J. Kocourek

Studies on lectins. XXXV. Water-soluble O-glycosyl polyacrylamide derivatives for specific precipitation of lectins.

By copolymerization of acrylamide and allyl glycosides of various sugars, O-glycosyl derivatives of polyacrylamide copolymers were prepared. The sugar content of the copolymers can be varied in the range 0--40%, their sedimentation coefficient shows the vales of 2.5-5.7 S; the molecular weight of an O-alpha-D-mannopyranosyl polyacrylamide copolymer (29% mannose, so20,w = 2.9 S) was estimated as 44 500. Copolymers with incorporated glycosyl residues interacting specifically with lectins yield precipitates with them upon immunodiffusion in cellulose acetate. The quantitative precipitin curves obtained with these copolymers are similar to those produced by quantitative precipitation of lectins with natural polysaccharides. The copolymers may serve as model substances of natural polysaccharides.

Studies on phytohemagglutinins III. Isolation and characterization of hemagglutinins from the pea (Pisum sativum L.)

Abstract A mixture of two closely related phytohemagglutinins have been isolated from crude water extracts of the garden pea seeds ( Pisum sativum L. var. Pyram) by (NH 4 ) 2 SO 4 precipitation of the active protein fraction, chromatography on hydroxylapatite and DEAE-cellulose as well as by specific adsorption on Sephadex. The two phytohemagglutinins were separated by further chromatography on DEAE-cellulose. Both are homogenous proteins exhibiting a single band by electrophoresis on starch and polyacrylamide gels as well as on cellulose-acetate strips at different pH values. Their electrophoretic mobility is similar to that of human IgG globulin, their isoelectric point lies at pH 7.7–7.9. They both reveal a single symmetrical peak on ultra-centrifugation and a s 20. w of 3.5 S. The molecular weights of 54 000 and 53 000 have been found for phytohemagglutinin 1 and phytohemagglutinin II, respectively, by the sedimentation equilibrium method. Both phytohemagglutinins show the same degree of nonspecific hemagglutinating activity against all types of human red blood cells of the ABO system. They have perceptible activity up to a dilution of 2.5 μg/ml. The amino acid compositions of both pea phytohemagglutinins are very similar. They contain high amounts of aspartic acid and threonine, but no cystine and methionine. The DNP method of N-terminal amino acid analysis of phytohemagglutinin H and the mixture of both phytohemagglutinins shows the presence of equal amounts of valine and threonine.

Studies on lectins. XXXI. Determination of dissociation constants of lectin. Sugar complexes by means of affinity electrophoresis.

A modification of affinity electrophoresis in polyacrylamide gels containing immobilized sugar residues is described. The immobilization of sugar residues is achieved by addition of a water soluble O-glycosyl polyacrylamide copolymer to the polymerization mixture, which serves for the preparation of gels for commonly used discontinuous polyacrylamide gel electrophoresis. The electrophoretic mobility of lectins in affinity gels containing specific immobilized sugar decreases with increasing sugar concentration. The addition of free sugar into the affinity gel abolishes retardation of the electrophoretic mobility caused by immobilized sugar. The relationship between electrophoretic mobility of lectins and concentration of immobilized and free sugar was used for the calculation of dissociation constants of the various complexes lectin-immobilized sugar and lectin-free sugar.

Studies of lectins XXXVI. Properties of some lectins prepared by affinity chromatography on O-glycosyl polyacrylamide gels☆

A number of lectins has been purified by affinity chromatography on O-glycosyl polyacrylamide gels. The lectins isolated (and the particular sugar ligands used in the affinity carriers) are as follows: Anguilla anguilla, serum (alpha-L-fucosyl-), Vicia cracca, seeds; Phaseolus lunatus, seeds; Glycine soja, seeds; Dolichos biflorus, seeds; Maclura pomifera, seeds; Sarothamnus scoparius, seeds; Helix pomatia, ablumin glands; Clitocybe nebularis, fruiting bodies (all N-acetyl-alpha-D-galactosaminyl-); Ricinus communis, seeds (beta-lactosyl-); Ononis spinosa, root; Fomes fomentarius, fruiting bodies; Marasmius oreades, fruiting bodies (all alpha-D-galactosyl-), Canavalia ensiformis, seeds, (i.e., concanavalin A) (alpha-D-glucosyl-). Physicochemical properties of Glycine soja, Dolichos biflorus, Phaseolus lunatus, Helix Pomatia and Ricinus communis lectins corresponded well to properties of the preparations studied earlier by other workers. For the other purified lectins the essential physiochemical data (sedimentation coefficient, molecular weight, subunit composition, electrophoretic patterns, amino acid composition, carbohydrate content, isoelectric point) were established and their precipitating, hemagglutinating and mitogenic activities determined.

Studies on phytohemagglutinins. IV. Isolation and characterization of a hemagglutinin from the lentil, Lens esculenta, Moench

Abstract The presence of two nonspecific phytohemagglutinins was shown in seeds of the lentil, Lens esculenta , Moench. One of them was isolated by water extraction, followed by ammonium sulfate fractionation and specific adsorption on Sephadex. Hemagglutinin exhibited a single symmetrical peak on ultracentrifugation and a single band on polyacrylamide and starch gel electrophoreses. It has a sedimentation coefficient s 20, w 3.78 S, as calculated from the sedimentation velocity data. A molecular weight of 42 000 was estimated by gel filtration at pH 7.4. In acid medium, dissociation into smaller subunits of uniform molecular weight occurs; the dissociation can be reversed under alkaline conditions. The lentil hemagglutinin contains high amounts of aspartic acid and threonine, about 1.5% of neutral sugar and no sulfur-containing amino acids. The presence of about 1 gatom of Mn per 66 500 g of agglutinin and 1 gatom of Ca per 11 000 g of agglutinin was shown. The minimal hemagglutinating dose of the purified preparation is 4.5 μg/ml. The hemagglutinating activity of the purified lentil agglutinin can be increased by some bivalent cations, especially by Mn 2+ .

Studies on phytohemagglutinins XII. O-glycosyl polyacrylamide gels for affinity chromatography of phytohemagglutinins

Abstract Copolymerization of alkenyl O -glycosides with acrylamide and N , N ′-methylene bisacrylamide produces neutral hydrophilic gels containing sugars having O -glycosidic links with the alkyl side chains of the polymer matrix. The preparation of these copolymers and their application as affinity adsorbents for the specific separation of phytohemagglutinins from other proteins is described.

Studies on phytohemagglutinins II. phytohemagglutinins ofPisum sativum L. andLens esculenta Moench: Specific interactions with carbohydrates

Die in den Samen der Erbse (Pisum sativum L.) und der Linse (Lens esculenta Moench) enthaltenen Phythämagglutinine reagieren spezifisch auf einige Kohlehydratsubstanzen. Mono- und Oligosaccharide, welche die Agglutinationswirkung dieser Phythämagglutinine hemmen, bewirken auch eine Erhöhung ihrer elektrophoretischen Wanderungsgeschwindigkeit auf Stärkegel. Diese Beschleunigung ist der Hemmwirkung direkt proportionell entgegengesetzt.

Studies on phytohemagglutinins: VII. Effect of Mn2+ and Ca2+ on hemagglutination and polysaccharide precipitation by phytohemagglutinin of Pisumsativum L

Abstract The “demetallization” procedure used to remove Mn and Ca from concanavalin A has been applied to pea phytohemagglutinin and was shown to decrease the Ca content but to leave the Mn content unaffected. “Demetallization” completely abolished the precipitation of mannan by the hemagglutinin and decreased hemagglutination to 25% of its original activity. Addition of Mn2+ and Ca2+ enhanced both the hemagglutinating and precipitating activities. Mn2+ was more active in promoting hemagglutination while Ca2+ showed a more pronounced stimulation of mannan precipitation. EDTA inhibited both hemagglutination and precipitation of mannan. Mannan precipitation by the phytohemagglutinin was more sensitive to the effect of EDTA than hemagglutination.

Studies on phytohemagglutinins XVIII. Affinity electrophoresis of phytohemagglutinins

Abstract A new separation technique has been developed combining the principles of a affinity chromatography and electrophoresis. On a polyacrylamide gel column composed of layers of large-pore gel, gel with covalently linked affinity ligands (affinity gel) and small-pore gel, it is possible to “filter off” on the affinity gel proteins with combining sites for the corresponding ligand and to separate the remaining components on the small-pore gel. Several examples are given for the separation of phytohemagglutinins from the plant protein mixtures using O-glycosyl polyacrylamide derivatives as affinity gels.

Studies on phytohemagglutinins XVII. Some properties of the anti-H specific phytohemagglutinin of the furze seeds (Ulex europaeus L.)

The anti-H specific hemagglutinin has been isolated from the seeds of Ulex europaeus L. by saline extraction, (NH4)2SO4 precipitation and affinity chromatography of the active protein fraction on an O-α-l-fucosyl polyacrylamide adsorbent. The phytohemagglutinin reveals a single symmetrical peak on ultracentrifugation and a s20,w of 4.1 S. Sedimentation equilibrium data and the results of the polyacrylamide electrophoresis in sodium dodecylsulfate medium indicate a molecular weight of 46 000 and 40 000, respectively. There is no observable dissociation in the sodium dodecylsulfate medium of the phytohemagglutinin treated with 4 M urea and/or mercaptoethanol. The substance appears homogeneous also by electrophoresis on cellulose acetate strips and its mobility indicates an approximate pl of 6.0–6.1 in the phosphate buffer. On disc polyacrylamide gel electrophoresis at pH 8.9 a minor component separates from the bulk of the substance, presumably an isophytohemagglutinin. The amino acid analysis demonstrates the presence of high amounts of aspartic acid, serine and glycine and of only small amounts of typptophan, cysteine and methionine. Serine was shown to be the only N-terminal amino acid detectable by the dansylation technique. The phytohemagglutinin contains 2 atoms of Ca and 1 atom of Zn or Mn per molecule. The minimum hemagglutinating dose of the phytohemagglutinin is 1.2 μg/ml against O-type erythrocytes, 10 μg/ml against A2 type, 40 μg/ml against B-type and 50 μg/ml against A1-type. It is only slightly activated by Zn2+ and Mn2+ and there is the inhibition of its activity by EDTA. Prolonged photooxidation in the presence of ethylene blue likewise shows no effect on the activity of the phytohemagglutinin.