BIOCHIMIE, 1976, 58, 1247-I253.

An improved method for purification of wheat germ agglutinin (lectin) by affinity chromatography.

Philippe BOUCHARD, Yvette Monovx (*), Ren6 TIxiEn (*), Jean-Paul PRIVAT and Michel MONSIGNY ~.

Centre de Biophysique Moldculaire, C.N.R.S., £5045 Orleans Cedex (France).

(28-7-1976).

Summary. - - A simple purification of wheat germ agglutinin fronl colnmereial wheat germ is described. From defatted ground wheat germ, the lectin was extracted and then purified in a single step by filtration on an ion exchange chromatography column and adsorption on an insolubilized N-aeetyl glucosamine derivative. The amount of lectin obtained from 1,000 g of ~vheat germ 'was larger than 500 rag. Although the yield was at least twice higher than that obtained with other methods, no impurities could be detected, and molecular charaeteristies are in good agreement with the protein purified by more sophisticated procedures.

INTRODUCTION.

Wheat germ agglut inin is a p lan t lectin which is capable of agglut inat ing erythrocytes and some other types of cells [1-31 [for reviews see ref. 4-7]. This lect in has been wide ly used in studies of cell surface s t ructure due to its proper ty of speci- fic b ind ing to complex carbohydra te con ta in ing N-acetyl-glucosamine [2, 3, 81. Several methods have been proposed to pur i fy this lectin, s tar t ing from wheat germ lipase [3, 8, 1043] from wheat germ acid phosphatase [11] or from raw wheat germ [9, 13-22]. Methods involved l ip id extract ions !9, 13-16 : 19-22], prec ip i ta t ion by ammon ium sul- fate [9, 11-2'0, 23], by low ionic s trength [13-21! or by heat dena tura t ion [10-12, 15, 17, 19-21], several centr i fugat ion steps [2, 8-12, 14-221 several dialysis steps [2, 8-12, 14-21~ ion exchange [8, 9, 11, 12, 15- 20], gel fi l tration I l l , 15, 17, 18], affinity chromato- graphy [2, 10, 11, 13-15, 17-22j and several cristalli- zation steps [12, 16].

Various supports for the affinity chromatogra- phy have been used : a) Ovomucoid on Kiesel- guhr [2], or on Sepharose [10, 11, 15, 17, 19, 201 ; b) derivatives of N-acetyl-glucosamine : O-glyco- sides [13, 22!, N-glycoside [14, 21j, S-glycosides [231 c) ch i t in [181. The bound proteins on the affi- ni ty matr ix have been eluted by free N-acetyl-glu- cosamine (5 mg/ml) [13, 21] by GlcNAc oligomers [18], by acetic acid 0,1 M [2, 10, 11, 14, 15, 17, 19, 20J or by HC1 0,02 N [221 or 0.05 [SJ.

U) Industrie Biologique Franqaise, 35, quai du Moulin de Cage 9,2230, Gennevilliers (France).

<> To whom all correspondence should be addressed.

Some procedures did not give an homogeneous prote in [2, 8, 107. In some cases, the prote in con- ta ined sugar [11, 12] ; in other cases, the prote in did not conta in any t ryp tophan [ l l l . Some proce- dures allow the isolation of chromatographica l ly dis t inct forms : three [9_~ or four [201 isolectins. More recent works showed that the lect in is a dimer at neutral pH [16, 19, 201 with a molecular weight of 36 000 daltons.

In this paper, a simplified puri f icat ion of wheat germ agglut inin is descr ibed us ing a S-benzyl-N- acetyl-glucosaminide derivat ive [231 as an affinity ligand. This procedure requires a single centr ifu- gation step, a single dialysis step and does not require heat denatura t ion of impuri t ies , ammo- n ium sulfate precipi ta t ion, low ionic s trength pre- c ipi tat ion or gel f i l t ra t ion chromatography.

MATERIAL AND METHODS.

Raw wheat germ was purchased from Grands Moulins de Paris or from Sigma Chemical Co. DEAE-Sephadex and Sephadex G 75 were from Pharmacia , Uppsala, Sweden ; Ultrogel A4 and Ultrogel ACA 44 from L.K.B., Br6ma, Sweden ; p -n i t rophenyl glycosides from Koch-Light.

Affinity ligand. The p-nitrobenzyl-l-thio-2-acetamido-2-deoxy-~3-

D-glucopyranoside (5.00 mg) prepared as pre- viously described [23] was dissolved in methanol- water (9 : 1 ; v /v) (100 ml) in the presence of HC1 (12 N, 0.1 ml) and reduced by hydrogen (1,1 atm, 6 h) with pa l lad ium-charcoa l (0,5 g) as catalyst.

1248 P. Bouchard and coll.

The suspension was filtered on a celite pad wh ich was washed wi th methanol . The solution was concent ra ted by evaporat ion unde r reduced pressure. The syrup was diluted wi th water (5 ml). This yellow solution was applied onto a Sephadex G-10 column (50 em × 2 cm) and eluted wi th water. The fract ions con ta in ing the p-amino ben- zyl- l - thio-2-acetamido-2-deoxy~- D -glueopyranosi- de were pooled and freeze-dried (250 rag).

Binding of the affinity ligand to the matrix.

Ultrogel A4 (50 ml) activated wi th cyanogen bromide [.24] was coupled with hexamethylene d iamine and then wi th succin ic anhydr ide . The free carboxyl groups, activated wi th CMCI [25] [N-cyclohexyl-N'- [ 2-(47~-morpholinyl)-ethyl I car- bodi imide-methyl-p- toluene sulfonate] , (Fluka) were allowed to react with p-amino-benzyl- l - th io- N-aeetyl-~-D-glucosaminide (170 my, 0.5 mmole). In order to lower th.e ion exchange effects, unreac- ted carboxyl groups in the extension arms ~ e r e blocked with 1.5 mmole of g lycinamide and 3.2 mmole of CMCI. The subst i tuted gel was washed wi th a 0.0~5 M Tris-HC1, 0.15 M NaCI buffer (pH 8.5) and poured into a column. After hydro- lysis (HC1 4 N, 4 h at 100°C), it was found that one ml of gel conta ined 2 ~tmoles of glucosamine. Cor- rect ions for dest ruct ion dur ing hydrolys is and for incomplete hydrolys is were made from the result of a parallel exper iment : hydrolys is of 2 ~moles of p-ni trobenzyNl-thio-2-acetamido-2-deozy -i~ - D- g lueopyranoside in presence of one ml of gel. One ml of gel was able to b ind more than 30 mg of wheat germ agglut inin .

Electrophoresis.

Analyt ical po lyacry lamide gel electrophoresis was per formed according to Davis [26], us ing a 7.5 per cent separat ing gel, at pH 9.4 (0.05 M Tris- HCI), at pH 5.0 (0.06 M Tris-acetate) and at pH 3.8 (0.2 M Tris-acetate). Gels were s tained wi th Coomassie blue G 250 or with Coomassie blue G 250 [27]. Sodium dodecylsnl fa te-polyacrylamide gel electrophoresis was carr ied out accord ing to the method of Shapiro et al [28]. Gels ~ 'ere polymer ised in glass tubes (0..6 X 10 cm) from a sodium phosphate 0.03 M buffer, pH 7.1 con ta in ing 12.5 per cent acrylamide, 0.3 per cent N-N'-me- thylene bis acrylamide, 0.13 per cent sodium do- decyl-sulfate and 0.03 per cent mercapto-ethanol . Molecular weight de te rmina t ion by dodecyl-sulfate po lyacry lamide gel electrophoresis was carr ied out according to Weber and Osborn [29]. Isoelec- t rofocalisat ion was per formed in po lyacry lamide gel according to Wrigley [30]. Gels were s ta ined wi th Coomassie blue G 2,50 (Sigma) according to Malik and Berrie [31].

hnmunoelectrophoresis.

Antiserum against pure wheat germ agglut inin was prepared in rabbits us ing 0.5 mg leet in sus- pended in complete F reund ' s adjuvant (1 ml). Booster doses were given after 1 and 2 months and the rabbits were bled 7 days after the last injec- tion. Immunoglobu l ins 'were par t ia l ly purif ied by (NH~)~SO 4 prec ip i ta t ion accord ing to Stelos [32]. Immuno-elec t rophores is was per formed in a 1 per cent gel plate of Agar Noble (Difeo) in a veronal buffer, pH 8.6 (I : 0.05).

Sedimentation studies.

Velocity sedimenta t ion measurements were per- formed at a speed of 60.000 rev /min . , on a Spinco- Beckman Model E u l t racent r i fuge equipped with a Schlieren optical system (concent ra t ion range higher than 1.5 mg/ml) or wi th an ul t raviolet op- t ical system at 280 nm (concentra t ion range 0.4-

1.5 mg/ml) . The S~o ' w was obtained by extrapola- t ion to zero concent ra t ion .

Amino acid and sugar analysis.

Samples of pro te in were hydrolyzed wi th redis- t i l led HC1 5.6 N in sealed tubes in oacuo, at 105°C for 24, 48 and 72 h. The amino acid content of the hydrolysate was de termined on a Beckman 12,0 C amino acid analyser according to the method of Spackman et aI. [33]. Quanti tat ive values for each amino acid were calculated from the average va- lues of three ident ica l t ime hydrolysates. Valine and isoleucine values were calculated from 72 h hydrolysates. The values of serine, th reon ine con- tent were corrected for dest ruct ion dur ing hydro- lysis. T ryp tophan was determined by 2-hydroxy-5 n i t robenzyl b romide react ion according to Bar- man and Koshland [34], and on the amino acid analyser from a sample hydro lysed wi th p-toluene sulfonic acid [35]. Amino sugars analysis was per- formed color imetr ical ly after hydrolysis (4 N HC1, 4 h at 100°C) by the Elson and Morgan procedure [36]. The phenol su lphur ic acid react ion [37] and the orcinol su lphur ic react ion [3.8] were used to determine the neutra l sugar content .

Agglutination assag.

A two-fold serial di lut ion of the pro te in was made in 5,0 vl 0.15 M NaG1 aqueous solution, 50 ~l of a 3 per cent suspens ion of huma n red blood cells (A group), washed 3 times wi th saline, were added, us ing a microt i t ra tor plate (system Cooke, M 220 4 A). After 1 h at room temperature, the degree of agglut inat ion was assessed on a serolo- gical scale (0 to 4) [39]. One uni t of agglut inat ing activity was defined as the amount of pro te in re- quired to cause half maximal agglutinat ion.

BIOCHIMIE, 1976, 58, n ° 10.

Purification of wheat germ lectin. 1249

Glgcosylhgdrolase activi t ies.

T h e g l y c o s y l h y d r o l a s e a c t i v i t i e s w e r e m o n i t o - r e d u s i n g p - n i t r o p h e n y l g l y c o s i d e s a c c o r d i n g to C o n c h i e [40]. T h e f o l l o w i n g g l y c o s i d e s w e r e u s e d : p - n i t r o p h e n y l d e r i v a t i v e s of ~-GlcNac , a-Man, aGlc , (3-Glc, f3-Gal, a-Gal, a -Fuc , ~-Fuc.

R E S U L T S .

Puri f icat ion of wheat germ agglutinin.

T h e p u r i f i c a t i o n s c h e m e is s h o w n in t a b l e I. W h e a t g e r m (1 kg) w a s d e f a t t e d w i t h m e t h y l e n e c h l o r i d e (1 1) a n d d r i e d i n a n a i r s t r e a m . T h e p r o - d u c t w a s g r o u n d i n a T u r m i x b l i n d e r , a n d f lour w a s c o l l e c t e d f r o m a s i eve D I N 4188. T h e f l ou r (860 g) w a s s t i r r e d w i t h 6 1 of 0.05 N HC1 a n d

o io

U cI -4" ,~o

Z --.+ t :1= CL O_

I I 1 /\.

\ . /-, ...... / ' , ~o ioo

F- ( D

-2000

-1000

Fro. 1. - - Aff ini ty chromatography of ehmte from Amberlite IRA /tOO, on Sepharose substituted by p-aminobenzyl-l-thio-2-acetamido-l,2-dideoxy-:~-D-glu- copyranoside column (7.5 cm X 3 c m ; flow r a t e : 300 m l / h r ) . The column was washed wi th a) 1 M NaC1, 0.05 M Tris-HC1 buffer pH 8.6 ; b) wi th 0.05 M sodium acetate pH 4.4. The lect in was eluted by c) 0.0'5 M HC1. Fract ions of 12 ml were collected. (-- ) Absorpt ion at 280 n m ; ( . . . . . . ) agglu t ina t ing act ivi ty ; f rac t ions wi th agglu t ina t ing act ivi ty were pooled.

Na2S204 (0.6 g) at 4°C o v e r n i g h t , c e n t r i f u g e d at 6.000 g fo r 20 nan.

T h e p H of s u p e r n a t a n t (5,8 1) w a s a d j u s t e d to 8.6 w i t h s o l i d T r i s a n d a p p l i e d to a c o l u n m (50 × 6 e n d of A m b e r l i t e IRA 400 (100-200 m e s h ) equ i l i - b r a t e d w i t h 0.05 M Tris-HC1 b u f f e r (pH 8.6). T h e e f f luen t w a s a p p l i e d c o n t i n u o u s l y to a c o h m m (3 × 5.0 cm) of 1 - t h i o - X - a c e t y l g l u e o s a m i n i d e - U l t r o g e l A4 e q u i l i b r a t e d at p H 8.6. B o t h c o l u m n s w e r e w a s h e d w i t h t h e s a m e b u f f e r u n t i l t h e a b s o r - b a n c e at 280 n m of t h e e f f l u e n t w a s l o w e r t h a n 0.05. The affinity colmnn was then washed sepa- rately wi th 1 M NaG1 (200 ml) wi th 0.05 M sodium acetate pH 4.4 (30.0 ml) (fig. 1). Final ly , wheat germ agglut inin was eluted with 0.05 N HC1 (90 nil). The pH of the last solution was adjusted to 7 wi th 0.5 M NaOH and the neutral solut ion was dialysed thoroughly against disti l led water and freeze-dried.

Solubil i ty .

T h e f r e e z e - d r i e d w h e a t g e r m a g g l u t i n i n w a s ea s i ly s o l u b l e in d i s t i l l e d w a t e r or i n n e u t r a l bu f - fers , u p to 20 m g / m l . I ts s o l u b i l i t y in 0.05 N HCI w a s e v e n h i g h e r .

Absorpt ion spec trum.

T h e l e c t i n i n n e u t r a l b u f f e r s s h o w e d a n a b s o r p - t i o n m a x i m m n at 278 n m a n d a m i n i m u m at 252 n m . I t s s p e c i f i c a b s o r b a n c e at 278 n m w a s 1.50 T- 0.05 p e r m g of p r o t e i n p e r ml (1 c m l i g h t p a t h , 0.15 M NaC1, Tr is -HC1 0.05 M, p H 7.5). T h e r a t i o of the a b s o r p t i o n at 278 n m to t h a t at 252 n in

w a s 1.95.

Emiss ion spec t rum. In a q u e o u s m e d i u m , f l u o r e s c e n c e w a s m a x i m u n l

at 348 m n ( e x c i t a t i o n w a v e l e n g t h : 265, 275 o r

TABLE I.

Puri f icat ion of w h e a t germ agglutinin.

Procedure

Crude extract . . . . . . . . . . . ~ i

Affinity column i - washes . . . . . . . . . . . . . . - NaCl 1 M eluate . . . . - acetate eluate . . . . . . . - HC1 eluate . . . . . . . . . . !

Volume (ml)

5 800

6 800 200 300

90

Total protein content (mg)

6 6 5OO

4 6 000 260 730 530 (*)

Agglutination i Specific titer (u/ml)(*')lactivity (u/mg)

8 0 . 7

2 1

650 110

The procedure s ta r ted w i th 1 kg of whea t germ. The prote in content was &278 n m - - 1 5 . es t imated pho tomet r ica l ly assuming a specific absorbance *'1 era, 1% --

(*) Weight a f te r freezedrying. (**) Human red blood cells A +.

BIOCHIMIE, 1976, 58, n ° 10.

1250 P. B o u c h a r d a n d col l .

FIG. 2. - - Polyacrylamide gel electrophoresis of wheat germ agglutinin.

a) in alkaline medium 0.05 M Tris-HC1 buffer pH 9.4. b) in acidic medium, 0.2 M Tris-acetate buffer pH 3~.8. c) with sodium dodecylsulfate 0.1 per cent.

295 nm). The f luorescence m a x i m u m was shif ted towards shor te r wavelengths ()~,,~x : 338 nm) in glycerol wa te r (95/5).

S e d i m e n t a t i o n analysis .

When the agglut inin was dissolved in 0.15 M NaC1 0.0.5 M Tris-HC1, pH 7.5, the agglut inin ap- peared as a single symmet r ica l peak in the ultra-

centrifuge. A value of s20 . ° w of 3.5 was obtained. However , in 0.05 N HC;1 solution, the sedimenta- t ion coefficient was 2.1. These values are in good agreement wi th those f rom other authors [16, 20].

Gel f i l t rat ion.

By ch roma tog raphy on Ulirogel ACA 44 or on Sephadex G 75 column, a single symmet r ica l peak was obtained and the specific agglut inat ing acti- vi ty was constant in each part of the peak. The elut ion of the lect in was sl ightly delayed on these columns, s ince it came off as a pro te in wi th a mo- lecular weight of 10.000. This abnormal feature has been observed by Allen et al. [9] w i th wheat germ aggIutinin and by I sh iyama and Uhlenbruck [41] wi th Hel i x poma t ia agglutinin.

P o l y a c r y l a m i d e gel e lec trophores is .

In basic med ium (pH 9.4) and in sl ightly a c i d i c med ium (pH 5.0) a unique band was obtained in po lyac ry lamide gel e lectrophoresis . However in ac id ic medium, three bands were detected corres- ponding to the three isolect ins (fig. 2). When poly- ac ry lamide gel e lec t rophores is was run in the pre- sence of sodium dodecylsulfate , a un ique band was detected. Fur thermore , by po lyac ry l amide gel

Fro. 3. --- lmmunoelectro- phoresis of iwheat germ agglu- tinilt.

A. crude extract ; upper trough : rabbit antiserum against crude extract of 'wheat germ ; lo,wer trough : rabbit antiserum against pure wheat germ agglutinin.

B. pure ,wheal germ agglu- tinin ; upper trough : rabbit serum; lo,wer trough rabbit antiserum against pure wheat germ agglutinin.

BIOCHIMIE, 1976, 58, n ° 10.

Puri f icat ion of wheat germ lectin. 1251

isoelectrofocal isa t ion, only one band was detected co r respond ing to a pH i of 8.5.

Immunoelectrophoresis.

When the lect in was a l lowed to diffuse against non- immunized rabbit serum, a diffuse prec ip i ta- t ion band was found. However , using the prote ins obtained by 45 per cent anmmnium sulfate (innnu- noglobul in fract ion) f rom non immunised rabbi t serum, no band could be seen. Using inmmnised rabbi t inmmnoglobul in fract ion, a single prec ip i - tation band was shown (fig. 3).

Amino acid composition.

Amino acid composi t ion of wheat germ agglu- t in in is given in table II. The data are presented in terms of molar ratios on the basis of 10 moles of alanine per mole of the lectin monomer (18.000). The total number of amino acid residues in one molecule of m o n o m e r was calcula ted to be 173 T- 10. The amino acid composi t ion is in good agreement wi th that repor ted by other authors [9, 11, 13, 16, 20]. The content of glycine and cys- t ine is very high, 24 per cent and 17 per cent rest-

TABLE 1I.

Amino acids composition of wheat germ aggluti- nin. Data are expressed as amino acid residues per protein of molecular weight 18 000.

Aspartic acid 15 Methionine 3 Threonine 5 Isolcucinc 1.5 Serine 12 Leucinc 5 Glutamie acid lfi Tyrosine 7 Proline 5 Phenylalanine 3

Glycine 41 Tryptophan 3 Alanine 10 Lysinc 8 Half cystine 30 Histidinc 2 VMinc 1.5 Arginine 5

dues respect ively . The content in h y d r o p h o b i c re- sidues (proline, valine, methionine , isoleucine, leu- cine, phenyla lan ine) is very low, hav ing only 10 residues per 100 on a molecu la r basis. Three residues of t ryp tophan per m o n o m e r were found by three different methods, in agreement wi th [16, 17] whi le LeVine et al [11] did not find any. No addi t ional n i n h y d r i n posi t ive peak was eluted after pheny la lan ine peak on the long or the short column in contrast w i th the findings of Nagata and Burger [16] using a three t imes crystal l ized lectin.

Carbohydrate content.

No detectable neutral or amino sugars were pre- sent in the purif ied lectin, using co lor imet r ic me-

BIOCHIMIE, 19,76, 58, n? 10.

thods. No t race of glucosamine or galactosamine was found by amino acid analysis.

Glycosylhydrolase activities.

Glycosylhydrolase act ivi t ies of the crude extract and of the purif ied lect in are shown in table III. No act ivi ty was re ta ined in the pur i f ied lecl in.

TABLE III.

Enzymatic activities (nmol × rain-2 × rag-1 pro- tein) assuming a protein absorbance of A ~SOcm, .... 1~,~ =15.

p-nitrophenyl glycosides

~-GIc NAc . . . . . . . . . . . . . . . ~-Gal . . . . . . . . . . . . . . . . . . . a-Gal . . . . . . . . . . . . . . . . . . ~.-Gtc . . . . . . . . . . . . . . . . . . . z-Man . . . . . . . . . . . . . . . . . . ~-Fuc . . . . . . . . . . . . . . . . . . .

Wheat germ extract

0.24 O. 94 0.32 O. 23 0.36 0.35

Pure WGA

Agglutination activity. Wheat germ agglutinin concent ra t ion requ i red

to agg,utinate unt rea ted e ry throcytes (human, rab- bit, horse, goat) was about 10 ~tg/ml. Normal hu- man and lno~¢se lymphocytes , routine plasmocyto- ma cells MOPC 173 were also agglutinated wi th low lect in concentra t ion. The agglut inat ing acti- vity did not depend upon the presence of d ivalent cation. Agglut inat ion was inh ib i ted by N-acetyl g lucosamine and chi t in ol igomers but not by N-acetyl neuranf in ic acid at a concent ra t ion up to 200 raM.

DISCUSSION.

Lipids were first r emoved with methylene chlo- r ide wh ich is non inf lammable and wh ich is at least as effective as l ight pe t ro leum ether. When non-defat ted wheat germ was used, the amount of sieved flour was low, the p ro te in ext rac t ion was less effective, and the colunm flow rate was slow. Ext rac t ions solutions conta ined I mM sodium di- th ioni te to p reven t oxida t ion react ions ; so, the extract did not turn to dark brown, and the puri- fied lect in was uncolored. Heat denatura t ion of con tamina t ing prote ins [10-1K 15-17, 19-21] w h i c h is tedious and t ime consuming, ammonium sulfate prec ip i ta t ions [9, 11-20, 22] or p rec ip i ta t ions of ma- ter ia l in low ionic s t rength [13-21] were avoided. Amberl i te IRA 400 ,~vas used instead of DEAE cel- lulose or DEAE Sephadex because the flow rate w i th this synthet ic ion exchange resin was much higher. At pH 8.6, the coIored compounds were

86

1252 P . B o u c h a r d a n d co l l .

a d s o r b e d on t he r e s i n , a n d no a g g l u t i n a t i n g ac t i - v i t y r e m a i n e d on t h e c o l u m n . I m p u r i t i e s a d s o r b e d o n t h e a f f in i ty c o l u m n w e r e p a r t i a l l y r e m o v e d b y a h i g h i o n i c s t r e n g t h b u f f e r w i t h o u t a n y loss of a g g l u t i n a t i n g a c t i v i t y . S o m e m o r e i m p u r i t i e s a n d a s m a l l a m o u n t of l e c t i n (0 to 5 p e r c e n t ) w e r e e l u t e d w i t h a n a c i d i c b u f f e r (pH 4.4). At las t , t h e l e c t i n w a s e l u t e d b y 0.05 M HCI, i n o r d e r to get a s h a r p e l u t i o n (500 m g in less t h a n 100 ml ) ; a c e t i c a c i d gave a s l o w e l u t i o n , e v e n u s i n g c o n c e n t r a - t i o n s as h i g h as 1 M.

U s i n g t h e a b o v e m e t h o d , o n e o b t a i n e d m o r e t h a n 500 m g of l e c t i n f r o m 1 kg of w h e a t g e r m . T h e ' w h o l e p r o c e s s c a n b e a c h e a v e d in less t h a n o n e w e e k w h e n t h e a f f i n i t y m a t r i x is a v a i l a b l e . T h e a f f i n i t y c o l u m n is v e r y s t ab l e , a n d t h e s a m e c o l u m n h a s b e e n u s e d m o r e t h a n 20 t i m e s w i t h o u t a n y loss of a c t i v i t y .

A l t h o u g h t h e y i e l d of p u r i f i e d l e c t i n is v e r y h i g h , no i m p u r i t i e s c o u l d be f o u n d a n d no t r a c e of n e u t r a l o r a m i n o s u g a r c o u l d b e d e t e c t e d i n c o n - t r a s t w i t h [16]. T h e l e e t i n c a n b e e a s i l y c r y s t a l l i - zed. T h e m i n i m u m a m o u n t of l e c t i n r e q u i r e d to a g g l u t i n a t e ce i l s is as lo'w as t h a t f o r t h e ] e c t i n f r o m o t h e r p u r i f i c a t i o n s [9, 17]. T h e s o l u b i l i t y of t h e p u r e l e c t i n w a s h i g h a n d t h e s p e c t r o s c o p i c p r o p e r t i e s w e r e s i m i l a r to t h e p r o t e i n p u r i f i e d b y n m r e s o p h i s t i c a t e d m e t h o d s [9, 17]. T h e t h r e e i so- l e c t i n s c o u l d b e d e t e c t e d b y p o l y a e r y l a m i d e gel e l e c t r o p h o r e s i s i n a c i d i c m e d i u m i n a g r e e m e n t w i t h p r e v i o u s r e s u l t s [20, 22].

T h u s , t h e m e t h o d d e s c r i b e d a b o v e is s i m p l e a n d q u i t e r a p i d . T h e o n l y o n e c e n t r i f u g a t i o n s t ep is s h a r e d w i t h al l t h e o t h e r t e c h n i q u e s , a n d c o u l d b e r e p l a c e d b y a f i l t r a t i o n w i t h a p r e s s - f i l t e r f o r l a r g e sca le p u r i f i c a t i o n . T h e o n l y o n e d i a l y s i s s t ep is u s e d at t h e v e r y e n d of t h e p r e p a r a t i o n , so t h a t t he t o t a l v o l u m e to b e d i a l y z e d is v e r y s m a l l : less t h a n 100 m l c o n t a i n i n g a b o u t 500 m g of l e c t i n f r o m o n e kg of w h e a t ge rm. T h i s m e t h o d c a n b e e a s i l y a d a p t e d fo r l a r g e sca le p u r i f i c a t i o n .

Acknowledgements .

This work was suppor ted by INSER.M granf n ° 7540.743, DGRST gran t n ° 74,70579 and a g ran t f rom Fonda t ion de la Recherche Mddicale Franqaise to M.M.

I~SUMI~,

Les au teurs d~crivent une m~thode simple de pur i - fication de la lect ine dn germe de bld h p a r t i r de germes trouvds dans le commerce. La lect ine est ext ra i te des g.ermes de bld hroyds et ddgraiss~s et pnrifide en une seule ~tape cons i s tan t en une f i l t ra t ion sur une colonne d '~ehangeurs d ' an ions et une adsorp- t ion sur un ddriv4 insolubl]is~ de la N-acdtyg]ncosa-

BIOCHIMIE, 1976, 58, n ° 10.

mine. La quant i td de leet ine obtenue h pa r t i r d 'un k i log ramme de germes est supdrieure h 500 rag. Bien que le rend.ement soit denx lois plus dlev4 que eelui obtenu e n u t i t i s an t d ' au t res mdthodes, aueune impuret~ n 'a pn ~tre ddeelde et les propridtds de la ]ectine ainsi purifi5e sont en bon accord avee celles de la prot~ine obtenue par des lndthodes plus longues et plus com- p]exes.

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