Biochimica et Biophysica Acta, 393 (.1975) 170-181 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 37047 STUDIES ON P H Y T O H E M A G G L U T I N 1 N S XXVI. A NEW TYPE OF A L E N T I L H E M A G G L U T I N I N ISOLATED FROM L E N S E S C U L E N T A MOENCH., SUBSP. M I C R O S P E R M A (BAUMG.) BARULINA
D. FIALOV,~, M. TICHA and J. KOCOUREK Department of Biochemistry, Charles University, Albertov 2030, Prague 2 (Czechoslovakia) (Received November 19th, 1974)
SUMMARY In seeds of the lentil Lens esculenta Moench., subsp, microsperma (Baumg.) Barulina, a nonspecific phytohemagglutinin was found, the properties of which differ from those of the previously described lentil hemagglutinins. The phytohemagglutinin is not adsorbed to the Sephadex matrix; its hemagglutinating activity is different towards erythrocytes of different human blood groups of the ABO system. The isolated phytohemagglutinin preparation is homogeneous by disc polyacrylamide and starch gel electrophoreses and by gel chromatography on Bio-Gel P-100. Ultracentrifugation of the phytohemagglutinin yields a single symmetrical peak with S~o.w of 3.9 S. From the sedimentation data, a molecular weight of 53 300 was calculated. Phytohemagglutinins of similar properties were shown to be present also in other cultivars of Lens esculenta, subsp, microsperma.
INTRODUCTION The presence of a nonspecific phytohemagglutinin in seeds of the lentil (Lens esculenta Moench. = Lens culinaris Med.) was first reported in 1908 by Landsteiner and Raubitschek [1]. Isolation and purification of lentil phytohemagglutinins and some of their properties were described independently by 3 groups of workers [2-4]. An exact specification of the variety or cultivar of the lentil seeds used for the isolation was described only by us in our previous communication [3]. In spite of some minor differences in physicochemical properties, very similar results were obtained by the 3 groups of workers: (i) the hemagglutinin from the lentil is bound to the Sephadex matrix and can be desorbed with D-glucose solution [3, 4, 6]; (ii) the hemagglutinin of the lentil agglutinates human red blood cells with equal activity irrespective to the blood type [4-6]; (iii) in the lentil seeds 2 isophytohemagglutinins are found [2, 3], their properties are very closely related [2]; Howard et al. [2] described that 6 different lentil "brands" tested varied in the proportion of the 2 isophytohemagglutinins. Our present paper describes the properties of a phytobemagglutinin isolated
171 from the lentil,Lens esculenta Moench., subsp, microsperma (Baumg.) Barulina, which manifests properties different from those (i-ill) of hitherto examined varities. Results of our present paper show the necessity and importance to identify the variety of the lentil seeds, which is employed as a source of phytohemagglutinin. MATERIALS AND METHODS Seeds of lentil, Lens esculenta Moench., subsp, microsperma (Baumg.) Barulina (in this paper called small-seed lentil), cv. SIovensk~ modrd were obtained from the Crop Breeding Station, Sl~idkovi6ovo-Nov2~ Dvor, cv. Moravskd drobnozrnn~ and cv. Trebigovsk~i - - from the Crop Breeding Station, Kagtice. Seeds of lentil Lens esculenta Moench., subsp, macrosperma (Baumg.) Barulina (in this paper called largeseed lentil), cv. Hrotovickd were provided by the Crop Breeding Station, Topolniky, Czechoslovakia. Phytohemagglutinin from the large-seed lentil cv. Hrotovick~ was prepared as described previously [3]. The two isophytohemagglutinins (phytohemagglutinin I and ll) were separated according to Howard et al. [2].
Extraction and purification Finely ground seeds (1 kg) were suspended in 10 I of deionized water; the pH was adjusted to 6.6 with 2 M NaOH. After continuous stirring for 1 h, the suspension was acidified with 2 M HCI to pH 4.5 and then left to settle at 4 °C for 20 h. The seed meal slurry together with precipitated nonactive proteins were removed by centrifugation and the supernatant was fractionated by salting out with solid (NH4)2SO4. "[he precipitate of the active protein obstained between 30 and 60 ~o saturation was dissolved in deionized water and dialyzed. The precipitate of inactive protein formed during dialysis was centrifuged off and the clear supernatant was freeze-dried. For preparative purposes, the active protein fraction (3 g) was dissolved in 0.15 M NaCI or deionized water (10 ml) and applied to a Sephadex G-150 column (3.5 cm × 60 cm) equilibrated with the same solvent. The column was first eluted either with water or 0.15 M NaC1 (see Fig. 2). A part of adsorbed proteins was released from the gel with 0.1 M D-glucose solution; at the end, the column was eluted with 1 M acetic acid; 5-ml fractions were collected at a flow rate of 10 ml/h. Tubes corresponding to Fractions II, Ill, and IV (see Fig. 2) were pooled. The protein Fraction lI, when the chromatography was carried out in water, and the Fraction IV were freeze-dried; the Fraction III was dialyzed against deionized water and then freeze-dried, as well as the Fraction II, when chromatography was done in 0.15 M NaCI. Small-scale experiments were carried out using a Sephadex G-150 column (1.5 c m x 30 cm) equilibrated either with 0.15 M NaCI, 0.15 M MnClz, or 0.075 M MnCI2 6- 0.075 M CaCI2. The active protein fraction (1 g) was dissolved in 3 ml of the solvent and applied to the column; 5-ml fractions were collected at a flow rate of 10 ml/h. Gel filtration on a Bio-Gel P-100 column (1 cm x 70 cm, 0.15 M NaCI, pH 7.0) was used to check homogeneity of the hemagglutinin preparation.
172
Electrophoretic methods Disc polyacrylamide gel electrophoresis was performed in an apparatus designed by Davis [7] in alkaline [8], or acidic [9] buffers. Vertical starch-gel electrophoresis was carried out according to the method of Smithies [10] in borate buffer, pH 8.6, or acetate buffer, pH 5.0. Polyacrylamide gel electrophoresis in dodecylsulfate medium was performed according to the procedure described by Weber and Osborn [11 ] in 5 ~ polyacrylamide gel. Molecular weights of the subunits were calculated from the comparison of their relative electrophoretic mobilities with those of known proteins [12, 13]: ovalbumin, subunits of concanavalin A [14], and oligomers of lysozyme [15]. UItracentrifugation analysis Measurements of the sedimentation velocity of the hemagglutinin were performed on a Spinco-Beckman Model E ultracentrifuge at a speed of 59 780 rev./min. Concentrations of the hemagglutinin tested were 0.2-0.9 % in 0.15 M NaCI. Sedimentation equilibrium studies were performed on the same ultracentrifuge at a speed of 20 410 rev./min, with a 0.03 % hemagglutinin solution in 0.15 M NaCI. The molecular weight was calculated from sedimentation equilibrium data according to the method of Yphantis [16] assuming a partial specific volume ~¢of 0.72. Carbohydrate analysis Total neutral sugar content was determined by the phenol/sulfuric acid method of Dubois et al. [17] with D-glucose as a standard. For amino sugar analysis, protein samples were hydrolyzed in sealed tubes at 110 °C with 4 M HCI for 14 and 20 h and analyzed on an automatic amino acid analyzer (Model AAA 881, Mikrotechna, Praha, Czechoslovakia). Metal content The metal content was determined by atomic absorption spectroscopy. A Varian Techtron Atomic Absorption spectrophotometer model AA4 equipped with hollow cathode lamps and ultraviolet sensitive HTVR-106 photomultiplier was used. Absorption was measured at the Ca 4226.7 A, Mn 2795.6 A, and Zn 2138.6 A analytical lines. Proiein and amino acid analyses Protein content in eluates was determined using absorption at 280 nm. For amino acid analysis, protein samples were hydrolyzed in sealed tubes under N2 at l l0 °C with 6 M HCI for 20 and 70 h and analyzed on an automatic amino acid analyzer (Model AAA 881, Mikrotechna). Values for serine and threonine were olztained by extrapolation to zero time. The highest values were taken for the remaining amino acids. The tryptophan content was determined independently [18]. Cystein was estimated as cysteic acid after oxidation of the sample by performic acid [19]. N-Terminal amino acids were identified by the dansylation technique according to Zanetta et al. [20] using thin-layer chromatography on silica gel (Silufol, Kavalier) for identification of the dansyl derivatives.
173
Determination of hemagglutinating activity Hemagglutinating activity was determined at 20 °C by the method referred to by Tobigka [21]. To 0.2-ml aliquots obtained by 2-fold serial dilution of an 1 hemagglutinin solution, an equal volume of a 2~o suspension of red blood cells, 3 times washed with 0.15 M NaCI was added. After 15 rain tubes (0.6 cm × 8 cm), were examined for agglutination. To study the effect of metal ions on the hemagglutinating activity of phytohemagglutinin, a solution containing 0.1 M MnC12 + 0.05 M NaCI or 0.075 M MnCI2 + 0.075 M CaCIz were used. Inhibitory activity of the sugars was estimated using a hemagglutinin solution 8 times more concentrated than the solution allowing the first perceptible hemagglutination. To 0.I ml of aliquots obtained by 2-fold serial dilution of an 1 ~o sugar solution, 0.1 ml of the phytohemagglutinin solution was added. After 15 min, hemagglutination was estimated as described above [21]. RESULTS
Interaction of the phytohemagglutinin with Sephadex One of the methods described for the isolation of the lentil phytohemagglutinin was based on its specific adsorption on the Sephadex gel followed by an elution with D-glucose or acidic buffer solutions [3-6]. When this procedure was applied to the isolation of hemagglutinin from the small-seed lentil, a major part of the active protein did not bind to the Sephadex matrix. The protein possessing hemagglutinating activity was eluted with 0.15 M NaCI after inactive proteins. The following elution of the Sephadex column with o-glucose solution resulted in a release of a small amount of a protein with hemagglutinating activity (see Fig. la). Under the exactly same conditions, hemagglutinin from large-seed lentil was completely bound to the Sephadex column and could be released with D-glucose solution (Fig. l b). An analogous experiment was carried out with additional 2 cultivars of the
A 1.0
0.5
1.0
QSJ 100
200
300
400 V(ml)
Fig. I. Comparison of behavior of phytohemagglutinins from small-seed and large-seed lentil in gel chromatography on Sephadex G-150. 1 g of active protein fl'action obtained by (NH4)2SO4 precipitation was applied to a Sephadex G-150 column (1.5 cm × 30 cm) equilibrated with 0.15 M NaCI. a. Active protein fraction from small-seed lentil, b. Active protein fraction from large-seed lentil. Arrows indicate start of the elution with 0.1 M D-glucose solution; ÷ denotes fractions with hemagglutinating activity.
174 small-seed lentil and another cultivar of large-seed lentil. Results were similar to those depicted in Fig. la and lb, respectively. The chromatography on a Sephadex column equilibrated with 0.15 M MnCIz or 0.075 M MnCI2 + 0.075 M CaC12 did not increase the amount of phytohemagglutinin which was adsorbed to the Sephadex matrix; this treatment only slightly increased the elution volume needed for the elution of the active protein,
Isolation o['phytohemagglutinin and its electrophoretic characterization For the isolation and further characterization of the hemagglutinin from smallseed lentil, which was not adsorbed to a Sephadex column, gel chromatography was performed on a Sephadex G-150 column on a preparative scale. A typical separation of the phytohemagglutinin is shown in Fig. 2 and Table I.
I1
1.o ¸
L
580
1000 1'500
20'00 2500
30'00
V(ml)
Fig. 2. Chromatography of active protein fraction obtained by (NH4)2SO4 precipitation on Sephadex G-150. Arrows denote solvent changes: a. 0.15 M NaCl; b. 0.1 M D-glucose;c. 1 M acetic acid; (see text for details). The elution of the column with 0.15 M NaCI or deionized water yielded two peaks (designated Fractions 1 and II) of proteins: Fraction 1 was inactive, Fraction 11 possessed hemagglutinating activity. As dialysis of the hemagglutinin solution resulted in a considerable loss of protein, the chromatography in deionized water was prefered. The adsorbed part of the hemagglutinin was released by elution of the column with 0.1 M D-glucose solution (Fraction II1). After this elution, a small part of proteins still remained bound to the Sephadex column, which was eluted with 1 M TABLE1 PURIFICATION OF LENTIL HEMAGGLUTININ Purification step
Titer of 1 ~ solution of lyophilized preparation*
Aqueous extraction (N 1-14)2SO4fractionation Chromatography on Sephadex G-150
4 32 512
Yield (g/kg of seeds)
4 0.20-0.25
* Hemagglutinating activity assayed against AI erythrocytes.
175 acetic acid. As this fraction was denaturated by the action of acetic acid, the hemagglutinating activity could not be examined. Our main interest was concerned with the properties of the phytohemagglutinin which was not adsorbed to the Sephadex gel, but was eluted with 0.15 M NaCI. The hemagglutinin preparation corresponding to Fraction lI (Fig. 2) proved to be homogeneous by polyacrylamide and starch gel electrophoresis both in alkaline and acidic buffers. The homogeneity of the hemagglutinin was confirmed also by gel filtration on a Bio-Gel P-100 column. The comparison of the electrophoretic behavior of the homogeneous hemagglutinin from small-seed lentil with that of a mixture of isophytohemagglutinins from large-seed lentil is shown on Fig. 3. It can be seen that the mobility of the studied hemagglutinin corresponds to the mobility of phytohemagglutinin II from the largeseed lentil, that forms a smaller part of the "native" phytohemagglutinin mixture.
1
2
3
4
Fig. 3. Polyacrylamide disc gel electrophoresis at pH 8.9 run at 4 mA per tube for 1 h. 1. Small-seed lentil hemagglutinin. 2. Mixture of isophytohemagglutinins from large-seed lentil. 3. Phytohemagglutinin I from large-seed lentil. 4. Phytohemagglutinin II from large-seed lentil. Phytohemagglutinin preparations, which were not adsorbed to the Sephadex gel, obtained from all 3 cultivars of small-seed lentil were homogeneous and their mobilities were identical. The protein fraction which was adsorbed to the Sephadex matrix (Fraction III, Fig. 2) and was eluted with D-glucose solution was electrophoretically indistinguishable from that which was not adsorbed. Both isophytohemagglutinins isolated from large-seed lentil were shown to be composed of two types of subunits differing in their molecular weights [22]. Polyacrylamide gel electrophoresis in dodecylsulfate medium showed that the hemagglutinin from small-seed lentil is composed also from two types of subunits (Fig. 4). The approximate molecular weights calculated by comparing mobilities of the subunits with those of known proteins were the same for both the large- and small-seed
176
Fig. 4. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Gels containing 5 % acrylamide, 0.13 ~ N,N'-methylene bisacrylamide and 0.1% sodium dodecylsulfate were used; protein samples were preincubated in a mixture containing 4 M urea, 1% mercaptoethanol, and 1% sodium dodecylsulfate for 45 rain at 45 °C, Electrophoresis was carried out at 7 mA per gel for 2 h, using 0.1 M phosphate buffer, pH 7.2, containing 0.1% sodium dodecylsulfate. 1. Small-seed lentil hemagglutinin eluted from Sephadex with 0.15 M NaCI (Fraction II, Fig. 2). 2. Protein Fraction III (Fig. 2) eluted from Sephadex with 0.1 M o-glucose solution. 3. Protein Fraction 1V (Fig. 2) eluted from Sephadex with 1 M acetic acid. 4. Phytohemagglutinin isolated from large-seed lentil. lentil hemagglutinins: 8000000and 18 0130. The electrophoretic behavior in the presence of dodecylsulfate of the hemagglutinin from small-seed lentil which was not adsorbed to Sephadex did not differ f r o m that of the hemagglutinin adsorbed to the dextran gel. The same electrophoretic pattern was given by Fraction IV (Fig. 2), eluted from Sephadex with 1 M acetic acid. Phytohemagglutinins from large-seed and small-seed lentils differ in their behavior on starch gel electrophoresis. The mobility of the hemagglutinin from smallseed lentil was significantly higher than that of large-seed lentil. An addition of omannose to the hemagglutinin from small-seed lentil affected its electrophoretic mobility significantly less than the mobility of the hemagglutinin from large-seed lentil (Fig. 5).
UltracentriJugal analysis Phytohemagglutinin yields a single symmetrical peak on ultracentrifugation. The sedimentation velocity data allow to calculate the s~0.w of 3.9 S. From the sedimentation equilibrium measurements, the molecular weight of the phytohemagglutinin was calculated as 53 300 ¢±25000).
177
4
Fig. 5. Vertical starch gel electrophoresis of hemagglutinins from large-seed and small-seed lentil and the effect of D-mannose on their mobility. Gels were prepared from Connaught starch in acetate buffer, pH 5.0, and run at 170 V/40 mA for 4.0 h. 1. Small-seed lentil hemagglutinin in acetate buffer containing 2 ~ D-mannose. 2. Small-seed lentil hemagglutinin in acetate buffer. 3. Large-seed lentil hemagglutinin in acetate buffer. 4. Large-seed lentil hemagglutinin in acetate buffer containing 2 ~ D-mannose. The top sharp lines are origins of electrophoresis.
Am&o acid composition and carbohydrate content The a m i n o acid c o m p o s i t i o n of the p h y t o h e m a g g l u t i n i n is presented in Table I1. It is similar to that of the h e m a g g l u t i n i n from large-seed lentil [2, 22]. A significant difference can be f o u n d in the tyrosine content. The hemagglutinin from small-seed lentil contains almost 4 times less of tyrosine t h a n the hemagglutinin from largeseed lentil. The dansylation method shows that the hemagglutinin isolated from small-
TABLE 1I AMINO ACID COMPOSITION OF SMALL-SEED LENTIL HEMAGGLUTININ Amino acid
Amino acid residues (tool per 53 300 g of protein)*
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine I/2 cystine Valine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
28.34 [28] 4.76 [5] 12.75 [I 3] 58.59 [59] 48.96 [49] 33.24 [33] 30.39 [30] 19.84 [20] 28.96 [29] 29.32 [29] 0.27** 40.40 [40] 19.02 [19] 18.62 [19] 3.45 [3] 25.38 [25] 8.87 [9]
* All values are corrected for a moisture content of 2.8 ~, numbers in parentheses indicate number of residues expressed in the nearest whole integer. ** Determination by the performic oxidation method [19].
178 seed lentil contains the same two N-terminal amino acids (valine and threonine), as the hemagglutinin of large-seed lentil [3]. The phytohemagglutinin isolated from small-seed lentil contains 1.2~ of neutral sugar and 0.32 ~o of glucosamine. Both values were lower in comparison with the hemagglutinin from large-seed lentil (for large-seed lentil 2.5~o of neutral sugar and 1.35 ~ of glucosamine). Metal content
From the metals examined" Mn, Zn, Fe, Cu, and Ca, only the presence of Ca, Mn, and Zn could be shown. Table IIi presents a comparison of metal content of the large- and small-seed lentil phytohemagglutinins. TABLE llI METAL CONTENT IN PHYTOHEMAGGLUT1NINS FROM SMALL-SEED AND LARGESEED LENTIL Source of phytohemagglutinin Lens esculenta subsp, microsperma subsp, macrosperma
Mn (%)
Ca (%)
(%)
0.19 0.21
0.44 0.34
0.015 0.006
Zn
H e m a g g l u t i n a t i n g activi O,
The hemagglutinin isolated from small-seed lentil exhibits different hemagglutinating activity towards human erythrocytes of different types, as can be seen in Table IV. Similar results were obtained with the hemagglutinins from all 3 cultivars of small-seed lentil examined. This finding differs from that one described by Howard and Sage [6], and Toyoshima et al. [4] and also from our previous results obtained for the hemagglutinin from large-seed lentil [5]. TABLE IV HEMAGGLUTINATING ACTIVITY OF PHYTOHEMAGGLUTININS FROM SMALLSEED AND LARGE-SEED LENTIL Source of phytohemagglutinin
Lens esculenta subsp, microsperma subsp, macrosperma
Titer of 1 ~ phytohemagglutinin At
A2
B
O
512
256
64
256
512
512
512
512
The hemagglutinating activity of the hemagglutinin from small-seed lentil was enhanced in the presence of Mn 2+, Ca 2+, and Mn 2+ + Ca 2+ approximately to the same degree as the activity of the hemagglutinin from large-seed lentil [23]. Similarly, the inhibition tests showed no difference between the two hemagglutinins (Table V).
179 TABLE V INHIBITION OF HEMAGGLUTINATION BY SUGARS Inhibitory activity of sugars is expressed in minimum amounts of sugar (mg/ml) necessary for inhibition of agglutination of O and A~ erythrocytes. Sugar
Methyl a-D-mannopyranoside Methyl a-D-glucopyranoside D-Mannose o-Glucose
Inhibition of phytohemagglutinin of Lens esculenta subsp, microsperma
subsp, macrosperma
0.68 1.25 1.25 2.5
0.68 1.25 1.25 2.5
DISCUSSION All lentil hemagglutinins hitherto described [3, 4, 6] were found to agglutinate equally erythrocytes of all types of the human ABO system. With exception of our papers [3, 5], the type of lentil used for the isolation has not been specified. The present paper shows that the hemagglutinin isolated from small-seed lentil (Lens esculenta, subsp, microsperma) differs in hemagglutinating activity in respect to different types of human erythrocytes. Similar results were obtained with hemagglutinin preparations isolated from all other small-seed lentil cultivars examined. It can be supposed that this property is one of the characteristics of phytohemagglutinins from the seeds of Lens esculenta, subsp, microsperma. In contradistinction to lentil hemagglutinins isolated previously, the hemagglutinin from small-seed lentil is not adsorbed to the Sephadex matrix; a weak interaction with the dextran causes only a retardation of the active protein on the column. This phenomenon is not a result of an insufficient saturation of the hemagglutinin with metals (Mn, Ca), as it was observed in the case of concanavalin A [24, 25]: the chromatography carried out on a Sephadex column equilibrated with Mn 2+ or Mn 2+ @ Ca z+ solution does not increase the amount of adsorbed hemagglutinin; moreover, the hemagglutinin which is not adsorbed to the dextran gel contains approximately the same amount of Ca z+ and Mn z+ as the hemagglutinin from largeseed lentil, which is adsorbed completely to Sephadex; similarly, the activation test of both hemagglutinins by metals does not reveal any difference. The observed behavior of the hemagglutinin from small-seed lentil on a Sephadex column is evidently not caused by exceeding the column capacity; in such a case, hemagglutinating activity would appear in the first fractions, together with inactive proteins. The decreased ability of the hemagglutinin from small-seed lentil to interact with polysaccharides containing D-glucopyranoside structures was confirmed also by its behavior in starch gel electrophoresis. The phytohemagglutinins from both types of lentil differ in their mobilities on starch gel in the absence and in the presence of an inhibiting sugar. The electrophoretic mobility of the phytohemagglutinin which interacts less with the starch gel was affected to a lower degree by the presence of an inhibiting sugar, than that one of the phytohemagglutinin which is characterized by a stronger interaction with the polysaccharide. The phenomenon of interaction
180 of a p h y t o h e m a g g l u t i n i n with the s u p p o r t i n g material in electrophoresis was described a l r e a d y in one of our previous c o m m u n i c a t i o n s [5] and could be utilized for analytical p u r p o s e s in the affinity electrophoresis of lectins [26]. In spite of the observed decreased ability of the hemagglutinin from smallseed lentil to interact with polysaccharides, no difference in inhibition by simple sugars and their glycosides was found, in c o m p a r i s o n with the hemagglutinin from large-seed lentil. This can be explained either by a low sensitivity o f the m e t h o d used for inhibition tests, or by the fact t h a t these two types of p h y t o h e m a g g l u t i n i n s differ in their interaction with m o r e complex sugar structures. Chemical c o m p o s i t i o n o f the hemagglutinin f r o m small-seed lentil was found to be similar to that o f the hemagglutinin from large-seed lentil; some difference, however, was revealed: the hemagglutinin from small-seed lentil contains lower a m o u n t s of glucosamine, neutral sugar, and especially, o f tyrosine. W h e t h e r these differences have s o m e t h i n g to do with a different a r r a n g e m e n t o f the binding site and are responsible for the decreased ability to interact with polysaccharides containing ~-D-glucopyranosyl units remains to be answered. O u r work in this direction is being continued. ACKNOWLEDGEMENT The a u t h o r s are indebted for the d e t e r m i n a t i o n o f metal content by a t o m i c a b s o r p t i o n spectroscopy to Dr M. Tich~, Institute o f Industrial Hygiene and Occ u p a t i o n a l Diseases, Praha.
REFERENCES 1 Landsteiner, K. and Raubitchek, H. (1908) Bakteriol. Zentr. 45, 660-671 2 Howard, 1. K., Sage, H. J., Stein, M. D., Young, N. M., Leon, M. A. and Dyckes, D. F. (197l) J. Biol. Chem. 246, 1590-1595 3 Tich;i, M., Entlicher, G., Kogti~, J. V. and Kocourek, J. (1970) Biochim. Biophys. Acta 221,282289 4 Toyoshima, S., Osawa, T. and Tonomura, A. (1970) Biochim. Biophys. Acta 221, 514-521 5 Entlicher, G., Tichfi, M., Kogti~, J. V. and Kocourek, J. (1969) Experientia 25, 17-19 6 Howard, 1. K. and Sage, H. J. (1969) Biochemistry 8, 2436-2441 7 Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 8 Steward, F. C., kyndon, R. F. and Barber, J. T. (1965) Am. J. Bot. 52, 155 164 9 Reisfeld, R. A., Lewis, U. J. and Williams, D. E. (1962) Nature 195, 281-283 10 Smithies, O. (1959) Biochem. J. 71, 585-587 I1 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 12 Dunker, A. K. and Rueckert, P. R. (1969) J. Biol. Chem. 244, 5074-5080 13 Maizel, T. V. (1966) Science 151,988 990 14 Abe, Y., Iwabuchi, M. and Ischii, S. (1971) Biochem. Biophys. Res. Commun. 45, 1271-1278 15 Payne, J. W. (1973) Bioehem. J. 135, 867-873 16 Yphantis, D. A. (1964) Biochemistry 3, 297-300 17 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. (1956) Anal. Chem. 28, 350-356 18 Spies, J. R. and Chambers, D. C. (1948) Anal. Chem. 20, 30-39 19 Moore, S. (1963) J. Biol. Chem. 238, 235-237 20 Zanetta, J. P., Vincendon, G., Mandell, P. and Gombos, O. (1970) J. Chromatogr. 51,441-453 21 Tobi~ka, J. (1964) Die Phyth~imagglutinine, H/imatologie und Bluttransfusionwessen, Vol. 3, pp. 169-176, Akademie Verlag, Berlin
181 22 Fliegerov~i,O., Salvetov~i, A., Tich~i, M. and Kocourek, J. (1974) Biochim. Biophys. Acta 351, 416-426 23 PaulovS., M., Tich~i, M., Entlicher, G., Ko~tiL J. V. and Kocourek, J. (1971) Biochim. Biophys. Acta 252, 388-395 24 Uchida, T. and Matsumoto, T. (1972) Biochim. Biophys. Acta 257, 230 234 25 Karlstam, B. (1973) Bicchim, Biophys. Acta 329, 295-304 26 Ho~ej~,i, V. and Kccourek, J. (1974) Biochim. 13iophys. Acta 336, 338-343
BBA 37047 STUDIES ON P H Y T O H E M A G G L U T I N 1 N S XXVI. A NEW TYPE OF A L E N T I L H E M A G G L U T I N I N ISOLATED FROM L E N S E S C U L E N T A MOENCH., SUBSP. M I C R O S P E R M A (BAUMG.) BARULINA
D. FIALOV,~, M. TICHA and J. KOCOUREK Department of Biochemistry, Charles University, Albertov 2030, Prague 2 (Czechoslovakia) (Received November 19th, 1974)
SUMMARY In seeds of the lentil Lens esculenta Moench., subsp, microsperma (Baumg.) Barulina, a nonspecific phytohemagglutinin was found, the properties of which differ from those of the previously described lentil hemagglutinins. The phytohemagglutinin is not adsorbed to the Sephadex matrix; its hemagglutinating activity is different towards erythrocytes of different human blood groups of the ABO system. The isolated phytohemagglutinin preparation is homogeneous by disc polyacrylamide and starch gel electrophoreses and by gel chromatography on Bio-Gel P-100. Ultracentrifugation of the phytohemagglutinin yields a single symmetrical peak with S~o.w of 3.9 S. From the sedimentation data, a molecular weight of 53 300 was calculated. Phytohemagglutinins of similar properties were shown to be present also in other cultivars of Lens esculenta, subsp, microsperma.
INTRODUCTION The presence of a nonspecific phytohemagglutinin in seeds of the lentil (Lens esculenta Moench. = Lens culinaris Med.) was first reported in 1908 by Landsteiner and Raubitschek [1]. Isolation and purification of lentil phytohemagglutinins and some of their properties were described independently by 3 groups of workers [2-4]. An exact specification of the variety or cultivar of the lentil seeds used for the isolation was described only by us in our previous communication [3]. In spite of some minor differences in physicochemical properties, very similar results were obtained by the 3 groups of workers: (i) the hemagglutinin from the lentil is bound to the Sephadex matrix and can be desorbed with D-glucose solution [3, 4, 6]; (ii) the hemagglutinin of the lentil agglutinates human red blood cells with equal activity irrespective to the blood type [4-6]; (iii) in the lentil seeds 2 isophytohemagglutinins are found [2, 3], their properties are very closely related [2]; Howard et al. [2] described that 6 different lentil "brands" tested varied in the proportion of the 2 isophytohemagglutinins. Our present paper describes the properties of a phytobemagglutinin isolated
171 from the lentil,Lens esculenta Moench., subsp, microsperma (Baumg.) Barulina, which manifests properties different from those (i-ill) of hitherto examined varities. Results of our present paper show the necessity and importance to identify the variety of the lentil seeds, which is employed as a source of phytohemagglutinin. MATERIALS AND METHODS Seeds of lentil, Lens esculenta Moench., subsp, microsperma (Baumg.) Barulina (in this paper called small-seed lentil), cv. SIovensk~ modrd were obtained from the Crop Breeding Station, Sl~idkovi6ovo-Nov2~ Dvor, cv. Moravskd drobnozrnn~ and cv. Trebigovsk~i - - from the Crop Breeding Station, Kagtice. Seeds of lentil Lens esculenta Moench., subsp, macrosperma (Baumg.) Barulina (in this paper called largeseed lentil), cv. Hrotovickd were provided by the Crop Breeding Station, Topolniky, Czechoslovakia. Phytohemagglutinin from the large-seed lentil cv. Hrotovick~ was prepared as described previously [3]. The two isophytohemagglutinins (phytohemagglutinin I and ll) were separated according to Howard et al. [2].
Extraction and purification Finely ground seeds (1 kg) were suspended in 10 I of deionized water; the pH was adjusted to 6.6 with 2 M NaOH. After continuous stirring for 1 h, the suspension was acidified with 2 M HCI to pH 4.5 and then left to settle at 4 °C for 20 h. The seed meal slurry together with precipitated nonactive proteins were removed by centrifugation and the supernatant was fractionated by salting out with solid (NH4)2SO4. "[he precipitate of the active protein obstained between 30 and 60 ~o saturation was dissolved in deionized water and dialyzed. The precipitate of inactive protein formed during dialysis was centrifuged off and the clear supernatant was freeze-dried. For preparative purposes, the active protein fraction (3 g) was dissolved in 0.15 M NaCI or deionized water (10 ml) and applied to a Sephadex G-150 column (3.5 cm × 60 cm) equilibrated with the same solvent. The column was first eluted either with water or 0.15 M NaC1 (see Fig. 2). A part of adsorbed proteins was released from the gel with 0.1 M D-glucose solution; at the end, the column was eluted with 1 M acetic acid; 5-ml fractions were collected at a flow rate of 10 ml/h. Tubes corresponding to Fractions II, Ill, and IV (see Fig. 2) were pooled. The protein Fraction lI, when the chromatography was carried out in water, and the Fraction IV were freeze-dried; the Fraction III was dialyzed against deionized water and then freeze-dried, as well as the Fraction II, when chromatography was done in 0.15 M NaCI. Small-scale experiments were carried out using a Sephadex G-150 column (1.5 c m x 30 cm) equilibrated either with 0.15 M NaCI, 0.15 M MnClz, or 0.075 M MnCI2 6- 0.075 M CaCI2. The active protein fraction (1 g) was dissolved in 3 ml of the solvent and applied to the column; 5-ml fractions were collected at a flow rate of 10 ml/h. Gel filtration on a Bio-Gel P-100 column (1 cm x 70 cm, 0.15 M NaCI, pH 7.0) was used to check homogeneity of the hemagglutinin preparation.
172
Electrophoretic methods Disc polyacrylamide gel electrophoresis was performed in an apparatus designed by Davis [7] in alkaline [8], or acidic [9] buffers. Vertical starch-gel electrophoresis was carried out according to the method of Smithies [10] in borate buffer, pH 8.6, or acetate buffer, pH 5.0. Polyacrylamide gel electrophoresis in dodecylsulfate medium was performed according to the procedure described by Weber and Osborn [11 ] in 5 ~ polyacrylamide gel. Molecular weights of the subunits were calculated from the comparison of their relative electrophoretic mobilities with those of known proteins [12, 13]: ovalbumin, subunits of concanavalin A [14], and oligomers of lysozyme [15]. UItracentrifugation analysis Measurements of the sedimentation velocity of the hemagglutinin were performed on a Spinco-Beckman Model E ultracentrifuge at a speed of 59 780 rev./min. Concentrations of the hemagglutinin tested were 0.2-0.9 % in 0.15 M NaCI. Sedimentation equilibrium studies were performed on the same ultracentrifuge at a speed of 20 410 rev./min, with a 0.03 % hemagglutinin solution in 0.15 M NaCI. The molecular weight was calculated from sedimentation equilibrium data according to the method of Yphantis [16] assuming a partial specific volume ~¢of 0.72. Carbohydrate analysis Total neutral sugar content was determined by the phenol/sulfuric acid method of Dubois et al. [17] with D-glucose as a standard. For amino sugar analysis, protein samples were hydrolyzed in sealed tubes at 110 °C with 4 M HCI for 14 and 20 h and analyzed on an automatic amino acid analyzer (Model AAA 881, Mikrotechna, Praha, Czechoslovakia). Metal content The metal content was determined by atomic absorption spectroscopy. A Varian Techtron Atomic Absorption spectrophotometer model AA4 equipped with hollow cathode lamps and ultraviolet sensitive HTVR-106 photomultiplier was used. Absorption was measured at the Ca 4226.7 A, Mn 2795.6 A, and Zn 2138.6 A analytical lines. Proiein and amino acid analyses Protein content in eluates was determined using absorption at 280 nm. For amino acid analysis, protein samples were hydrolyzed in sealed tubes under N2 at l l0 °C with 6 M HCI for 20 and 70 h and analyzed on an automatic amino acid analyzer (Model AAA 881, Mikrotechna). Values for serine and threonine were olztained by extrapolation to zero time. The highest values were taken for the remaining amino acids. The tryptophan content was determined independently [18]. Cystein was estimated as cysteic acid after oxidation of the sample by performic acid [19]. N-Terminal amino acids were identified by the dansylation technique according to Zanetta et al. [20] using thin-layer chromatography on silica gel (Silufol, Kavalier) for identification of the dansyl derivatives.
173
Determination of hemagglutinating activity Hemagglutinating activity was determined at 20 °C by the method referred to by Tobigka [21]. To 0.2-ml aliquots obtained by 2-fold serial dilution of an 1 hemagglutinin solution, an equal volume of a 2~o suspension of red blood cells, 3 times washed with 0.15 M NaCI was added. After 15 rain tubes (0.6 cm × 8 cm), were examined for agglutination. To study the effect of metal ions on the hemagglutinating activity of phytohemagglutinin, a solution containing 0.1 M MnC12 + 0.05 M NaCI or 0.075 M MnCI2 + 0.075 M CaCIz were used. Inhibitory activity of the sugars was estimated using a hemagglutinin solution 8 times more concentrated than the solution allowing the first perceptible hemagglutination. To 0.I ml of aliquots obtained by 2-fold serial dilution of an 1 ~o sugar solution, 0.1 ml of the phytohemagglutinin solution was added. After 15 min, hemagglutination was estimated as described above [21]. RESULTS
Interaction of the phytohemagglutinin with Sephadex One of the methods described for the isolation of the lentil phytohemagglutinin was based on its specific adsorption on the Sephadex gel followed by an elution with D-glucose or acidic buffer solutions [3-6]. When this procedure was applied to the isolation of hemagglutinin from the small-seed lentil, a major part of the active protein did not bind to the Sephadex matrix. The protein possessing hemagglutinating activity was eluted with 0.15 M NaCI after inactive proteins. The following elution of the Sephadex column with o-glucose solution resulted in a release of a small amount of a protein with hemagglutinating activity (see Fig. la). Under the exactly same conditions, hemagglutinin from large-seed lentil was completely bound to the Sephadex column and could be released with D-glucose solution (Fig. l b). An analogous experiment was carried out with additional 2 cultivars of the
A 1.0
0.5
1.0
QSJ 100
200
300
400 V(ml)
Fig. I. Comparison of behavior of phytohemagglutinins from small-seed and large-seed lentil in gel chromatography on Sephadex G-150. 1 g of active protein fl'action obtained by (NH4)2SO4 precipitation was applied to a Sephadex G-150 column (1.5 cm × 30 cm) equilibrated with 0.15 M NaCI. a. Active protein fraction from small-seed lentil, b. Active protein fraction from large-seed lentil. Arrows indicate start of the elution with 0.1 M D-glucose solution; ÷ denotes fractions with hemagglutinating activity.
174 small-seed lentil and another cultivar of large-seed lentil. Results were similar to those depicted in Fig. la and lb, respectively. The chromatography on a Sephadex column equilibrated with 0.15 M MnCIz or 0.075 M MnCI2 + 0.075 M CaC12 did not increase the amount of phytohemagglutinin which was adsorbed to the Sephadex matrix; this treatment only slightly increased the elution volume needed for the elution of the active protein,
Isolation o['phytohemagglutinin and its electrophoretic characterization For the isolation and further characterization of the hemagglutinin from smallseed lentil, which was not adsorbed to a Sephadex column, gel chromatography was performed on a Sephadex G-150 column on a preparative scale. A typical separation of the phytohemagglutinin is shown in Fig. 2 and Table I.
I1
1.o ¸
L
580
1000 1'500
20'00 2500
30'00
V(ml)
Fig. 2. Chromatography of active protein fraction obtained by (NH4)2SO4 precipitation on Sephadex G-150. Arrows denote solvent changes: a. 0.15 M NaCl; b. 0.1 M D-glucose;c. 1 M acetic acid; (see text for details). The elution of the column with 0.15 M NaCI or deionized water yielded two peaks (designated Fractions 1 and II) of proteins: Fraction 1 was inactive, Fraction 11 possessed hemagglutinating activity. As dialysis of the hemagglutinin solution resulted in a considerable loss of protein, the chromatography in deionized water was prefered. The adsorbed part of the hemagglutinin was released by elution of the column with 0.1 M D-glucose solution (Fraction II1). After this elution, a small part of proteins still remained bound to the Sephadex column, which was eluted with 1 M TABLE1 PURIFICATION OF LENTIL HEMAGGLUTININ Purification step
Titer of 1 ~ solution of lyophilized preparation*
Aqueous extraction (N 1-14)2SO4fractionation Chromatography on Sephadex G-150
4 32 512
Yield (g/kg of seeds)
4 0.20-0.25
* Hemagglutinating activity assayed against AI erythrocytes.
175 acetic acid. As this fraction was denaturated by the action of acetic acid, the hemagglutinating activity could not be examined. Our main interest was concerned with the properties of the phytohemagglutinin which was not adsorbed to the Sephadex gel, but was eluted with 0.15 M NaCI. The hemagglutinin preparation corresponding to Fraction lI (Fig. 2) proved to be homogeneous by polyacrylamide and starch gel electrophoresis both in alkaline and acidic buffers. The homogeneity of the hemagglutinin was confirmed also by gel filtration on a Bio-Gel P-100 column. The comparison of the electrophoretic behavior of the homogeneous hemagglutinin from small-seed lentil with that of a mixture of isophytohemagglutinins from large-seed lentil is shown on Fig. 3. It can be seen that the mobility of the studied hemagglutinin corresponds to the mobility of phytohemagglutinin II from the largeseed lentil, that forms a smaller part of the "native" phytohemagglutinin mixture.
1
2
3
4
Fig. 3. Polyacrylamide disc gel electrophoresis at pH 8.9 run at 4 mA per tube for 1 h. 1. Small-seed lentil hemagglutinin. 2. Mixture of isophytohemagglutinins from large-seed lentil. 3. Phytohemagglutinin I from large-seed lentil. 4. Phytohemagglutinin II from large-seed lentil. Phytohemagglutinin preparations, which were not adsorbed to the Sephadex gel, obtained from all 3 cultivars of small-seed lentil were homogeneous and their mobilities were identical. The protein fraction which was adsorbed to the Sephadex matrix (Fraction III, Fig. 2) and was eluted with D-glucose solution was electrophoretically indistinguishable from that which was not adsorbed. Both isophytohemagglutinins isolated from large-seed lentil were shown to be composed of two types of subunits differing in their molecular weights [22]. Polyacrylamide gel electrophoresis in dodecylsulfate medium showed that the hemagglutinin from small-seed lentil is composed also from two types of subunits (Fig. 4). The approximate molecular weights calculated by comparing mobilities of the subunits with those of known proteins were the same for both the large- and small-seed
176
Fig. 4. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Gels containing 5 % acrylamide, 0.13 ~ N,N'-methylene bisacrylamide and 0.1% sodium dodecylsulfate were used; protein samples were preincubated in a mixture containing 4 M urea, 1% mercaptoethanol, and 1% sodium dodecylsulfate for 45 rain at 45 °C, Electrophoresis was carried out at 7 mA per gel for 2 h, using 0.1 M phosphate buffer, pH 7.2, containing 0.1% sodium dodecylsulfate. 1. Small-seed lentil hemagglutinin eluted from Sephadex with 0.15 M NaCI (Fraction II, Fig. 2). 2. Protein Fraction III (Fig. 2) eluted from Sephadex with 0.1 M o-glucose solution. 3. Protein Fraction 1V (Fig. 2) eluted from Sephadex with 1 M acetic acid. 4. Phytohemagglutinin isolated from large-seed lentil. lentil hemagglutinins: 8000000and 18 0130. The electrophoretic behavior in the presence of dodecylsulfate of the hemagglutinin from small-seed lentil which was not adsorbed to Sephadex did not differ f r o m that of the hemagglutinin adsorbed to the dextran gel. The same electrophoretic pattern was given by Fraction IV (Fig. 2), eluted from Sephadex with 1 M acetic acid. Phytohemagglutinins from large-seed and small-seed lentils differ in their behavior on starch gel electrophoresis. The mobility of the hemagglutinin from smallseed lentil was significantly higher than that of large-seed lentil. An addition of omannose to the hemagglutinin from small-seed lentil affected its electrophoretic mobility significantly less than the mobility of the hemagglutinin from large-seed lentil (Fig. 5).
UltracentriJugal analysis Phytohemagglutinin yields a single symmetrical peak on ultracentrifugation. The sedimentation velocity data allow to calculate the s~0.w of 3.9 S. From the sedimentation equilibrium measurements, the molecular weight of the phytohemagglutinin was calculated as 53 300 ¢±25000).
177
4
Fig. 5. Vertical starch gel electrophoresis of hemagglutinins from large-seed and small-seed lentil and the effect of D-mannose on their mobility. Gels were prepared from Connaught starch in acetate buffer, pH 5.0, and run at 170 V/40 mA for 4.0 h. 1. Small-seed lentil hemagglutinin in acetate buffer containing 2 ~ D-mannose. 2. Small-seed lentil hemagglutinin in acetate buffer. 3. Large-seed lentil hemagglutinin in acetate buffer. 4. Large-seed lentil hemagglutinin in acetate buffer containing 2 ~ D-mannose. The top sharp lines are origins of electrophoresis.
Am&o acid composition and carbohydrate content The a m i n o acid c o m p o s i t i o n of the p h y t o h e m a g g l u t i n i n is presented in Table I1. It is similar to that of the h e m a g g l u t i n i n from large-seed lentil [2, 22]. A significant difference can be f o u n d in the tyrosine content. The hemagglutinin from small-seed lentil contains almost 4 times less of tyrosine t h a n the hemagglutinin from largeseed lentil. The dansylation method shows that the hemagglutinin isolated from small-
TABLE 1I AMINO ACID COMPOSITION OF SMALL-SEED LENTIL HEMAGGLUTININ Amino acid
Amino acid residues (tool per 53 300 g of protein)*
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine I/2 cystine Valine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
28.34 [28] 4.76 [5] 12.75 [I 3] 58.59 [59] 48.96 [49] 33.24 [33] 30.39 [30] 19.84 [20] 28.96 [29] 29.32 [29] 0.27** 40.40 [40] 19.02 [19] 18.62 [19] 3.45 [3] 25.38 [25] 8.87 [9]
* All values are corrected for a moisture content of 2.8 ~, numbers in parentheses indicate number of residues expressed in the nearest whole integer. ** Determination by the performic oxidation method [19].
178 seed lentil contains the same two N-terminal amino acids (valine and threonine), as the hemagglutinin of large-seed lentil [3]. The phytohemagglutinin isolated from small-seed lentil contains 1.2~ of neutral sugar and 0.32 ~o of glucosamine. Both values were lower in comparison with the hemagglutinin from large-seed lentil (for large-seed lentil 2.5~o of neutral sugar and 1.35 ~ of glucosamine). Metal content
From the metals examined" Mn, Zn, Fe, Cu, and Ca, only the presence of Ca, Mn, and Zn could be shown. Table IIi presents a comparison of metal content of the large- and small-seed lentil phytohemagglutinins. TABLE llI METAL CONTENT IN PHYTOHEMAGGLUT1NINS FROM SMALL-SEED AND LARGESEED LENTIL Source of phytohemagglutinin Lens esculenta subsp, microsperma subsp, macrosperma
Mn (%)
Ca (%)
(%)
0.19 0.21
0.44 0.34
0.015 0.006
Zn
H e m a g g l u t i n a t i n g activi O,
The hemagglutinin isolated from small-seed lentil exhibits different hemagglutinating activity towards human erythrocytes of different types, as can be seen in Table IV. Similar results were obtained with the hemagglutinins from all 3 cultivars of small-seed lentil examined. This finding differs from that one described by Howard and Sage [6], and Toyoshima et al. [4] and also from our previous results obtained for the hemagglutinin from large-seed lentil [5]. TABLE IV HEMAGGLUTINATING ACTIVITY OF PHYTOHEMAGGLUTININS FROM SMALLSEED AND LARGE-SEED LENTIL Source of phytohemagglutinin
Lens esculenta subsp, microsperma subsp, macrosperma
Titer of 1 ~ phytohemagglutinin At
A2
B
O
512
256
64
256
512
512
512
512
The hemagglutinating activity of the hemagglutinin from small-seed lentil was enhanced in the presence of Mn 2+, Ca 2+, and Mn 2+ + Ca 2+ approximately to the same degree as the activity of the hemagglutinin from large-seed lentil [23]. Similarly, the inhibition tests showed no difference between the two hemagglutinins (Table V).
179 TABLE V INHIBITION OF HEMAGGLUTINATION BY SUGARS Inhibitory activity of sugars is expressed in minimum amounts of sugar (mg/ml) necessary for inhibition of agglutination of O and A~ erythrocytes. Sugar
Methyl a-D-mannopyranoside Methyl a-D-glucopyranoside D-Mannose o-Glucose
Inhibition of phytohemagglutinin of Lens esculenta subsp, microsperma
subsp, macrosperma
0.68 1.25 1.25 2.5
0.68 1.25 1.25 2.5
DISCUSSION All lentil hemagglutinins hitherto described [3, 4, 6] were found to agglutinate equally erythrocytes of all types of the human ABO system. With exception of our papers [3, 5], the type of lentil used for the isolation has not been specified. The present paper shows that the hemagglutinin isolated from small-seed lentil (Lens esculenta, subsp, microsperma) differs in hemagglutinating activity in respect to different types of human erythrocytes. Similar results were obtained with hemagglutinin preparations isolated from all other small-seed lentil cultivars examined. It can be supposed that this property is one of the characteristics of phytohemagglutinins from the seeds of Lens esculenta, subsp, microsperma. In contradistinction to lentil hemagglutinins isolated previously, the hemagglutinin from small-seed lentil is not adsorbed to the Sephadex matrix; a weak interaction with the dextran causes only a retardation of the active protein on the column. This phenomenon is not a result of an insufficient saturation of the hemagglutinin with metals (Mn, Ca), as it was observed in the case of concanavalin A [24, 25]: the chromatography carried out on a Sephadex column equilibrated with Mn 2+ or Mn 2+ @ Ca z+ solution does not increase the amount of adsorbed hemagglutinin; moreover, the hemagglutinin which is not adsorbed to the dextran gel contains approximately the same amount of Ca z+ and Mn z+ as the hemagglutinin from largeseed lentil, which is adsorbed completely to Sephadex; similarly, the activation test of both hemagglutinins by metals does not reveal any difference. The observed behavior of the hemagglutinin from small-seed lentil on a Sephadex column is evidently not caused by exceeding the column capacity; in such a case, hemagglutinating activity would appear in the first fractions, together with inactive proteins. The decreased ability of the hemagglutinin from small-seed lentil to interact with polysaccharides containing D-glucopyranoside structures was confirmed also by its behavior in starch gel electrophoresis. The phytohemagglutinins from both types of lentil differ in their mobilities on starch gel in the absence and in the presence of an inhibiting sugar. The electrophoretic mobility of the phytohemagglutinin which interacts less with the starch gel was affected to a lower degree by the presence of an inhibiting sugar, than that one of the phytohemagglutinin which is characterized by a stronger interaction with the polysaccharide. The phenomenon of interaction
180 of a p h y t o h e m a g g l u t i n i n with the s u p p o r t i n g material in electrophoresis was described a l r e a d y in one of our previous c o m m u n i c a t i o n s [5] and could be utilized for analytical p u r p o s e s in the affinity electrophoresis of lectins [26]. In spite of the observed decreased ability of the hemagglutinin from smallseed lentil to interact with polysaccharides, no difference in inhibition by simple sugars and their glycosides was found, in c o m p a r i s o n with the hemagglutinin from large-seed lentil. This can be explained either by a low sensitivity o f the m e t h o d used for inhibition tests, or by the fact t h a t these two types of p h y t o h e m a g g l u t i n i n s differ in their interaction with m o r e complex sugar structures. Chemical c o m p o s i t i o n o f the hemagglutinin f r o m small-seed lentil was found to be similar to that o f the hemagglutinin from large-seed lentil; some difference, however, was revealed: the hemagglutinin from small-seed lentil contains lower a m o u n t s of glucosamine, neutral sugar, and especially, o f tyrosine. W h e t h e r these differences have s o m e t h i n g to do with a different a r r a n g e m e n t o f the binding site and are responsible for the decreased ability to interact with polysaccharides containing ~-D-glucopyranosyl units remains to be answered. O u r work in this direction is being continued. ACKNOWLEDGEMENT The a u t h o r s are indebted for the d e t e r m i n a t i o n o f metal content by a t o m i c a b s o r p t i o n spectroscopy to Dr M. Tich~, Institute o f Industrial Hygiene and Occ u p a t i o n a l Diseases, Praha.
REFERENCES 1 Landsteiner, K. and Raubitchek, H. (1908) Bakteriol. Zentr. 45, 660-671 2 Howard, 1. K., Sage, H. J., Stein, M. D., Young, N. M., Leon, M. A. and Dyckes, D. F. (197l) J. Biol. Chem. 246, 1590-1595 3 Tich;i, M., Entlicher, G., Kogti~, J. V. and Kocourek, J. (1970) Biochim. Biophys. Acta 221,282289 4 Toyoshima, S., Osawa, T. and Tonomura, A. (1970) Biochim. Biophys. Acta 221, 514-521 5 Entlicher, G., Tichfi, M., Kogti~, J. V. and Kocourek, J. (1969) Experientia 25, 17-19 6 Howard, 1. K. and Sage, H. J. (1969) Biochemistry 8, 2436-2441 7 Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 8 Steward, F. C., kyndon, R. F. and Barber, J. T. (1965) Am. J. Bot. 52, 155 164 9 Reisfeld, R. A., Lewis, U. J. and Williams, D. E. (1962) Nature 195, 281-283 10 Smithies, O. (1959) Biochem. J. 71, 585-587 I1 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 12 Dunker, A. K. and Rueckert, P. R. (1969) J. Biol. Chem. 244, 5074-5080 13 Maizel, T. V. (1966) Science 151,988 990 14 Abe, Y., Iwabuchi, M. and Ischii, S. (1971) Biochem. Biophys. Res. Commun. 45, 1271-1278 15 Payne, J. W. (1973) Bioehem. J. 135, 867-873 16 Yphantis, D. A. (1964) Biochemistry 3, 297-300 17 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. (1956) Anal. Chem. 28, 350-356 18 Spies, J. R. and Chambers, D. C. (1948) Anal. Chem. 20, 30-39 19 Moore, S. (1963) J. Biol. Chem. 238, 235-237 20 Zanetta, J. P., Vincendon, G., Mandell, P. and Gombos, O. (1970) J. Chromatogr. 51,441-453 21 Tobi~ka, J. (1964) Die Phyth~imagglutinine, H/imatologie und Bluttransfusionwessen, Vol. 3, pp. 169-176, Akademie Verlag, Berlin
181 22 Fliegerov~i,O., Salvetov~i, A., Tich~i, M. and Kocourek, J. (1974) Biochim. Biophys. Acta 351, 416-426 23 PaulovS., M., Tich~i, M., Entlicher, G., Ko~tiL J. V. and Kocourek, J. (1971) Biochim. Biophys. Acta 252, 388-395 24 Uchida, T. and Matsumoto, T. (1972) Biochim. Biophys. Acta 257, 230 234 25 Karlstam, B. (1973) Bicchim, Biophys. Acta 329, 295-304 26 Ho~ej~,i, V. and Kccourek, J. (1974) Biochim. 13iophys. Acta 336, 338-343
