially available from various sources, the biological properties of SBA are known to vary from batch to batch. Radical departures in the chemical composition of SBA have also been documented ( 9 ) and attributed to the different source of soybean meal used. Soybean lines showing wide variation in the content of 120,000-Dalton lectin are also shown (10). Therefore, there is considerable interest in a convenient lahoratory procedure for the isolation of SBA. Several procedures for the isolation of SBA are available in the literature. Liener (11) was the first to describe the isolation of "Soyin" or SBA in the acidified extract of the soybean meal. Subsequently, the method was modified by Lis and co-workers (12) and adopted by Nachbar and Oppenheim (13). Later Lis and Sharon (14) described a further revised procedure. The aim of this study is to describe a straightfor-

Steps Involved in the isolation of Purlfied SBA SOYBEAN MEAL suspended in water pH adjusted t o 4.6

AS-1 (acid soluble proteins) saturated to 0.4 [(NH,),SO,, 28 d l 0 0 mll

RESIDUE discarded

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AS-2 saturated from 0.4-0.8 [(NH,),SO,, 28 g1100 mll i

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SUPERNATANT discarded

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RESIDUE discarded

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RESIDUE (0.4-0.8 proteins) suspended in water dialyzed I

SUPERNATANT re-precipitated [(NH&),SO,, 56 g1100 mll

RE~DUE discarded

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SUPERNATANT

RES~DUE

discarded

suspended in phosphate buffer, dialyzed against 60% ethanol (-8 "C) dialyzed against water

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SUPERNATANT lyophilized

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RESIDUE discarded

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CRUDE SBA chromatographed on Bio-Gel HTP

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FRACTIONS A,B (Fig. 1 ) discarded

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FRACTION C dialyzed, lyophilized

I PURIFIED SBA

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ward and relatively inexpensive method for routine isolation of purified SRA, suitable to be used as a starting material i n most studies.

Experimental A schematic representation of the isolation procedure is shown in the table. All isolation steps were carried out a t 5 OC unless otherwise stated. A Sorvall RC2-B refrigerating centrifuge and a New Brunswick lyophilizer were used to centrifuge and lyophilize. A Bio-Rad system was employed for gel electrophoresis. Hydraxylapatite, Bio-Gel HTP, was used in column chromatography. Soybean meal, 1-200, was a gift from the A. E. Staley Manufacturing Co., Decatur, Illinois. The N acetyl-D-galactosamine was purchased from the Sigma Chemical Company, and the rabbit blood in Alsever'ssolutiun, from the Carolina Biological Supply Company.

Isolation of Crude SBA Soybean meal (250 g) was added Dadually to 3 L of water over a period of 1br and under vigorous magnetic stirring. The pH of the suspension was adjusted from near 6.6 to 4.6 on a pH meter, using about 10 ml of concentrated hvdrochloric acid. The color of the suspension turned markedly light during acidification herause of the preripitat~onof the a r ~ dinsoluble proteins. The mixture was stirred tor anvther hour at room tempernlure and kept overnight in a a d d room. The clear yellow supernatant was decanted; the fine suspension was centrifuged (10 min, 6000 rpm), and the coarse suspension was filtered under suction. Supernatants and filtrates were combined to vield 2500-2600 ml solution of acid soluble nroteins AS-1. and the iesiduss were discarded. AS-1 was set to sti;magnetieallyin an ice bath, and 700-750 g of ammonium sulfate (28 g1100 ml) was added gradually to bring it to0.4saturation. Stirring was continued for another 2 hr in the cold rwm, and the suspension wasallowed tosettle overnight. Proteins precipitated at 0.4 saturation were removed by centrifugation (10 min, 6000 rpm), and the supernatant AS-2 collected. An equal amount of ammonium sulfate was added to the AS-2 in the manner descrihed above to bring it to 0.8 saturation. After allowing it to settle overnight in the cold room, the suspension was centrifuged (15 min, 10,000 rpm) to collect 0.4-0.8 fraction of soy proteins. The supernatant was discarded. The residue was suspended in 75 mlof water and dialyzedfreeof sulfate ions by 3 changes of water in the cold room. Much yellow coloring matter is lost during this proceA9. A mall amount of r e d u e that had prec~pitawdwas removed by rrntr~fugation115mln. I0,Utiil rpmi. I'nnteins were re-precipitated from the summatant bv 0.8saturatiun 10.;161! m l ~with ammonium sulfate. ~ u h n the g re-precipitation the supernatant that was translucent initially became a clear solution with the addition of first few crystals. This is perhaps the result of increased solubility of soy proteins in an ionic medium. However, further precipitation took place smoothly as more ammonium sulfate was added. The suspension was allowed to stir for another hour in the cold rwm. The precipitate was collected hy centrifugation (10 min, 6000rpm), dissolved in 25ml of 0.05 M phosphate buffer pH 6.1, and dialyzed against 60% ethanol in the freezer comoartment of a refrieerator (-8 "C) for 48 hr when all the supernatant dialyzed out, lea"& onliaresidue. The dialysis hag was transferred to a 2-L beaker containing water and dialyzed for another 24 hr with 2 changes of water, or until the dialyzate became free of phosphate ions. The insoluble residue precipitated was removed by centrifugation (20 min, 15,000 rpm), and the supernatant was freeze dried to obtain 0.4-0.8 g of crude SBA. Column Chromatography of Crude SBA Bio-Gel H T P (35 g) was packed in 0.005 M phosphate huffer pH 6.8, according to manufacturer's instructions, to obtain a column (2.5 X 17 cm) with 40-60 m l h r flow rate, depending upon the level of eluting buffers. Crude SBA (1 g) was dissolved in 25-30 ml of the starting buffer, filtered to remove insoluble matter, and applied to the column. The column was eluted first with 400 ml of the starting huffer, followed by 1WO ml of 0.072 M phosphate buffer pH 6.8. Final elution of the purified SBA was achieved using a linear 1000 ml gradient from 0.072 M to 0.250 M phosphate buffer pH 6.8. Entire chromatography was conducted in the cold room. Fractions were collected at 9 min-intervals and monitored for UV absorbance at 280 nm to obtain three distinct fractions, A, B, and C (Fig. 1). Only fraction C exhibited hemagglutinating propertiesand was pooled, dialyzed free of phosphate ions, and lyophilized to yield 300-350 mg of the purified SBA. 978

Journal of Chemical Education

Polyacrylamide Gel Electrophoresis Crude SBA and purified SBA were examined on 7.5% polyacrylamide gel under standard alkaline conditions using Tris-glycine buffer pH 8.6, according to the method of Davis (15). Samples (1 mglml) were prepared in the starting huffer; 125fil of this was mixed with 25 of the bromophenol blue solution in 40% sucrose. Sample volumes of 30-60 fil, containing approximately 2 5 4 0 fig of protein, were applied on to the stacking gel. Samples were stacked a t 50 V, and separated at 3 mamp per tuhe. After electrophoresis, gels were suspended in 12.5% trichloroacetic acid (TCA) for 30 min, removed, and immersed in freshly prepared staining solution (1:20 dilution of a 1% aqueous stock solution of Coomassie brilliant blue by 12.5% TCA). After 1hr of staining, gels were washed once with 10% TCA and stored in it for photography after 24 hr (16). SDS-PAGE This was conducted according to Laemmli's system (17), with 7.5% separating gel and 3%stacking gel. Aqueous samples (5 mglml) were diluted 1:5 with sample buffer and heated in a boilingwater bath for 2 min. An aliquot of this sample, having 50 fig of protein, was stacked at 50 V and separated at 3 mamp per tuhe. After electrophoresis gels were stained with Coomassie brilliant blue, according to Weber and Osborn (18). HemagglutinationAssays Standard ervthrocvte susoension was nreoared accordine to the is-and ~ i a r o nl.j 4 ,L The matkrial to he tested was diamethod of -~ -~ rdved in saline. Serial 2.fdd ddutions of the start in^ solution were made in a final vulume of 2 ml in Speitronic-20cuvrts ( I 2.5 mm). To each cuvet 2 ml of the standard erythrocyte suspension was added, mixed by inversion, and left in a rack in vertical position at room temperature. In each set 4 contml tubes, containing 2 ml of saline and 2 ml of the standard ervtbrocvk susnension. were included. After 2.5 hr absorbance was readat 620nm. T; main& consistent results, care was taken to transfer the tubes into Soectronie-20 with minimum di.;turhanre and tu read the ahrorbance without allowing the sample to rrdistribute.'Shr ipec~fich~magglutinatingactivity o i the material rested. I I U mg, was calculatrd nccordina to the formula descrihed by Lis and Sharon (14) ~

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h'apten Inhibition Assays This was performed along similar lines as the hemagglutination assay descrihed above. Aqueous solutions of SBA (1 mglml) and were prepared. To N-acetyl-D-galactmamine or NAG (1mM-lo*) a mixture of 100 ul of SBA and 100 ul of NAG solutions. 3.80 ml of watcr war addrd'tu prepare 4.00 miof the starting sulu~ion.Sprial 2-fold dilutions of the *tarling solution wpre made and tested for inhibit~onof hemagglutinatina activitv. Protein assays Protein content of various samples was determined according to Lowry's method (19).

VOLUME llilsrl

Figure 1. Elution profileof crude SEA on a Bio-Oei HTP column (2.5 X 17 cm). Crude SEA was dissolved in 0.005 Mphosphate buffer (1 9/30 mi) pH 6.8 and applied to me column. Elution was achieved with varying concentrations of DhoSDhate buffers DH 6.8 in steDwlsel400 mlof 0.005 M. followedbv 1OOOml bf 0.072 Ml and linear gradient (from 01072 Mto 0.250 Ml modes (-) at 5 OC. Fractions were monitored for absorbance st 280 nm (-0-0.).

Soybean meal was suspended in water, the suspension was acidified. and the extract was seoarated to obtain the mixture of acid sbluhle proteins AS-1 shown in the tahle. An agdutination exoeriment Derformed on s a m ~ l e sof oroteins. obtained by iractional brecipitation of AS-1b; 0.0-0.4; 0.4-0.6. 0.6-0.8.0.%1.0 saturation with ammonium sulfate. indicated that lectins were present in fractions 0.4-0.6 and 0.60.8. Proteins r~reripitatinrat 0.4 saturation of AS-1 were removed to obtain acid solurble proteins AS-2 from which crude SBA was precipitated bv 0.8 saturation, dialvzed aminst water and re-precipitated b; 0.8 saturation under n&tral conditiuns. There was much yellow coloring matter present in AS-1 and AS-2. most of which was lost during ~ - -the ~ - first ~~-~~ ~ - dialysis of the crude'^^^. To get rid of final traces of the yellow colorine matter and other alcohol soluble imourities. the re~~~. precipcated protein was dissolved in phosphate buffer and dialvzed against 60% ethanol when all the sunernatant dialyzed ouCThe dialysis bag was transferred h e c t l y to an aaueous medium for further dialvsis. followed hv filtration and lyophilization to yield the c k d k SBA. ~ o l f o w i nthis ~ ~rocedure.the initial vield of the crude SBA was found to be twice as much as thaireported by earlier workers. However, as the supply of soybean meal aged, the yield of the crude SBA decreased sharply. After 2 years the yield had decreased to 50%of the original. This observation has not been investigated further; however, the use of fresh soybean meal is strongly advocated. Chromatography of crude SBA on an affmity column would yield a chemically nonhomogeneous product, consisting of a mixture of different molecuiar weight species, and is, therefore, not suitable. Column chromatography on hydroxylapatite has been most commonly applied for this purpose. Varying concentrations of phosphate huffer pH 6.8 have been employed as the eluting solvent in a stepwise or gradient elution mode or a combination. The choice of an eluting solvent devends on the orooerties of the hvdroxvlaoatite used. which. in turn, is a finetion of its method of pieparation (20). ~ h u s ; to have consistent results the decision was made to use standard Bio-Gel HTP. Columns were prepared in 0.005 M phosphate buffer, and the sample was applied in and eluted with the starting buffer to elute fraction A (Fig. 1).Use of a 2000-ml linear madient from 0.005 M to 0.250 M ~ h o s ~ h a t e huffer, however, did not result in proper separatibn oifractions B and C. The most suitable method for the elution of soy proteins at 5 O C on the Bio-Gel H T P appears to he a combination of stepwise and linear gradient modes (Fig. 1). Fraction C was pooled, dialyzed, and lyophilized to obtain the purified SRA. Pure SRA gave a single hand on 7.8% polyacrylamide gel (Fig. 2) and on SDS-PACE. As expected the mol~culrvweiaht of the monomer corresponded ti30,000-~altons. The hemagglutination procedure was adopted to use Spectronic-20 without any modification, and the SBA was found to have 5000-6000 HUImg hemagglutinating activity, which was inhibited by low concentrations of N-acetyl-D-galactosamine. The product ohtained by this procedure was adequate for most a~olications,es~eciallvas a starting material for fluore~centiabelin~ e&eriments"where it is purified subsequently on a Bio-Gel DEAE column. If needed, pure SBA, free of accompanying isolectins, could be obtained by chromatography on a DEAE cellulose column (14). This research is now used as one of the projects available to CHEM 389 students a t this campus to provide advanced

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Figure 2. Polyawlamide gels of cNde SEA and purifiedSEA, cantaining 50 pg of proteins under standard Trisglycine alkaline conditions ( 151, stained with Coomassie brilliant blue (16).

lab training at the two year college level. The fadty-student interaction is beautifully symbiotic and provides the student with an opportunity to he part of an active research team. Acknowledgment

This research was supported by grants from Fayette Campus Advisory Board and PSU Faculty Scholarship Support Fund. Authors acknowledge further fmancial support and encouragements from Mr. Hugh Barclay, Director, and Dr. Stephen M. Priselac, Associate Director a t this institution. We thank Professors Joseph A. Dixon, and Eugene S. Lindstrom a t the PSU, University Park, PA, for their continued support and encouragement and. also our students Edward G. Zapach and Eric Sheetz for their participation in this research a t various stages. We also recognize tremendous support from the campus library and business office. Literature Clted (1) Goldstein, 1.J., Hughca,R. C., Momigny,M..Oaawa,T..andSharon,N,Noture,Z85 INn Frifini CC IIPm> ",

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Sharon. N %.ml.l.r Amrrirun 236 $1"61, 108 19-71, !?I d.dd.kln.1 J . a n d linyr~.(:.E .Aa C o r b Chrm Rmmem .36.127t117R ( 4 1 1.rncr.l E..Ann hsc Punr Pn)..oI .21,241 197. t i , Ls. H .andih.ron. Z ."rhhA"tfze"s,"%b M E l ror,.Acndrmtr i',ru,New Y Y Y ~ , 1%)

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is, H.;~ h & n , ~ . , & d ~ a t c h a l s k i ,J~ . id. , cham.,zr~,68a (1966). (131 Naehbar, M. S., and Oppmheim, J. D., Biochim. Biophyr. Aefo.. 320.494 (1973). 114) Lis, H.,snd Sharon. N.,"Methalr inEnzymolom,*Ginaburg,V.. (Editor), Academic Press, New York. 1912,VolXXVIII. p 3EQ.

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(171 Laemmli. U.K.,Noture, 227,680 11970). (16) Weber, K..and Oahorn, M., J B i d . Chem.. 244.4406 11969). (191 Lawry, 0. H., Rasebmugh. N. J.,Ferr,A.L.,and Randall, R. J.. J B i d . Chsm., 193. 265 (19511. (20) Bernardi. G., "Methods in hmnhmnloki,"Aeadnnie Press,Nev Vork, 1971,Vol XXII, p 325.

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