Studies on lectins XL. O-glycosyl derivatives of spheron in affinity chromatography of lectins

518

Biochimica et Biophysica Acta, 539 (1978) 518--528

© Elsevier/North-Holland Biomedical Press

BBA 28470 STUDIES ON LECTINS XL. O-GLYCOSYL DERIVATIVES OF SPHERON IN A F F I N I T Y CHROMATOGRAPHY OF LECTINS

K. FILKA a, j. COUPEK b and J. KOCOUREK a a Department of Biochemistry, Charles University, 128 40 Praha 2 and b Chemical Research and Development Department, Laboratory Instrument Works, 162 03 Praha 6 (Czechoslovakia)

(Received July 26th, 1977)

Summary Free monosaccharides can be used for direct glycosylation of Spheron, a spherical macroporous h y d r o x y a l k y l methacrylate-ethylene dimethacrylate copolymer, in a reaction that proceeds at room temperature in dioxane medium under catalysis of dry HC1 or BF3. Derivatives of L-fucose, ;~ -galactose, D-glucose, D-mannose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine thus prepared from Spheron beads have been shown to be efficient affinity carriers in isolation of lectins from seeds of Canavalia ensiformis D.C. (concanavalin A), D o l i c h o s biflorus L., Glycine so]a (L.) Sieb. et Zucc., L e n s esculenta Moench, R i c i n u s c o m m u n i s L., Ulex e u r o p a e u s L. and from albumin glands of the garden snail Helix p o m a t i a L.

Introduction Recent years brought a rapid development of various affinity techniques using different carriers for isolation of lectins. According to their origin the materials applied can be divided into (1) unmodified or partially modified natural substances, usually polysaccharides or glycoproteins or (2) synthetic products. With the exception of some polysaccharide substances like chitin [1], cross-linked dextrans (Sephadex) [2--4] or modified agarose (Sepharose) [5,6] that can be used directly in their commercially available form, all other materials require more or less complicated modifications, e.g., desolubilization by crosslinking [7,8] of natural polysaccharides or activation of the matrix [9-11] followed by coupling with a ligand which, in most cases, must also be synthetized. Similarly, also the various glycoproteins, the carbohydrate part ot

519 CH20H

HO --CH2

/CH2--OH

\CH 2 NHAc CH 2

CH 2

~CH 2 7

CO CH~ I I --CH 2 - C - CH 2 - C - CH 2 I I CH3 CO 7

CO I C--CH 2I CH 3

O\

HCI or E,F3 Spheron

+

o-GalNAc

(dioxane)

/CH2 CH2

•

/ ° CH 3 I

CO I

CO ~O

CH 3

C~H2 jCH2 HO -- CH 2

CH 3 I CO O ~

.

\

jCH2 CH2

OH

, ~ o I~

H ~1

CH 2 -

Fig. 1. Partial tentative structure o f an O-(N-acetyl-<~-D-galactosaminyl) S p h e r o n derivative.

which can interact with lectins, must be coupled to a suitable carrier [7,12,13]. The synthetic materials, based usually on a polyacrylamide matrix [14,16], posses a number of advantages over materials derived from natural substances. All of them show a higher stability against various chemical agents and microbial attack, can be kept indefinitely in dry condition at room temperature and are easily regenerated for further use. Their ligands have eligible anomeric configuration, spacer length and ring size and in most cases their sugar content as well as pore size can be regulated. Preparation in bead form ensures a rapid elution in column techniques. Their disadvantage is a relatively low mechanical stability causing plugging of columns operated at higher pressures and limited exclusion limits of molecular weights. The present paper deals with preparation and use in affinity chromatography of lectins [17] of a new fully synthetic glycosyl adsorbent based on poly(hydroxyalkyl methacrylate) matrices commercially available under the designation Spheron *. Recently Spheron [18,19] has been successfully used in the form of different derivatives, both in affinity chromatography of enzymes [20--22] and in ion exchange chromatography [23] techniques. The glycosylation of Spheron described in this paper starts from free sugars and can be achieved under very mild conditions by catalysis of dry HC1 or BF3. Additional experiments dealing with O-glycosyl Spheron synthesis [17,24] and application [17,25] have been described by us elsewhere; a preliminary communication of this paper has been published [26]. A partial tentative structure of an O-glycosyl Spheron adsorbent is shown in Fig. 1. * Distributed b y L a c h e m a , Brno, C z e c h o s l o v a k i a , H y d r o n Laboratories Inc. N e w B r u n s w i c k , N.J., U.S.A. and Koch-Light Ltd., C o l n b r o o k . Bucks, U.K.

520 Materials and Methods

Material for preparation of the affinity sorbents The p o l y ( h y d r o x y a l k y l methacrylate) gels, Spheron P-300, of particle size 100--150 pm were prepared as described earlier [18,19], or were obtained from Lachema, Brno, Czechoslovakia. Monosaccharides used for glycosylation were standard commercial products.

O-Glycosylation of Spheron After optimum conditions of the glycosylation reaction had been established in preliminary experiments [17] the following procedures were used. (a) Glycosylation catalyzed by HCI. Spheron beads (5 g) were left to swell for 12 h at room temperature in dry dioxane (60 ml) containing 6% (w/v) of dry HC1. Then 3 g of neutral sugar or 1.1 g N-acetylated aminosugar was added and the suspension was shaken for 10 h at 35°C. The beads were then thoroughly washed with water on a sintered glass funnel until the reaction of washings gave a negative reaction for carbohydrates (or a negative ninhydrine reaction in the case of amino sugars). Finally, the beads were washed with ethanol and diethyl ether and dried at 40°C. (b) Glycosylation catalyzed by boron trifluoride. Spheron beads (5 g) were left to swell at room temperature in 100 ml of dry dioxane containing 3--5% (w/v) of dry BF3; 3 g of hexose was then added and the suspension was shaken at 50°C for 24 h. The washing and drying of the beads was performed as described in the preceding paragraph.

Lectin materials used for purification by affinity chromatography The seed protein fractions with erythroagglutinating activity were obtained by extraction of the homogenized seeds by saline, saturation with amonium sulphate, dialysis and freeze-drying. Numbers in parentheses indicate (NH4)2SO4 saturation range: Dolichos biflorus L. (50--60%), Glycine so]a (L.) Sieb. et Zucc. (0--100%), Lens esculenta Moench. (30--60%), Ricinus communus L. (0--30%), Ulex europaeus L. (0--40%). For the isolation of concanavalin A from jack bean meal (Canavalia ensiformis D.C.) seeds a 10% (w/v) saline extract obtained by 24 h extraction was used directly. Similarly, the extract from Helix pomatia albumin glands was prepared by 48 h extraction of the homogenized tissue (1.5 g) by saline (5 ml). The term saline is used throughout this paper for a 0.9% NaC1 solution.

Determination of carbohydrate content Neutral carbohydrate c o n t e n t covalently bound to Spheron matrix was determined according to Dubois et al. [27]. In glycosyl Spheron derivatives the determination was carried out as follows. To 100 mg of glycosyl Spheron, 10 ml of 5 N H~SO4 was added and the suspension was heated under reflux on a boiling water bath for 6 h. After cooling the Spheron beads were centrifuged down and the carbohydrate c o n t e n t was determined in the supernatant. Amino sugar content in the N-acetylglycosaminyl Spheron derivatives was determined after hydrolysis on an automatic amino acid analyzer (Model AAA 881, Mikrotechna, Praha, Czechoslovakia). A sample of 10 mg was hydrolyzed

521 in 2 ml of 4 N HC1 for 14 h at 110°C in a sealed tube. After separation of Spheron beads by centrifugation, the hydrolyzate was evaporated to dryness in vacuo then a volume of distilled water corresponding to the original hydrolyzate volume was added and again evaporated. The last operation was repeated, the dry residue dissolved in citrate buffer, pH 2.2, and applied to the amino acid analyzer.

Determination of protein content Protein content in chromatographic fractions was followed by measurements of optical absorbance at 280 nm on a MOM 202 spectrophotometer (MOM, Budapest, Hungary).

Affinity chromatography Chromatography on glycosyl Spheron adsorbents was carried out in columns equilibrated with saline. The same solvent was used for application of the crude protein material and to elution of inactive proteins. Adsorbed lectins were displaced with a 0.2 M solution of the appropriate haptenic sugar. Instead of saline, 0.15 M phosphate buffer was used in chromatography of the U. europaeus lectin. Both protein content and erythroagglutinating activity were followed continuously during elution. Details to procedures in individual chromatographic runs are given in legend to Fig. 2.

Estimation of lectin binding capacity of glycosyl Spherons Glycosyl Spheron (1 g) was left to swell in saline for 12 h, poured onto a column (1 X 10 cm) and washed with 50 ml of saline. Aliquots of 50 mg lectin in 1 ml saline were successively applied and the inactive proteins eluted by saline at a rate of 8 ml/h. The elution of protein was monitored by spectrometry and erythroagglutination assay. As soon as the effluent showed erythroagglutinating activity, the last 50 mg portion of the active protein fraction was applied, the column was thoroughly washed with saline and the bound lectin eluted with a 0.2 M solution of the haptenic sugar in saline at a rate 8 ml/h. Fractions containing proteins were combined, dialyzed for 72 h against five changes each of 4 1 of distilled water, and freeze-dried. The yield indicates the capacity of the specific sorbent for the corresponding lectin. In case of concanavalin A, where a saline extract of jack bean meal was directly applied, the elution of inactive proteins was carried out after application of 3 ml of the extract.

Assay of nonspecific interactions of the sorbents with proteins A freeze-dried active protein fraction was applied to a column (1 X 30 cm) prepared from 4 g of glycosyl Spheron that had been swollen for 12 h in saline and was washed with 200 ml of saline. After entering the column the inactive proteins were eluted by saline and their content in the eluate was measured spectrometrically and by agglutination assay. When the elution with saline was completed, the column was washed with a solution (0.2 M) of the ligand sugar in saline and, finally, with glycine buffer (0.05 M) pH 2.7.

Discontinuous polyacrylamide electrophoresis Discontinuous polyacrylamide electrophoresis was carried out in alkaline

522 medium (pH 8.9) according to Davis [28] using a buffer system described originally by Steward [29].

Estimation o f erythroagglutinating activity Erythroagglutinating activity was assayed macroscopically, in test tubes using a 2% saline suspension of three times washed red cells and a 1% solution of the lectin material as a starting concent r a t i on in the doubling dilution technique [30]. After 15 min at r oom temperature, the test tubes were centrifuged for 1 min at 1000 rev./min and observed. Suspension of trypsinized red cells was prepared from 0.5 ml of e r y t h r o c y t e s washed three times with saline. After washing 10 mg of trypsin was added and the suspension brought with saline to a total volume of 2 ml. The mixture was incubated 45 min at 37~C, then washed again three times with saline, after which the volume was adjusted with the same solution to 20 ml. The titer has been expressed as highest dilution of a 1% (w/v) sample solution showing detectable erythroagglutination. Results

Preparation and properties of the O-glycosyl Spheron derivatives The procedure for glycosylation of Spheron described in the Materials and Methods section yields a p r o d u c t visually undistinguishable from the starting n o n m o d i f i e d Spheron. Neither does microscopical examination reveal any change on the surface of the spherical particles or in their size distribution. According to gel permeation c h r o m a t o g r a p h y measurements using standard p o l y d e x t r a n e series (Pharmacia Fine Chemicals, Uppsala) the exclusion limit of original Spheron was lowered by 15--20%. The inner surface area measured by nitrogen desorption m e t h o d [18] revealed a slight increase of original values from 45 m2/g up to 55 m2/g. Conditions of glycosylation described had been chosen after a systematic quantitative study [17]. By extending the reaction time to about 70 h, a carboh y d rate c o n t e n t of 5--7% w/w has been achieved even at room t em perat ure and by only occasional shaking of the suspension of the reactants in solution of dry HC1 in dioxane. The application of BF3 catalyst led to an increase of the sugar c o n t e n t in copolymers up to 20% w/w. Carbohydrate contents of the individual O-glycosyl Spherons used in our work are summarized in Table I.

Affinity chromatography of lectins on O-glycosyl Spherons Elution patterns of affinity c h r o m a t o g r a p h y of lectins on giycosyl Spherons are shown in Fig. 2, the yields and erythroaggiutinating activities of the purlfled substances are given in Table I. Nonspecific interactions of lectins and o t h e r proteins with the Spheron matrix were also checked. All the proteins applied appeared in the eluate w i t h o u t retardation in one peak and with unchanged erythroagglutinating activity. In two instances the activity was observed also in a few fractions before starting elution with the haptenic sugar. This was the case in c h r o m a t o g r a p h y of lectins of C. ensiformis (fraction 3--6, n o t shown in Fig. 2) and G. so]a (fractions 3--5 Fig. 2d). However, the titer of these fractions was very low and cor r es pon ded to an approxi m at e lectin con-

I

CARRIERS

USED, YIELDS

AND ACTIVITY

OF LECTINS

ISOLATED

ON O-GLYCOSYL

SPHERONS

* Trypsinized

erythrocytes.

cz-D-Glc a-D-GalNAc c~-D-Gal ~-D-GIc-NAc a-D-Glc a-D-Man a-D-Gal a-L-Fuc

Canavalia e n s i f o r m i s Dolichos biflorus G l y c i n e soja Helix p o m a t i a Lens esculenta L e n s esculanta Ric~nus c o m m u n i s Ulex e u r o p a e u s

8.2 7.3 7.6 8.1 8.2 9.0 7.6 5.4

O-Glycosyl Spheron and its sugar content in % (w/w)

Lectin

20-ml extract 230 400 5-ml extract 200 250 300 700

Active fraction applied (rag)

41.1 17.4 6.2 16.3 5.8 7.4 77.2 7.1

Lectin yield (mg)

512 *

O

512 * 128 64 * 512 64 64 16 384 0

512 * n.d. n.d. 1 024 64 64 8 192 16 0 32 32 16 384 8

n.d. n.d.

512 * n.d. n.d. 0 64 64 8 192 32

2048 * 1 024 4 096 * 32 768 256 256 65 536 2

2048 * n.d. n.d. 65 536 256 256 32 768 1 024

A2

A 1

B

A1

A2

Purified lectin

Active fraction or extract

Titer

2048 * n.d. n.d. 0 128 128 65 536 256

B

2048 * n.d. n.d. 0 256 256 32 768 4 096

O

All lectins are of seed origin except for Helix p o m a t i a where albumin glands of the animal were used. Data on active fractions and extracts are given in Materials and Methods. Titer refers to 1% sample solutions or undiluted extract; n.d. = not determined.

AFFINITY

TABLE

CJ1 tO

c~

k

o

c~

f

k

~ I

m

_

Q

,

uI

k

o

,

o

~J

:X

~

~

k

d

0.2M o-Galactose

525

2.0-

1.0

$0

100

'~(ml)

0.2 M N-Aceryl-o-glucosamine

A 2.0-

e

l pH 2.7

1.0~

L. 1~

l~l V(ml)

0, 2 M N - Acetyl- D - lahictOlamine 2.0

f

l pH 2.7

1.0-

Fig. 2. A f f i n i t y c h r o m a t o g r a p h y o f l e c t i n s o n O - g l y c o s y l S p h e r o n d e r i v a t i v e s . If n o t s t a t e d o t h e r w i s e , a p p r o x . 1 0 % ( w / v ) s o l u t i o n s o f p r o t e i n s i n saline w e r e a p p l i e d t o O - g l y c o s y l S p h e r o n c o l u m n s (1 × 3 0 c m ) e q u i l i b r a t e d w i t h saline a n d e l u t i o n p e r f o r m e d w i t h saline a t f l o w r a t e 8 m l / h ; f r a c t i o n s o f 4 m l were collected. Arrows indicate start of application of 0.2 M haptenic eluant (solution of sugar corres p o n d i n g t o t h e g l y c o s y l l i g a n d ) o r a p p l l c a t i o n o f 0 . 0 5 M g l y c i n e b u f f e r , p H 2 . 7 . N u m b e r s in b r a c k e t s indicate fractions that were combined, dialyzed and freeze-dried. Amounts of crude material applied and y i e l d s o f p u r i f i e d l e c t i n s a r e given in T a b l e I. (a) Ulex e u r o p a e u s ; l e c t i n s o l u t i o n a p p l i e d in 0 . 1 5 M p h o s phate buffer, pH 7.9, to column equilibrated with the same buffer. Flow rate 15 ml/h, 5-ml fractions [ 4 3 - - 4 8 ] ; (b) R i c i n u s c o m r n u n i s [ 2 4 - - 2 8 ] ; (c) L e n s e s c u l e n t a [ 3 1 - - 3 5 ] ; (d) G l y c i n e soja [ 2 2 - - 2 4 ] ; (e) H e l i x p o m a t i a ; saline e x t x a c t ( 5 m l ) o f a l b u m i n g l a n d s a p p l i e d t o a c o l u m n ( 1 . 2 X 2 0 c m ) c o n t a i n i n g 3.1 g o f t h e c a r r i e r [ 2 6 - - 2 9 ] : (f) D o l i c h o s biflorus; f l o w r a t e 1 2 m l / h ; 4 - m l f r a c t i o n s [ 2 7 - - 3 1 ] .

526

"i

d

a

b

c

d

e

f

Fig. 3. Disc p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f c r u d e f r a c t i o n s a n d a f f i n i t y c h r o m a t o g r a p h y p u r i f i e d lectins. E l e c t r o p h o r e t i c e x p e r i m e n t s w e r e p e r f o r m e d a t p H 8.9, 4 m A p e r t u b e , for 7 0 - - 7 5 h. S o l u t i o n s (1%) o r e x t r a c t s a p p l i e d in t h e a m o u n t s (pl) given b e l o w ; left c r u d e f r a c t i o n s , r i g h t p u r i f i e d lectins: (a) Ulex europaeus, 1 0 - 1 0 ; (b) Ricinus communis, 2 0 - 1 0 ; (c) Lens esculenta, 4 0 - 2 0 ; (d) Glycin¢' soja, 20-40; (e) Helix pomatia, 1 0 - 1 0 ; (f) Dolichos biflorus, 10-10. D a t a o n c r u d e f r a c t i o n s a n d e x t r a c t s given in Materials a n d M e t h o d s .

tent of 0.17 mg and 0.029 mg, respectively, i.e. 0.3% and 0.2% of the total lectin content. This p h e n o m e n o n was not observed if the once-isolated lectins were rechromatographed under identical conditions. Fig. 3 shows electrophoretic patterns of starting protein fractions and of those purified by affinity chromatography. The presence of only one isolectin in the purified L. esculenta substance (Fig. 3c, left) is due to special cultivar of seeds (Lens culinaris Moench, cv. Lenka) that contains only one isolectin (Tich~, M., unpublished results}.

Capacity o f the sorbents By the procedure given in Materials and Methods binding capacity of two different carriers was determined. The O-a-D-glucosyl Spheron showed a capacity of 31.2 mg/g for concanavalin A (i.e.C. ensiformis lectin) and the O-a-D-galactosyl Spheron a capacity of 42.5 mg for the R. cornmunis lectin. Discussion O-Glycosyl derivatives of Spheron P introduced in this paper as efficient affinity carriers for lectin isolations are new functional derivatives of a series of macroporous copolymers prepared from h y d r o x y a l k y l methacrylate and ethylene dimethacrylate. Spheron beads are commercially available in five different porosities. Their macroporous inner architecture guarantees very large pore diameters together with extensive inner surface area values. Structure of Spheron matrix (Fig. 1) shows a striking resemblance to pivalic acid esters that are known to be most resistant to both acid and alkaline hydrolysis. Spheron P shows both these properties in a still more pronounced degree and is, more-

527 over, extremely stable mechanically and thermally (up to 230°C). These properties allow its use in high-pressure liquid chromatography techniques as well as in gas chromatography in form of a series o~f derivatives. Another characteristic feature important for practical use are its relatively small bed volume changes between dry and swollen conditions. In aqueous media no changes result over a wide range of pH and ionic strength values.The free h y d r o x y l groups of Spheron manifest a reactivity equal to h y d r o x y l groups of alcohols or polysaccharides and thus render a series of derivatizations possible. O-Glycosyl Spheron derivatives resemble in many ways O- or S-glycosyl polyacrylamide derivatives described earlier [14,16], especially with regard to their stability, resistance to microbial attack and ease of regeneration for repeated use. However, they exceed them in their advantageous mechanical properties, chemical stability and the simplicity of preparation that allows direct use of free monosaccharides of different types. At present, they represent the most easily accessible synthetic affinity carriers for lectins described. The results of affinity chromatography of lectins as summarized above show that both the composition of the active substances and their properties as well as their yields are in good agreement with findings of previous workers [27,11, 12,14--16]. Excellent mechanical properties of the carriers allow a relatively high speed of elution without impairing purity of the products or the yield. The reasons for appearance of very small amounts of the active substances at the start of elution with saline are not clear. However, the same phenomenon was observed before in applications of other affinity sorbents for lectins (e.g. Sephadex [4]) and does not seem to have any relation to the structure of the sorbent matrix or to the flow rate of the eluant. The covalent attachment of sugars in the O-glycosyl Spheron derivatives results in a high degree of hydrophilization of the sorbent inner surface with relatively small decrease of the specific pore volume [24]. Thus the advantageous combination of high stability of the poly-(hydroxyalkyl methacrylate) structure with hydrophilic properties of polysaccharide carriers makes the new Spheron derivatives very promissing for applications in chromatography of biopolymers and their fragments in general. Investigations in this direction are now under progress in our laboratories. Acknowledgment The authors are deeply indebted to Dr. V. Ho[ej~i and Dr. M. Tich~, Department of Biochemistry, Charles University, Prague for their generous gifts of some of the lectin-containing protein fractions. References 1 2 3 4 5 6

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