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[44] Soy bean (Glycine max) agglutinin
360
PURIFICATIONOF CARBOHYDRATE-BINDINGPROTEINS
[44]
protein at saturation. The equivalent weight of protein which binds 1 mole of L-fucose at saturation is therefore 1IN. The association constants, K, at 4 ° for binding of L-fucose by protein A is 0.9 to 1.2 × 104M -1, by protein B is 0.5 to 0.6 × 104M -1, and by protein C is 2.1 to 3.7 × 104M -1. The equivalent binding weights are: A, 2.8 to 3.1 × l0 w g; B, 2.9 to 3.1 × 104 g; and C, 3.0 to 3.2 × 104 g.
[44]
Soy Bean
(Glydne
max)
Agglutinin
B y HALINA LIS and NATHAN SHARON The ability of soybean (Glycine max) seeds to strongly agglutinate erythrocytes has been known since the beginning of the century. 1 The protein responsible for this hemagglutinating activity was first isolated in purified form and characterized by Liener and his co-workers, 2-4 who named it soyin and subsequently renamed it soybean hemagglutinin. 4 More recently, with the finding t h a t this protein also agglutinates cells other than erythrocytes, its name was changed to soybean agglutinin (SBA)2 SBA is one of many cell agglutinating proteins, or lectins, which are widely distributed in nature and which are finding increasing use in investigations of the structure of the cell surface and of carbohydrate-containing biopolymers2
Assay M e t h o d Principle. The activity of soybean agglutinin is assayed by measuring its ability to agglutinate trypsinized rabbit erythrocytes. This can be done by the conventional procedure of serial 2-fold dilution, with visual estimation of the degree of agglutination, either in test tubes 7 or with the T a k h s y microtitrator, s which provides a method for making rapid, accurate dilutions using minute quantities of material. However, the G. C. Toms and A. Western, in "Chemotaxonomy of the Leguminosae" (J. Itarborne, D. Boulter and B. L. Turner, eds.), p. 369. Academic Press, New York, 1971. I. E. Liener and M. J. Pallansch, J. Biol. Chem. 197, 29 (1952). I. E. Liener, J. Nutr. 49, 527 (1953). 4S. Wada, M. J. Pallansch, and I. E. Liener, J. Biol. Chem. 233, 395 (1958). ~H. Lis, B. A. Sela, L. Sachs, and N. Sharon, Biochim. Biophys. Acta 211, 582 (1970). 6N. Sharon and H. Lis, Science 177, 949 (1972). E. A. Kabat and M. M. Mayer, "Experimental Immunochemistry," 2rid ed., pp. 114-115. Thomas, Springfield, Illinois (1961). s j. L. Sever, J. Immunol. 88~ 320 (1962).
[44]
SOY BEAN AGGLUTININ
361
Visual method is not very precise and does not permit the detection of small differences in hemagglutinating activity. A quantitative procedure has been developed by Liener, 9 in which the degree of agglutination is evaluated photometrically, by measuring the absorbance of the layer of unsedimented erythrocytes. This method is described below.
Reagents Saline: 0.9% solution of NaC1 Phosphate-buffered saline (PBS) : 0.006M K Na phosphate buffer, pH 7.4, in saline Alsever's solution: 2.05 g of glucose, 0.8 g of sodium citrate, and 0.42 g of NaC1, dissolved in 100 ml of H..,O and brought to pH 6.1 by the addition of solid citric acid Anticoagulant: 8 g of sodium citrate, 54 ml of 37% formaldehyde, 100 ml saline Trypsin: 1% solution of Bacto-trypsin 1:250 in PBS Stock blood suspension: venous whole rabbit blood, added to an equal volume of Alsever's solution containing 1/30 volume of the anticoagulant. This suspension can be stored as long as 2 weeks at 4 °.
Preparation of Standard Trypsinized Erythrocyte Suspension. Trypsinized erythrocytes should be prepared on the day of the assay. Erythrocytes are collected from the stock blood suspension by centrifugation at room temperature in a clinical table centrifuge (2000 rpm, 5 minutes) and washed 3-4 times with saline (5 ml of saline for each milliliter of packed erthrocytes). The washed erythrocytes are added to PBS (about 4 ml of cells per 100 ml of PBS) to give a suspension with an absorbance of 2 at 620 nm. To 10 parts of this suspension is added 1 part of 1% trypsin solution, and the mixture is incubated at 37 ° for 1 hour. The trypsinized erythrocytes are then washed 4-5 times with saline as above to remove the last traces of trypsin and are finally suspended in sufficient saline to give a standard erythrocyte suspension with an adsorbanee of 1 at 620 nm (1.2-1.5 ml packed cells/100 ml). About 80 ml of standard erythrocyte suspension is obtained from 5 ml of stock blood suspension. Hemagglutination Assay. The material to bc tested is dissolved in saline. Serial 2-fold dilutions of the starting solution are made in a final volume of 1 ml of saline in 10 × 75 mm test tubes. To each tube is added 1 ml of the standard erythrocytc suspension, the contents of each tube are mixed by inversion and the tubes are placed in 9I. E. Liener, Arch. Biochem. Biophys. 54, 223 (1955).
362
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
[44]
a rack that holds them in an exactly vertical position. After 2.5 hours at room temperature, the tubes are read in the photometer, due care being taken not to agitate the contents. Each experiment should include a set of 2-4 control tubes, containing 1 ml of saline and 1 ml of standard blood suspension. Measurements o] Absorbance. All measurements of absorbance are performed at 620 nm in a Coleman Junior spectrophotometer, Model 6A, equipped with a special adapter to hold the 10 × 75 mm test tubes. The opening of the adapter should be masked with black plastic tape to leave an aperture of 1 cm 2 only, the center of the aperture being located 5 cm from the top of the adapter. The size and position of the aperture are so calculated that in the control tubes the absorbance of the erythrocyte suspension at the level of the aperture remains unchanged after 2.5 hours, thus eliminating the effect of the spontaneous sedimentation of the erythrocytes. Calculation o] Hemagglutinating Activity. One hemagglutinating unit (HU) is arbitrarily defined as that amount of material which is required to cause a decrease of 50% in the absorbance of the erthrocyte suspension in 2.5 hours under the conditions described above. The reciprocal of dilution (x) corresponding to one H U is calculated from the readings of the two tubes that have optical densities nearest to half the absorbance of the control (Eso ~ 0.25), one of the readings (EA) being lower and the other (EB) being higher than Eso. The following equation (where A is the reciprocal of dilution of the tube with EA) is then used: Eso - EA. log 2 logx = l o g A + E B B - - E A
The specific hemagglutinating activity of the material tested, H U / m g protein, is calculated from the value of x and from the concentration in the starting solution. The method is highly reproducible (within ___5%) when the assay is done with the same preparation of standard erythrocyte suspension. With different preparations of erythrocytes, the variations can be higher. It is therefore advisable to include in each assay of activity a sample of SBA with known specific activity, for comparison. Purification of Soybean Agglutinin-Method I Soybean meal contains four closely related agglutinins (isolectins), one major and three minor ones. 1°,11 The procedure described below is I°H. Lis, C. Fridman, N. Sharon, and E. Katchalski, Arch. Biochem. Biophys. 117, 301 (1966). 1~N. Catsimpoolas and E. W. Meyer, Arch. Biochem. Biophys. 132, 279 (1969).
[44]
SOY BF.J~N AGGLUTININ
363
for the purification of the m a j o r component and is based on the method described by Liener 3 and modified by us. 1~ I s o l a t i o n o1 Crude S B A . Five hundred grains of untoasted defatted soybean meal 13 are suspended in 6 liters of distilled water at room temperature and extracted for 1 hour with constant stirring. The suspension is acidified to p H 4.6 with concentrated HC1 (18 ml) and allowed to settle overnight at 4 °. Most of the clear yellow supernatant fluid is siphoned off, and the remainder is collected by centrifugation (10 minutes at 6000 r p m in a Sorval R C 2 centrifuge). To each liter of supernatant, 300 g of (NH~)2S04 are gradually added while stirring; the precipitate is removed by filtration TM and discarded. To each liter of the supernatant is added 270 g of (NH4)2SO~ while stirring. After allowing the precipitate to settle overnight at 4 °, most of the supernatant is siphoned off and the precipitate is collected by centrifugation (10 minutes, 5000 rpm). I t is suspended in 150-200 ml of water, and the suspension is dialyzed against distilled water for 24 hours in the cold room with two changes of water. Any insoluble material which is present after dialysis is removed by centrifugation and discarded. The p H of the solution is adjusted to 4.6 with 1 N HC1. After addition of ( N H 4 ) : S 0 4 (56 g/100 ml of solution), the precipitate is collected by centrifugation (10 minutes, 5000 rpm) and dissolved in 50 ml of 0.05 M phosphate buffer, p H 6.1. The above solution is dialyzed against 60% ethanol at - 1 5 ° for 48 hours. The precipitate which forms inside the dialysis bag is collected by centrifugation in the cold~; it is suspended in approximately 25 ml of distilled water and dialyzed overnight against a large volume of distilled water in the cold. After removal by centrifugation of any insoluble material, the solution is lyophilized to give 700-800 mg of crude SBA. C h r o m a t o g r a p h y on C a l c i u m P h o s p h a t e . A column (2.8 × 15 cm) 12H. Lis, N. Sharon, and E. Katchalski, J. Biol. Chem. 241, 684 (1966). ~3We now use meal kindly supplied by Etz Hazait factory, Petah Tikva, Israel. The meal is taken out from the processing line after extraction of the oil with hexane. Removal of solvent is carried out in the laboratory by drying in a hood at room temperature. 14Sometimes difficulty is encountered at this stage: upon addition of (NH4)~SO, very fine suspension may be formed, which sediments only after prolonged high speed centrifugation. This occurred, for example, in our laboratory when certain batches of Soyafluff 20 were used as starting material for the isolation of SBA The reason for this is not clear, but may in part be the result of the industrial procedures used for the production of the meal, the exact details of which are not ahvays available. In such cases, it is advisable to try other sources of untoasted meal. ~ The supernatant is enriched with respect to the minor soybean agglutinins. They can be obtained from this supernatant in purified form as described by Lis et al.~°
364
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
[44]
of calcium phosphate (hydroxylapatite), prepared according to Tiselius et al., 16,17 is equilibrated with 0.001 M phosphate buffer, pH 6.8, at room temperature. Crude SBA (ca. 750 mg) is dissolved in 35 ml of the same buffer and applied to the column. The column is washed with 200 ml of the 0.001 M buffer, followed by 0.072 M buffer. Elution with this buffer continues until no material absorbing at 280 nm is detected in the effluent (about 500 ml). The purified agglutinin is then eluted from the column with 0.2M phosphate buffer, pH 6.8. Fractions of 10 ml are now collected at a flow rate of 40 ml/hour. The fractions are analyzed for protein by measuring the absorbance at 280 mn and are assayed for hemagglutinating activity. The fractions containing the activity (tubes 9-15) are pooled, dialyzed against distilled water at 4 ° and lyophilized, to give 100-150 mg of purified SBA with a specific activity of 5000-6000 units per milligram of protein. At this stage the product is sufficiently pure for most purposes. However, it still contains trace amounts of the three other agglutinins, which can be removed by chromatography on DEAE-cellulose. C h r o m a t o g r a p h y on D E A E - C e l l u l o s e . SBA (50 mg) is dissolved in 10 ml of 0.01 M phosphate buffer, pH 6.8 (starting buffer), and applied to a column (1.2 × 40 cm) of DEAE-cellulose in equilibrium with the same buffer. The column is washed with 200 ml of starting buffer, whereupon the three minor agglutinins are eluted. The column is then connected to a closed mixing chamber containing 200 ml of starting buffer, and elution of the main agglutinin is performed by passing into the mixing chamber 0.4 M NaC1 in starting buffer. Five milliliter fractions are collected at a rate of 25-30 ml per hour, monitored at 280 nm and assayed for hemagglutinating activity. The fractions containing activity (tubes 10-25 after application of the gradient) are pooled, dialyzed and lyophilized. Yield 35 mg; specific activity 5000-6000 HU/mg.
Properties Chemical Properties. ~2 SBA is a glycoprotein with a molecular weight
of about 110,000. It contains 4.5% of neutral sugars and about 1% of amino sugars. The neutral sugar has been identified as D-mannose and the amino sugar as N-acetyl-D-glucosamine. The amino acid composition of SBA is characterized by its relatively high content of aspartic acid, serine, and threonine, and by the absence of cysteine and the very low content of methionine. SBA is stable in dry form at room temperature and in solution in frozen form. I'A. Tiselius, S. H]ert~n, and G. Levin, Arch. Biochem. Biophys. 65, 132 (1956). IT0. Levin, see Vol. 5 [2].
[44]
soy BEAN AGGLUTININ
365
Biological Properties. SBA agglutinates different types of cell; however, the concentrations of SBA required for the agglutination v a r y markedly with the type of cell2 Thus, rabbit erythrocytes which have been treated with trypsin are highly sensitive, requiring only 0.1-0.2 t~g of SBA per milliliter for detectable agglutination. These cells are used therefore for the routine assay of SBA. Untrypsinized rabbit erythrocytes are much less sensitive, the concentration of SBA required for their agglutination being 100-200-fold higher than that for the trypsinized cells. SBA agglutinates human erythrocytes of all types (A > 0 > B), although the concentrations required for their agglutination are rather high (0.2-1 mg/ml). In this case, too, the susceptibility to agglutination is increased about 100-fold by trypsinization. In addition to erythrocytes, SBA also agglutinates somatic cells, grown in culture, which have been transformed by viral or chemical carcinogens or by irradiation; the untransformed parent cells are not agglutinated under the same conditions, unless they have been treated with trypsin, is In all cases, the agglutination is specifically inhibited by N-acetyl-D-galactosamine and to a lesser extent by D-galactose ~,ls indicating that N-acetyl-D-galactosamine-like residues are present on cell surfaces. SBA can therefore be used for the detection and quantitation of such residues ~9 and for the study of the changes that cell surfaces undergo upon malignant transformation. The toxicity of SBA has been studied under a variety of conditionsY ,3,2° The LDso of SBA administered intraperitoneally to young rats is about 50 mg/kg. When administered to rats by stomach tube no lethal effect was observed up to a level of 500 mg/kg.
ADDENDUM M e t h o d II: Purification of S B A by Affinity Chromatography 21 B y JVLIUS A. GORDON, SHMARYAHU BLUMBERG,HALINA LIS, and NATHAN SHARON Principle. The four soybean agglutinins are adsorbed from solution to a column made of D-galactose covalently attached to Sepharose. The
~B. A. Sela, H. Lis, N. Sharon, and L. Sachs, J. Membrane Biol. 3, 267 (1970). 1'B. A. Sela, It. Lis, N. Sharon, and L. Sachs, Biochim. Biophys. Acta 249, 564 (1971). 2oI. E. Liener and J. E. Rose, Proc. Soc. Exp. Biol. Med. 83, 539 (1953). 21j. A. Gordon, S. Blumberg, H. Lis, and N. Sharon, FEBS Letters, in press (1972).
PURIFICATIONOF CARBOHYDRATE-BINDINGPROTEINS
[44]
protein at saturation. The equivalent weight of protein which binds 1 mole of L-fucose at saturation is therefore 1IN. The association constants, K, at 4 ° for binding of L-fucose by protein A is 0.9 to 1.2 × 104M -1, by protein B is 0.5 to 0.6 × 104M -1, and by protein C is 2.1 to 3.7 × 104M -1. The equivalent binding weights are: A, 2.8 to 3.1 × l0 w g; B, 2.9 to 3.1 × 104 g; and C, 3.0 to 3.2 × 104 g.
[44]
Soy Bean
(Glydne
max)
Agglutinin
B y HALINA LIS and NATHAN SHARON The ability of soybean (Glycine max) seeds to strongly agglutinate erythrocytes has been known since the beginning of the century. 1 The protein responsible for this hemagglutinating activity was first isolated in purified form and characterized by Liener and his co-workers, 2-4 who named it soyin and subsequently renamed it soybean hemagglutinin. 4 More recently, with the finding t h a t this protein also agglutinates cells other than erythrocytes, its name was changed to soybean agglutinin (SBA)2 SBA is one of many cell agglutinating proteins, or lectins, which are widely distributed in nature and which are finding increasing use in investigations of the structure of the cell surface and of carbohydrate-containing biopolymers2
Assay M e t h o d Principle. The activity of soybean agglutinin is assayed by measuring its ability to agglutinate trypsinized rabbit erythrocytes. This can be done by the conventional procedure of serial 2-fold dilution, with visual estimation of the degree of agglutination, either in test tubes 7 or with the T a k h s y microtitrator, s which provides a method for making rapid, accurate dilutions using minute quantities of material. However, the G. C. Toms and A. Western, in "Chemotaxonomy of the Leguminosae" (J. Itarborne, D. Boulter and B. L. Turner, eds.), p. 369. Academic Press, New York, 1971. I. E. Liener and M. J. Pallansch, J. Biol. Chem. 197, 29 (1952). I. E. Liener, J. Nutr. 49, 527 (1953). 4S. Wada, M. J. Pallansch, and I. E. Liener, J. Biol. Chem. 233, 395 (1958). ~H. Lis, B. A. Sela, L. Sachs, and N. Sharon, Biochim. Biophys. Acta 211, 582 (1970). 6N. Sharon and H. Lis, Science 177, 949 (1972). E. A. Kabat and M. M. Mayer, "Experimental Immunochemistry," 2rid ed., pp. 114-115. Thomas, Springfield, Illinois (1961). s j. L. Sever, J. Immunol. 88~ 320 (1962).
[44]
SOY BEAN AGGLUTININ
361
Visual method is not very precise and does not permit the detection of small differences in hemagglutinating activity. A quantitative procedure has been developed by Liener, 9 in which the degree of agglutination is evaluated photometrically, by measuring the absorbance of the layer of unsedimented erythrocytes. This method is described below.
Reagents Saline: 0.9% solution of NaC1 Phosphate-buffered saline (PBS) : 0.006M K Na phosphate buffer, pH 7.4, in saline Alsever's solution: 2.05 g of glucose, 0.8 g of sodium citrate, and 0.42 g of NaC1, dissolved in 100 ml of H..,O and brought to pH 6.1 by the addition of solid citric acid Anticoagulant: 8 g of sodium citrate, 54 ml of 37% formaldehyde, 100 ml saline Trypsin: 1% solution of Bacto-trypsin 1:250 in PBS Stock blood suspension: venous whole rabbit blood, added to an equal volume of Alsever's solution containing 1/30 volume of the anticoagulant. This suspension can be stored as long as 2 weeks at 4 °.
Preparation of Standard Trypsinized Erythrocyte Suspension. Trypsinized erythrocytes should be prepared on the day of the assay. Erythrocytes are collected from the stock blood suspension by centrifugation at room temperature in a clinical table centrifuge (2000 rpm, 5 minutes) and washed 3-4 times with saline (5 ml of saline for each milliliter of packed erthrocytes). The washed erythrocytes are added to PBS (about 4 ml of cells per 100 ml of PBS) to give a suspension with an absorbance of 2 at 620 nm. To 10 parts of this suspension is added 1 part of 1% trypsin solution, and the mixture is incubated at 37 ° for 1 hour. The trypsinized erythrocytes are then washed 4-5 times with saline as above to remove the last traces of trypsin and are finally suspended in sufficient saline to give a standard erythrocyte suspension with an adsorbanee of 1 at 620 nm (1.2-1.5 ml packed cells/100 ml). About 80 ml of standard erythrocyte suspension is obtained from 5 ml of stock blood suspension. Hemagglutination Assay. The material to bc tested is dissolved in saline. Serial 2-fold dilutions of the starting solution are made in a final volume of 1 ml of saline in 10 × 75 mm test tubes. To each tube is added 1 ml of the standard erythrocytc suspension, the contents of each tube are mixed by inversion and the tubes are placed in 9I. E. Liener, Arch. Biochem. Biophys. 54, 223 (1955).
362
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
[44]
a rack that holds them in an exactly vertical position. After 2.5 hours at room temperature, the tubes are read in the photometer, due care being taken not to agitate the contents. Each experiment should include a set of 2-4 control tubes, containing 1 ml of saline and 1 ml of standard blood suspension. Measurements o] Absorbance. All measurements of absorbance are performed at 620 nm in a Coleman Junior spectrophotometer, Model 6A, equipped with a special adapter to hold the 10 × 75 mm test tubes. The opening of the adapter should be masked with black plastic tape to leave an aperture of 1 cm 2 only, the center of the aperture being located 5 cm from the top of the adapter. The size and position of the aperture are so calculated that in the control tubes the absorbance of the erythrocyte suspension at the level of the aperture remains unchanged after 2.5 hours, thus eliminating the effect of the spontaneous sedimentation of the erythrocytes. Calculation o] Hemagglutinating Activity. One hemagglutinating unit (HU) is arbitrarily defined as that amount of material which is required to cause a decrease of 50% in the absorbance of the erthrocyte suspension in 2.5 hours under the conditions described above. The reciprocal of dilution (x) corresponding to one H U is calculated from the readings of the two tubes that have optical densities nearest to half the absorbance of the control (Eso ~ 0.25), one of the readings (EA) being lower and the other (EB) being higher than Eso. The following equation (where A is the reciprocal of dilution of the tube with EA) is then used: Eso - EA. log 2 logx = l o g A + E B B - - E A
The specific hemagglutinating activity of the material tested, H U / m g protein, is calculated from the value of x and from the concentration in the starting solution. The method is highly reproducible (within ___5%) when the assay is done with the same preparation of standard erythrocyte suspension. With different preparations of erythrocytes, the variations can be higher. It is therefore advisable to include in each assay of activity a sample of SBA with known specific activity, for comparison. Purification of Soybean Agglutinin-Method I Soybean meal contains four closely related agglutinins (isolectins), one major and three minor ones. 1°,11 The procedure described below is I°H. Lis, C. Fridman, N. Sharon, and E. Katchalski, Arch. Biochem. Biophys. 117, 301 (1966). 1~N. Catsimpoolas and E. W. Meyer, Arch. Biochem. Biophys. 132, 279 (1969).
[44]
SOY BF.J~N AGGLUTININ
363
for the purification of the m a j o r component and is based on the method described by Liener 3 and modified by us. 1~ I s o l a t i o n o1 Crude S B A . Five hundred grains of untoasted defatted soybean meal 13 are suspended in 6 liters of distilled water at room temperature and extracted for 1 hour with constant stirring. The suspension is acidified to p H 4.6 with concentrated HC1 (18 ml) and allowed to settle overnight at 4 °. Most of the clear yellow supernatant fluid is siphoned off, and the remainder is collected by centrifugation (10 minutes at 6000 r p m in a Sorval R C 2 centrifuge). To each liter of supernatant, 300 g of (NH~)2S04 are gradually added while stirring; the precipitate is removed by filtration TM and discarded. To each liter of the supernatant is added 270 g of (NH4)2SO~ while stirring. After allowing the precipitate to settle overnight at 4 °, most of the supernatant is siphoned off and the precipitate is collected by centrifugation (10 minutes, 5000 rpm). I t is suspended in 150-200 ml of water, and the suspension is dialyzed against distilled water for 24 hours in the cold room with two changes of water. Any insoluble material which is present after dialysis is removed by centrifugation and discarded. The p H of the solution is adjusted to 4.6 with 1 N HC1. After addition of ( N H 4 ) : S 0 4 (56 g/100 ml of solution), the precipitate is collected by centrifugation (10 minutes, 5000 rpm) and dissolved in 50 ml of 0.05 M phosphate buffer, p H 6.1. The above solution is dialyzed against 60% ethanol at - 1 5 ° for 48 hours. The precipitate which forms inside the dialysis bag is collected by centrifugation in the cold~; it is suspended in approximately 25 ml of distilled water and dialyzed overnight against a large volume of distilled water in the cold. After removal by centrifugation of any insoluble material, the solution is lyophilized to give 700-800 mg of crude SBA. C h r o m a t o g r a p h y on C a l c i u m P h o s p h a t e . A column (2.8 × 15 cm) 12H. Lis, N. Sharon, and E. Katchalski, J. Biol. Chem. 241, 684 (1966). ~3We now use meal kindly supplied by Etz Hazait factory, Petah Tikva, Israel. The meal is taken out from the processing line after extraction of the oil with hexane. Removal of solvent is carried out in the laboratory by drying in a hood at room temperature. 14Sometimes difficulty is encountered at this stage: upon addition of (NH4)~SO, very fine suspension may be formed, which sediments only after prolonged high speed centrifugation. This occurred, for example, in our laboratory when certain batches of Soyafluff 20 were used as starting material for the isolation of SBA The reason for this is not clear, but may in part be the result of the industrial procedures used for the production of the meal, the exact details of which are not ahvays available. In such cases, it is advisable to try other sources of untoasted meal. ~ The supernatant is enriched with respect to the minor soybean agglutinins. They can be obtained from this supernatant in purified form as described by Lis et al.~°
364
PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS
[44]
of calcium phosphate (hydroxylapatite), prepared according to Tiselius et al., 16,17 is equilibrated with 0.001 M phosphate buffer, pH 6.8, at room temperature. Crude SBA (ca. 750 mg) is dissolved in 35 ml of the same buffer and applied to the column. The column is washed with 200 ml of the 0.001 M buffer, followed by 0.072 M buffer. Elution with this buffer continues until no material absorbing at 280 nm is detected in the effluent (about 500 ml). The purified agglutinin is then eluted from the column with 0.2M phosphate buffer, pH 6.8. Fractions of 10 ml are now collected at a flow rate of 40 ml/hour. The fractions are analyzed for protein by measuring the absorbance at 280 mn and are assayed for hemagglutinating activity. The fractions containing the activity (tubes 9-15) are pooled, dialyzed against distilled water at 4 ° and lyophilized, to give 100-150 mg of purified SBA with a specific activity of 5000-6000 units per milligram of protein. At this stage the product is sufficiently pure for most purposes. However, it still contains trace amounts of the three other agglutinins, which can be removed by chromatography on DEAE-cellulose. C h r o m a t o g r a p h y on D E A E - C e l l u l o s e . SBA (50 mg) is dissolved in 10 ml of 0.01 M phosphate buffer, pH 6.8 (starting buffer), and applied to a column (1.2 × 40 cm) of DEAE-cellulose in equilibrium with the same buffer. The column is washed with 200 ml of starting buffer, whereupon the three minor agglutinins are eluted. The column is then connected to a closed mixing chamber containing 200 ml of starting buffer, and elution of the main agglutinin is performed by passing into the mixing chamber 0.4 M NaC1 in starting buffer. Five milliliter fractions are collected at a rate of 25-30 ml per hour, monitored at 280 nm and assayed for hemagglutinating activity. The fractions containing activity (tubes 10-25 after application of the gradient) are pooled, dialyzed and lyophilized. Yield 35 mg; specific activity 5000-6000 HU/mg.
Properties Chemical Properties. ~2 SBA is a glycoprotein with a molecular weight
of about 110,000. It contains 4.5% of neutral sugars and about 1% of amino sugars. The neutral sugar has been identified as D-mannose and the amino sugar as N-acetyl-D-glucosamine. The amino acid composition of SBA is characterized by its relatively high content of aspartic acid, serine, and threonine, and by the absence of cysteine and the very low content of methionine. SBA is stable in dry form at room temperature and in solution in frozen form. I'A. Tiselius, S. H]ert~n, and G. Levin, Arch. Biochem. Biophys. 65, 132 (1956). IT0. Levin, see Vol. 5 [2].
[44]
soy BEAN AGGLUTININ
365
Biological Properties. SBA agglutinates different types of cell; however, the concentrations of SBA required for the agglutination v a r y markedly with the type of cell2 Thus, rabbit erythrocytes which have been treated with trypsin are highly sensitive, requiring only 0.1-0.2 t~g of SBA per milliliter for detectable agglutination. These cells are used therefore for the routine assay of SBA. Untrypsinized rabbit erythrocytes are much less sensitive, the concentration of SBA required for their agglutination being 100-200-fold higher than that for the trypsinized cells. SBA agglutinates human erythrocytes of all types (A > 0 > B), although the concentrations required for their agglutination are rather high (0.2-1 mg/ml). In this case, too, the susceptibility to agglutination is increased about 100-fold by trypsinization. In addition to erythrocytes, SBA also agglutinates somatic cells, grown in culture, which have been transformed by viral or chemical carcinogens or by irradiation; the untransformed parent cells are not agglutinated under the same conditions, unless they have been treated with trypsin, is In all cases, the agglutination is specifically inhibited by N-acetyl-D-galactosamine and to a lesser extent by D-galactose ~,ls indicating that N-acetyl-D-galactosamine-like residues are present on cell surfaces. SBA can therefore be used for the detection and quantitation of such residues ~9 and for the study of the changes that cell surfaces undergo upon malignant transformation. The toxicity of SBA has been studied under a variety of conditionsY ,3,2° The LDso of SBA administered intraperitoneally to young rats is about 50 mg/kg. When administered to rats by stomach tube no lethal effect was observed up to a level of 500 mg/kg.
ADDENDUM M e t h o d II: Purification of S B A by Affinity Chromatography 21 B y JVLIUS A. GORDON, SHMARYAHU BLUMBERG,HALINA LIS, and NATHAN SHARON Principle. The four soybean agglutinins are adsorbed from solution to a column made of D-galactose covalently attached to Sepharose. The
~B. A. Sela, H. Lis, N. Sharon, and L. Sachs, J. Membrane Biol. 3, 267 (1970). 1'B. A. Sela, It. Lis, N. Sharon, and L. Sachs, Biochim. Biophys. Acta 249, 564 (1971). 2oI. E. Liener and J. E. Rose, Proc. Soc. Exp. Biol. Med. 83, 539 (1953). 21j. A. Gordon, S. Blumberg, H. Lis, and N. Sharon, FEBS Letters, in press (1972).