Eur. J. Biochem. 77, 419-426 (1977)
a-D-Galactosidase from Soybeans Destroying Blood-Group B Antigens Purification by Affinity Chromatography and Properties Noam HARPAZ, Harold M. FLOWERS, and Nathan SHARON Department of Biophysics, The Weizmann Institute of Science, Rehovot (Received March 24, 1977)
a-D-Galactosidase was isolated from untoasted soybean meal and purified to homogeneity by affinity chromatography on N-eaminoacaproyl a-D-galactopyranosylamine-Sepharose. The purified enzyme destroyed the B-specificity of human ovarian cyst B-glycoprotein with an accompanying increase in H-specificity, and converted human type-B erythrocytes to type 0. The enzyme consists primarily of a tetramer, molecular weight 150000 f 5000 at pH 4.0, and of a monomer, molecular weight 40000 & 3000 at pH 8.0. Polyacrylamide gel electrophoresis in dodecyl sulfate at pH 7.2 distinguished between two types of monomeric unit of similar molecular weight. N-terminal alanine was identified as the sole N-terminal amino acid residue. The enzyme was shown to be devoid of carbohydrate.
a-D-Galactosidases have been isolated from a variety of animal, plant and microbial sources, but most are inactive towards high molecular-weight substrates [l]. Hydrolysis of the terminal am-galactopyranosyl moieties of blood-group-B substances has been reported for a limited number of enzymes, including those from Trichomonas foetus [2], Clostridium maehashi [ 3 ] ,figs [4], Streptomyces spp. [5], and coffee beans [6]. We recently described the isolation and purification by affinity chromatography of coffee bean a-D-galactosidase [7], which converts human type-B erythrocytes to type 0 [8]. This paper deals with some of the properties of a a-u-galactosidase which destroys blood-group B antigens and is isolated from soybean meal and purified by affinity chromatography. MATERIALS AND METHODS Sepharose 4B and Sephadex G-200 (Fine) were purchased from Pharmacia (Uppsala, Sweden) ; Dgalactose, acrylamide, N,N’-methylene bisacrylamide and sodium dodecyl sulfate from B.D.H. (Poole, England) ; ammonium sulfate and cyanogen bromide from Fluka (Buchs, Switzerland) ;p-nitrophenyl glycopyranosides from Koch-Light (Colnbrook, England) ; Ulex europeus anti-H lectin from Dade (Miami, Florida) ; bovine serum albumin, catalase, cytochrome c (horse heart), dithiothreitol and dansyl chloride Abbreviation. Dansyl, 5-dimethylaminonaphthalene-1-sulfonyl. Enzynzes. 1-o-Galactoside galactohydrolase or a-galactosidase (EC 3.2.1.22).
from Sigma (St Louis, Missouri); soybean trypsin inhibitor, fi-galactosidase (Escherichia coli), chymotrypsinogen and ovalbumin from Worthington Biochemical (Freehold, New Jersey) ; lactate dehydrogenase (rabbit muscle) and alcohol dehydrogenase from Boehringer (Mannheim, W. Germany) ; polyamide sheets from Cheng Chin (Taipei, Taiwan); and NaB3H4 from New England Nuclear (Boston, Massachusetts). Ovarian cyst blood-group substances were kindly provided by Prof. Winifred M. Watkins and eel anti-H serum by Prof. George F. Springer. Hexaneextracted, untoasted soybean oil meal was a gift of Etz Hazait (Petah Tikva, Israel). cc-D-Galactosidase was assayed as described previously [7], except that albumin and McIlvaine buffer [9], pH 6.0, were replaced by McIlvaine buffer, pH 5.0, or by 0.2 M sodium acetate buffer, pH 5.0. One unit of activity is defined as the amount of enzyme catalyzing the hydrolysis of 1 pmole of p-nitrophenyl a-Dgalactopyranoside per min under these conditions. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed on 1.6-mmthick slab gels (12.5 x 12.5 cm), using a locally manufactured apparatus. Phosphate-buffered gels containing 7.5 % acrylamide, pH 7.2, were prepared according to Weber and Osborn [lo]. Samples containing 510 pg of protein in 20 ”/, glycerol, 0.01 M sodium phosphate, pH 7.0, 1 sodium dodecyl sulfate, and 1 mM dithiothreitol, final volume 20 - 60 pl, were heated at 100 ’C for 3 min prior to electrophoresis. Bromphenol blue was used as the tracking dye. Proteins were allowed to enter the gels at a constant current of 20 mA, and
420
the current was then raised to 90 mA for the duration of the electrophoresis (about 5 h). Protein was stained with Coomassie blue [lo] and carbohydrate with the periodic acid/Schiff reagent [ll]. N - E - Aminocaproyl a- u -galactopyranosylamine conjugated to Sepharose (about 9 pmol per g of resin) was prepared as described [7]. Hydrolysis of protein samples (0.4 - 0.5 mg) was performed in evacuated vials in 1 ml of 6 M HC1 containing 50 p1 of 5 % phenol at 110 T for 24 h. HCI was removed by rotary evaporation, and amino acids were determined on a Beckman-Spinco amino acid analyzer (model 120B or model 121) [12], using DL-norleUCine and L-a-aminoguanidinopropionicacid as internal standards for the long and short columns, respectively. r w t e i n e waq determined according to Spencer and Wold [13], and tryptophan according to Matsubara and Sasaki [14]. For the analysis of carbohydrates, the hydrolysis was performed for 3 h in 2 M HC1. Protein was determined by the method of Lowry rt al. [15], using bovine serum albumin as standard. N-terminal amino acids were determined by a modification of the procedure of Gray [16]. The dansyl protein derivative was prepared by dissolving 0.5 mg of the protein in 0.1 ml of 0.1 M sodium bicarbonate containing 1 % sodium dodecyl sulfate, and adding 0.1 ml of 2.5 7; dansyl chloride in acetone. Turbidity was eliminated by the addition of several drops of dimethylformamide. After incubation at 37 “C for 3 h, the mixture was dialyzed sequentially against 0.1 M ammonium hydroxide, 90 % acetone and distilled water, and hydrolyzed in 6 M HC1 at 110 “C for 16 h. The hydrolysate was evaporated in vucuo over NaOH pellets, taken up in 10 pl of 50”/;1pyridine, and 1-pl aliquots analyzed by two-dimensional chromatography on 7.5x7.5-cm polyamide sheets [17]. The dansyl amino acids were identified by co-chromatography with appropriate standard markers. Free sugars were determined by a modification of the NaB3H4 procedure of Takasaki and Kobata [18]. To 4 pg of the protein hydrolysate was added 40 pg of NaB3& (330.1 Ci/mol) in 50 p1 of 0.1 M NaOH. After incubation at room temperature for 16 h, 100 pl of 1 M acetic acid was added, and the solution evaporated to dryness. Three further 100-p1 portions of 1 M acetic acid were added and, after each addition, the solution was evaporated to dryness. The dried product was then taken up in 50 pl of water and spotted on Whatman No. 3 paper (70 x 46 cm) in parallel with unlabelled sugar alcohol standards ( 5 - 10 pg). High voltage paper electrophoresis was performed in 0.04 M borate buffer, pH 9.5, at 40 V/cm for 3.75 h, and radioactivity determined by counting 0.5-cm horizontal strips in 3.5 ml of toluene scintillation fluid. Radioactive and suitable background regions were then eluted with water [19] and counted in 10 ml of Bray’s scintillation fluid. Unlabelled sugar alcohols were
Soybean a-Galactosidase
stained with silver nitrate [20]. The radioactivity incorporated in known quantities of D-galactose was approximately 450 counts x min-’ x ng-’ and was linear over the range tested (from 1.O to 10.5 ng). Velocity sedimentation was performed in a Beckman ultracentrifuge (model E) equipped with an ultraviolet monochromator and scanning optics. The protein (350 pg/ml, Azso = 0.7) in McIlvaine or 0.1 M sodium phosphate buffers of pH 4.0, 6.0 and 8.0, containing 0.1 mM Na2EDTA, was centrifuged at 52000 rev./min, 20 C, and scanned at 8-min intervals at 280 nm. Serological tests and the incubation of human erythrocytes with a-D-galactosidase were performed as described previously [8].
RESULTS AND DISCUSSION Preliminary Isolation The soybean meal (500 g) was powdered in a blender and extracted overnight at 4 “ C in saline (2.5 1). The mixture was centrifuged at 10000 x g for 20 min at 4 ’C, the precipitate discarded, and the supernatant (1575 ml) brought to 70 ”/, ammonium sulfate saturation by gradual addition of the salt at room temperature. The precipitate was isolated by centrifugation as above, redissolved in a minimal volume of distilled water. dialyzed exhaustively against distilled water at room temperature, and the ammonium sulfate precipitation was repeated. After dissolving the precipitate in distilled water, the pH was lowered to 4.0 by the dropwise addition of saturated citric at 0 ’C. The insoluble residue was removed by centrifugation at 15000 x g for 20 min, and the supernatant dialyzed against four changes of ‘chromatography’ buffer (McIlvaine buffer, pH 4.0, containing 0.1 niM NazEDTA and 0.01 NaN3).
Ajfin ity Chromat ogrup hy The solution obtained above (465 ml) was passed through a column (8.5 x 2.6) of N-&-aminocaproyl a-D-galactopyranosylamine-Sepharoseconjugate preequilibrated with chromatography buffer and the column washed until the absorbance of the effluent at 280 nm was less than 0.01. Elution of the a-D-galactosidase was achieved with 50 ml of 0.2 M D-galactose in chromatography buffer. Fractions absorbing at 280 nm were pooled, dialyzed thoroughly against chromatography buffer, and stored at 4 C in polyethylene containers. The results of a typical experiment in which the enzyme-containing fractions were dialyzed individually, is shown in Fig. 1. As indicated, the specific activity across the enzyme peak was constant. The purification scheme described above is summarized in Table 1. As can be seen, the ammonium
42 1
N. Harpaz, H. M. Flowers, and N. Sharon
sulfate precipitation step removed material (presumably oligosaccharides and polysaccharides) which inhibited a-D-galactosidase activity. When affinity chromatography was performed directly on the crude extract, even after prolonged dialysis, less than 50% of the enzyme was retained on the column. Retention was improved significantly after one ammonium sulfate precipitation but was complete only after the second precipitation step. The final yield of the enzyme was high (over 90 based on the first ammonium sulfate precipitation step), and the product was highly pure, as shown below. Soybean agglutinin [21] was not bound to the affinity adsorbent, presumably because of its low galactose-binding activity at room temperature and low pH (H. Lis and N. Sharon, unpublished results), and the final enzyme preparation was free of the lec-
Fraction number
Fig. 1. A , f f i / ~ i t jchroniutogrciph~ < oj cc-u-guluc./o.FidcisL.. Partially purified r-o-galactosidase (4200 mg protein in 230 ml of McIlvaine buffer, pH 4.0, containing 0.1 m M Na2EDTA and 0.01 'I;, NaN3) was passed through a column (8.5 x 2.6 cm) of N-6-aminocaproyl a-D-galactopyranosylamine-Sepharoseconjugate pre-equilibratcd with the same buffer. Arrow indicates elution with buffer containing 0.2 M o-galactose. Fractions (6 ml) were monitored at 280 nm and assayed for r-o-galactosidase activity. Fractions containing D-gdlaCtoSe were dialyzed individually against the chromatography buffer and the specific activity of the enzyme fractions determined by assay of protein 1151 and of enzyme activity. (-) Absorbance at 280 n m ; (0-- --0) enzyme activity; ( x ) spccific activity
tin as judged by the failure of a 175 pg/ml solution to agglutinate trypsinized rabbit erythrocytes under microscopic inspection.
Cutalytic Properties of Soybean a-D-Guluctosidase The purified enzyme preparation showed no activity towards the p-nitrophenyl glycopyranosides of fl-D-galactoSe, a-D-glucose, fl-D-glucose, a-L-fucose, a-D-mannose, N-acetyl b-D-glucosamine, N-acetyl a-D-galactosamine and N-acetyl/l-D-galactosamine. In these tests, the a-D-galactosidase (0.2 U, 2 pg) was incubated with 1.25 mM solutions of substrate in McIlvaine buffers of pH 5.0 and 6.0 for 1 h at 37 "C. The enzyme showed a broad pH dependence towards p-nitrophenyl a-D-galactopyranoside, as shown in Fig.2, with an optimum at pH 5.0. At this pH, the K,, determined by a Lineweaver-Burk plot, was 0.57 mM, and the corresponding V was 141 pmol x min-' x mg of protein-' (Fig. 3). Coffee bean a - ~ galactosidase, in comparison, has a pH optimum at pH 6.0; its K , is 0.3 mM and corresponding V 124 pmol x min-l x mg-' (authors' unpublished results). As shown in Fig. 4, concentrations ofp-nitrophenyl a-D-galactopyranoside above 1.25 mM partially inhibited the enzyme. Substrate inhibition by aryl a-Dgalactopyranosides is a feature common to coffee bean 2-D-galactosidase (authors' unpublished results) as well as other a-D-galactosidases [I]. The pH dependence of the enzymic destruction of blood-type-B specificity in ovarian cyst B substance is shown in Fig. 5 ; as indicated, maximal loss occurred at pH 4.0. Blood-type-H specificity, as determined with U . europeus lectin (Fig.6) and with eel anti-H agglutinin, was increased 16-fold when B specificity was completely abolished. Of the various sugars tested, only D-galactose and methyl a-D-galactopyranoside inhibited this reaction (Fig. 7). Treatment of intact human type-B erythrocytes with 50 U (0.5 mg) of soybean a-D-galactosidase for 2.5 h resulted in the loss of their B specificity and in the enhancement of their H specificity to a level identical to that of normal 0 erythrocytes (Fig. 8).
Table 3 . Purifi'cation of soybean % - D - g ~ h C t o s i C h S P For details of the purification procedures and definition of enzyme units, see text. Yields and degrees ofpurification are given relative to the first ammonium sulfate precipitation, since activity in the crude extract is inhibited by galactose-containing compounds Procedure
Crude extract from 500 g soybean meal 1st ammonium sulfate precipitate 2nd ammonium sulfate precipitate Acidification Affinity chromatography
Volume
Protein
Enzyme activity
ml
1%
U
1575 650 520 465 40
91 000 45 500 38000 8 500 10
800 1030 1005 965 945
Yield
Specific activity
Purification
0,
/
U/mg
-fold
78 100 98 94 92
0.009 0.023 0.026 0.114 94.5
~
1
1.1 5.0 4100
422
Soybean r-Galactosidase I
I
I
I
7
I
i
0.60
0.50
5
Fl--
LD .;f 0
0.40
%
em
ss: 0.20 I
0
I
5O .
4.0
6.0
7.0
PH
Fig.2. Egret of p H on the activity of a-D-galactosidase towards p-nitrophenyl cc-D-~alactopyranoside. 50 pl of purified enzyme (0.60 U/ml) was incubated with 50 pl of substrate (5.0 mM) in the presence of 0.1 ml of McIlvaine buffers ol‘ various pH values, at 37 “C. Reactions were terminated after 0.5, 1.0 and 1.5 min by the addition of 0.8 ml of Na2C03(0.5 M) and thc increase per min of absorbance at 405 nm plotted as a function of pH
Fig. 4. Suhsrrate inhihition of n-n-~alactosidu.rc. Reactions were performed as described in the legend to Fig. 3, and reaction rates plotted as a function of substrate concentration
i
. .
c ._
E
0 c
64
-
._ r ._ c
._ 0
80-
. -
16
2 ._
-
3
-
b
m 0)
40
3
-
,
4 g
I ,
,’ , ’
I
,, ‘
/,
g
tl
4.0
4.5
5.0
5.5
6.0
6.5
7.0
0
1 / [ S ] ( mM”)
Fig. 3. Lineweavrr-Burk plot of rn;.vmic’ 1 i y d r . 0 1 ~oj ~ 1p-nitrophenyl ~ x-a-galrctopyranoside. 50 p1 of purified enzyme (0.44 U m l ) was incubated with 0.1 ml of Mcllvaine buffer, pH 5.0, and 50 pl of substrate at various concentrations, at 37 ’C. Reactions were terminated as in the legend to Fig.2, and the reciprocals of the reaction rates plotted as a function of the reciprocal of substrate concentrations
I
1
I
I
Fig. 5. F;/fiJcfcc.r of p H on the activitj. of a-n-jicrloc,to.sidusr activit.c toward~?ovarian cyst B-suhstance. Reaction mixtures containing purified x-n-galactosidase (2.5 U/ml). B substance. 50 pg:ml, and Mcllvaine buffers of various pH values. final volume 50 pl, were incubated at 37 “C for 24 h. After the addition of 50 pl of phosphatebuffered saline, pH 7.0, serial dilutions (50 pl) were treated with an equal volume of blood-grouping anti-B serum (four hemdgglutinating doses) at room temperature. After 30 min, 50 p1 of a 2“,, suspension of saline-washed, type-B erythrocytes was added, and the mixtures inspected for complete agglutination after 3 h [8]
I
I
-a-Galactosidase
I
I
I
1 -
+a-Galactosidase
0
t
I
I
I
I
2
8
32
I
I 1 1 I I 128 512 2 8 Hemagglutination inhibition titer
I
I 32
I 128
I
I I 512
Fig. 6. Destruction of B .specificity and enharzcrmmt of H specificity in ovarian c ~ . . s rB-suhstuiicc~by x-a-jial(ic,ro,sidase. Reaction mixtures containing purified x-D-galactosidase (2.5 U/ml),H substance, 440 pgjml, and Mcllvaine buffer, pH 4.0, final volume 0.1 ml, were incubated at 37 ”C for 48 h. After the addition of 0.1 ml of plio~phate-bufferedsaline, pH 7.0, one half of each mixture was tested for B activity and one half for H activity, using blood-grouping anti-B serum and B erythrocytes, and U. ruroprus lectin and 0 erythrocytes, respectively, as described in the legend to Fig. 5
423
N Harpaz, H. M. Flowers, and N. Sharon
1 512
-g L
a,
._ -32
._ c c m
._
I
3
-8
2 - 2.
I
-0 B
I
I
I
H
Fig. 8. Destruction of B specificity and enhancement of H .specificity of human type-B erythrocytes by a-o-guluctosidase. 40 p1 of freshlydrawn, packed, type-B erythrocytes was incubated with 90 p1 of purified x-D-galactosidase, 50 U (0.5 mg), in 0.1 M citric acid/0.2 M Na2HP04,p H 5.0. containing 3 ( w h ) glycerol. A parallel control was incubated without the enzyme. After 2.5 h, samples were brought to about 1 ml by the gradual addition of phosphate-buffered saline, pH 7.0, and washed twice with buffered saline by centrifugation. 2 :d suspensions of erythrocytes in buffered saline were tested for complete agglutination by serial dilutions of blood-grouping anti-R serum and of U . europeus lectin
Fig. I . Inhibition by succliurides of a-D-guluctosiduse activity ton.urc/s ovarian cyst B-substance. Reaction mixtures containing purified x-D-galactosidase (4.0 U/ml), ovarian cyst B-substance, 50 pgjml, McIlvaine buffer, p H 4.0, and various saccharides, 0.1 M, final volume, 50 pl, were incubated at 37 'T for 36 h. Hemagglutination inhibition activities were determined as described in the legend to Fig. 5
1
B
H
I
I
se
Fraction number
Fig. 9. Gelfiltration ofpurified x-u-galuctosiduse un S e p h u d a G-200 ut p H 4.0 ( A ) undpt-l 6.0 ( B ) . Approximately 2 ml of cc-D-galactosidase (0.3 mg/ml) in McIlvaine buffer, p H 4.0, containing 0.1 m M Na2EDTA, 0.01 NaN3 and 10% sucrose, was applied to a column (53.5 x 2.3 cm) of Sephadex G-200 (fine) and eluted at room temperature with McIlvaine buffer, p H 4.0 or 6.0, containing 0.1 mM Na2EDTA and 0.01 yo NaN3, at a constant hydrostatic pressure of 15 cm. Fractions (2.5 ml) were collected and monitored for protein at 280 nm (-) and for a-D-galactosidase activity at 405 nm ( - - ~ ). The peak maxima of dextran blue 2000 and of sucrose are indicated ~~
Molecular Weight and Subunit Composition Gel filtration of the purified enzyme on a column (53.5 x 2.3 cm) of Sephadex G-200 at pH 4.0 resulted in a symmetrical enzyme peak (Fig.9A), whose elution volume corresponded to a molecular weight of 150 000 i 5000 (Fig. 10) [22]. Chromatography at pH 6.0 re-
sulted in an additional enzyme peak (Fig. 9 B) of molecular weight 40000 -t 3000 (Fig. 10). Upon velocity sedimentation the enzyme was shown to dissociate reversibly from a predominantly (about 90 %) high-molecular-weight form ( s , , ~ o = 7.45 S) to a low-molecular-weight form (sw, z0 = 4.15 S) as the pH was increased from 4.0-8.0
424
Soybean a-Galactosidase
I
7.45
sl
0.35
I'
o
I 0.2
I 0.6
I 0.4
I
0.8
1.0
K
Fig. 10. Molwulur weight determinutions (?Jsc-n-gnlucto.Fiduse oligonier und monomer by gel fi'lfration. The column of Sephadex G-200
described in the legend to Fig. 9 was calibrated with the following proteins at p H 4.0 (molecular weights in parentheses): (1) rabbit immunoglobulin G (160000); (2) lactate dehydrogenase (144000); (3) soybean agglutinin (120000); (4) bovine serum albumin (68000); (5) ovalbumin (43000); (6) chymotrypsinogen (25700); (7) cytochrome c (12300). The logarithms of the molecular weights were plotted as a function of K , defined as (V,- Vo)/(V,- Vo),where Ve = elution volume of protein; V , = elution volume of dextran blue 2000; V , = elution volume of sucrose, and are indicated by circles. K values of soybean a-D-galactosidase oligomer and monomer were obtained from the data shown in Fig.8. A second column (119.0 x 1.0 cm) of Sephadex G-200 (fine) was equilibrated with saline containing 0.1 2, sodium dodecyl sulfate and calibrated with the following proteins (subunit molecular weights in parentheses): ( I ) ovalbumin (43000); (2) alcohol dehydrogenase (41 000); (3) lactate dehydrogenase (36000); (4) soybean trypsin inhibitor (20500). Proteins were boiled for 3 min in the presence of 1 sodium dodecyl sulfate and 10 mM dithiothrcitol prior to chromatography. The data. indicated by triangles, are plotted as above
/, polyacrylamide gels, pH 7.2 [lo]. Gels stained with Cooniassie blue were photographed as transparencies and scanned spectrophotometrically at 580 nm. The relative migration of bromphenol blue is taken as 1.0
* Determined as cysteic acid.
Corrected value, based on yield of 79Y0 obtained with lysozyme.
amino acid analysis of 0.5 mg of enzyme protein. By the NaB3H4 procedure, less than 2 ng of sugar was observed in 4 pg of the protein. The enzyme is thus not a glycoprotein. Concluding Remarks
, 1
o
0.2
0.4 0.6 Relative migration
0.8
1.0
Fig. 13. Molecular tcvighl determiriation ofx-n-guluctosidu.,e 12). polyacrylutnide gel electrophoresis in dadecyl .sn&re. Electrophoresis of about 10 pg of purified enzyme was performed for 5 h at 90 mA in parallel with the following protein standards (subunit molecular weights in parentheses): (1j P-gdhctosidase (1 30000); (2) bovine serumalbumiti(68000); (3)catalase(58000); (4) ovalbumin(43000): (5) alcohol dehydrogenase (41 000); (6) lactate dehydrogenase (36000); (7) soybean agglutinin (30000); (8) chymotrypsinogen (25 700). Logarithms of molecular weights are plotted as a function of migrations relative to bromphenol blue. Arrow indicates relative migration of ?-D-galactosidase
The presence of multiple forms of a-11-galactosidase in soybeans germinated for 3 days has been previously reported [23]. One enzyme had a molecular weight of 130000- 150000, and two others molecular weights of 40000, as determined by gel filtration. Since the pH conditions under which these results were obtained were not specified, it is possible that one or both of the lower-molecular-weight species might have been related to the high-molecular-weight enzyme by a monomer-oligomer relationship. The redistribution of 1-D-galactosidases from high to low-molecularweight forms during germination has been reported in a variety of seeds [24], and it would be interesting to compare the chemical properties of purified enzyme species as a function of germination. Affinity chromatography may provide a convenient method for purifying such enzymes. Soybean a-11-galactosidase, whose facile purification from a cheap and readily available source has been described, may be useful in studies of blood-group substances or other cell surface glycoconjugates. This work was supported by grant number 548 from the United States - Israel Binational Foundation.
426
N . Harpaz, H. M. Flowers. and N. Sharon: Soybean cc-Galactosidase
REFERENCES 1. Dey, P. M. & Pridham, J. B. (1972) Adv. Enzymol. 36, 91 - 129. 2. Watkins, W. M. (1956) Biochem. J . 64, 21P. 3. Iseki, S., Furukawa, K. & Yamamoto, S. (1959) Proc. Jap. Acad. 35, 507-512. 4. Hakomori, S. I., Siddiqui, B., Li, Y.-T., Li, S.-C. & Hellerqvist, C. G. (1971) J . Bid. Chem. 246, 2271 -2271. 5. Oishi, K. & Aida, K. (1972) Agric. Biol. Chem. 36, 578 - 587. 6. Zarnitz, M. L. & Kabat, E. A. (1960) J . Am. Chenz. Soc. 82, 3953 3957. 7. Harpaz, N., Flowers, H. M. & Sharon, N. (1974) Biochim. Biophys. Acta, 341, 213-221. 8. Harpaz, N., Flowers, H. M. & Sharon, N. (1975) Arch. Biochem. Biophys. 170,676-683. 9. Mcllvaine, T. C. (1921) J . Biol. Chem. 49, 183- 186. 10. Weber, K. & Osborn, M. (1969) J . Biol. Chem. 244,4406-4412. 11. Zacharius, R. M., Zell, T. E., Morrison, J. H. & Woodlock, J. J. (1969) Anal. Biochem. 30, 148- 152. 12. Spackman, D. H., Stein, W. H. & Moore, S. (1958) Anal. Chem. 30,1190- 1206. -
13. Spencer, R. L. &Wold, F. (3969) Anal. Biochem. 32, 185-189. 14. Matsubara, H. & Sasaki, R. M. (1969) Biochem. Biophys. Res. Commun. 35, 175-181. 15. Lowry, 0. J., Rosebrough, N . J., Farr, A. L. & Randall. R. J. (1951) J . Bid. Chem. 193, 265-275. 16. Gray, W. R. (1967) Methods Enzymol. 11, 139-151. 17. Woods, K. R. & Wang, K . T. (1967) Biochim. Biophys. Acto, 133, 369-370. 18. Takasaki, S. & Kobaia, A. (1974) J . Biochem. 76, 783-789. 19. Eshdat, Y. & Mirelman, D. (1972) .I. Chromatogr. 65,458-459. 20. Trevylan, W. E., Procter, D. P. & Harrison, J. S. (1950) Nature (Lond.) 166,444-445. 21. Gordon, J. A,, Blumberg, S., Lis, H. &Sharon, N. (1972) F E B S Lett. 24, 193-196. 22. Andrews. P. (1964) Biochem. J . 91, 222-233. 23. McCleary, B. V. & Matheson, N. K. (1974) Phytoclzernistry, 13, 1747- 1757. 24. Barham, D., Dey, P. M., Griffiths, D. & Pridham, J. B. (1971) Phytochemistry, 10, 1759 - 1763.
N Harpaz, H M Flowers, and N Sharon*, Department of Biophysics, The Weizmann Institute of Science, P 0 Box 26, Rehovot, Israel ~
* To whom correspondence should be addressed
a-D-Galactosidase from Soybeans Destroying Blood-Group B Antigens Purification by Affinity Chromatography and Properties Noam HARPAZ, Harold M. FLOWERS, and Nathan SHARON Department of Biophysics, The Weizmann Institute of Science, Rehovot (Received March 24, 1977)
a-D-Galactosidase was isolated from untoasted soybean meal and purified to homogeneity by affinity chromatography on N-eaminoacaproyl a-D-galactopyranosylamine-Sepharose. The purified enzyme destroyed the B-specificity of human ovarian cyst B-glycoprotein with an accompanying increase in H-specificity, and converted human type-B erythrocytes to type 0. The enzyme consists primarily of a tetramer, molecular weight 150000 f 5000 at pH 4.0, and of a monomer, molecular weight 40000 & 3000 at pH 8.0. Polyacrylamide gel electrophoresis in dodecyl sulfate at pH 7.2 distinguished between two types of monomeric unit of similar molecular weight. N-terminal alanine was identified as the sole N-terminal amino acid residue. The enzyme was shown to be devoid of carbohydrate.
a-D-Galactosidases have been isolated from a variety of animal, plant and microbial sources, but most are inactive towards high molecular-weight substrates [l]. Hydrolysis of the terminal am-galactopyranosyl moieties of blood-group-B substances has been reported for a limited number of enzymes, including those from Trichomonas foetus [2], Clostridium maehashi [ 3 ] ,figs [4], Streptomyces spp. [5], and coffee beans [6]. We recently described the isolation and purification by affinity chromatography of coffee bean a-D-galactosidase [7], which converts human type-B erythrocytes to type 0 [8]. This paper deals with some of the properties of a a-u-galactosidase which destroys blood-group B antigens and is isolated from soybean meal and purified by affinity chromatography. MATERIALS AND METHODS Sepharose 4B and Sephadex G-200 (Fine) were purchased from Pharmacia (Uppsala, Sweden) ; Dgalactose, acrylamide, N,N’-methylene bisacrylamide and sodium dodecyl sulfate from B.D.H. (Poole, England) ; ammonium sulfate and cyanogen bromide from Fluka (Buchs, Switzerland) ;p-nitrophenyl glycopyranosides from Koch-Light (Colnbrook, England) ; Ulex europeus anti-H lectin from Dade (Miami, Florida) ; bovine serum albumin, catalase, cytochrome c (horse heart), dithiothreitol and dansyl chloride Abbreviation. Dansyl, 5-dimethylaminonaphthalene-1-sulfonyl. Enzynzes. 1-o-Galactoside galactohydrolase or a-galactosidase (EC 3.2.1.22).
from Sigma (St Louis, Missouri); soybean trypsin inhibitor, fi-galactosidase (Escherichia coli), chymotrypsinogen and ovalbumin from Worthington Biochemical (Freehold, New Jersey) ; lactate dehydrogenase (rabbit muscle) and alcohol dehydrogenase from Boehringer (Mannheim, W. Germany) ; polyamide sheets from Cheng Chin (Taipei, Taiwan); and NaB3H4 from New England Nuclear (Boston, Massachusetts). Ovarian cyst blood-group substances were kindly provided by Prof. Winifred M. Watkins and eel anti-H serum by Prof. George F. Springer. Hexaneextracted, untoasted soybean oil meal was a gift of Etz Hazait (Petah Tikva, Israel). cc-D-Galactosidase was assayed as described previously [7], except that albumin and McIlvaine buffer [9], pH 6.0, were replaced by McIlvaine buffer, pH 5.0, or by 0.2 M sodium acetate buffer, pH 5.0. One unit of activity is defined as the amount of enzyme catalyzing the hydrolysis of 1 pmole of p-nitrophenyl a-Dgalactopyranoside per min under these conditions. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed on 1.6-mmthick slab gels (12.5 x 12.5 cm), using a locally manufactured apparatus. Phosphate-buffered gels containing 7.5 % acrylamide, pH 7.2, were prepared according to Weber and Osborn [lo]. Samples containing 510 pg of protein in 20 ”/, glycerol, 0.01 M sodium phosphate, pH 7.0, 1 sodium dodecyl sulfate, and 1 mM dithiothreitol, final volume 20 - 60 pl, were heated at 100 ’C for 3 min prior to electrophoresis. Bromphenol blue was used as the tracking dye. Proteins were allowed to enter the gels at a constant current of 20 mA, and
420
the current was then raised to 90 mA for the duration of the electrophoresis (about 5 h). Protein was stained with Coomassie blue [lo] and carbohydrate with the periodic acid/Schiff reagent [ll]. N - E - Aminocaproyl a- u -galactopyranosylamine conjugated to Sepharose (about 9 pmol per g of resin) was prepared as described [7]. Hydrolysis of protein samples (0.4 - 0.5 mg) was performed in evacuated vials in 1 ml of 6 M HC1 containing 50 p1 of 5 % phenol at 110 T for 24 h. HCI was removed by rotary evaporation, and amino acids were determined on a Beckman-Spinco amino acid analyzer (model 120B or model 121) [12], using DL-norleUCine and L-a-aminoguanidinopropionicacid as internal standards for the long and short columns, respectively. r w t e i n e waq determined according to Spencer and Wold [13], and tryptophan according to Matsubara and Sasaki [14]. For the analysis of carbohydrates, the hydrolysis was performed for 3 h in 2 M HC1. Protein was determined by the method of Lowry rt al. [15], using bovine serum albumin as standard. N-terminal amino acids were determined by a modification of the procedure of Gray [16]. The dansyl protein derivative was prepared by dissolving 0.5 mg of the protein in 0.1 ml of 0.1 M sodium bicarbonate containing 1 % sodium dodecyl sulfate, and adding 0.1 ml of 2.5 7; dansyl chloride in acetone. Turbidity was eliminated by the addition of several drops of dimethylformamide. After incubation at 37 “C for 3 h, the mixture was dialyzed sequentially against 0.1 M ammonium hydroxide, 90 % acetone and distilled water, and hydrolyzed in 6 M HC1 at 110 “C for 16 h. The hydrolysate was evaporated in vucuo over NaOH pellets, taken up in 10 pl of 50”/;1pyridine, and 1-pl aliquots analyzed by two-dimensional chromatography on 7.5x7.5-cm polyamide sheets [17]. The dansyl amino acids were identified by co-chromatography with appropriate standard markers. Free sugars were determined by a modification of the NaB3H4 procedure of Takasaki and Kobata [18]. To 4 pg of the protein hydrolysate was added 40 pg of NaB3& (330.1 Ci/mol) in 50 p1 of 0.1 M NaOH. After incubation at room temperature for 16 h, 100 pl of 1 M acetic acid was added, and the solution evaporated to dryness. Three further 100-p1 portions of 1 M acetic acid were added and, after each addition, the solution was evaporated to dryness. The dried product was then taken up in 50 pl of water and spotted on Whatman No. 3 paper (70 x 46 cm) in parallel with unlabelled sugar alcohol standards ( 5 - 10 pg). High voltage paper electrophoresis was performed in 0.04 M borate buffer, pH 9.5, at 40 V/cm for 3.75 h, and radioactivity determined by counting 0.5-cm horizontal strips in 3.5 ml of toluene scintillation fluid. Radioactive and suitable background regions were then eluted with water [19] and counted in 10 ml of Bray’s scintillation fluid. Unlabelled sugar alcohols were
Soybean a-Galactosidase
stained with silver nitrate [20]. The radioactivity incorporated in known quantities of D-galactose was approximately 450 counts x min-’ x ng-’ and was linear over the range tested (from 1.O to 10.5 ng). Velocity sedimentation was performed in a Beckman ultracentrifuge (model E) equipped with an ultraviolet monochromator and scanning optics. The protein (350 pg/ml, Azso = 0.7) in McIlvaine or 0.1 M sodium phosphate buffers of pH 4.0, 6.0 and 8.0, containing 0.1 mM Na2EDTA, was centrifuged at 52000 rev./min, 20 C, and scanned at 8-min intervals at 280 nm. Serological tests and the incubation of human erythrocytes with a-D-galactosidase were performed as described previously [8].
RESULTS AND DISCUSSION Preliminary Isolation The soybean meal (500 g) was powdered in a blender and extracted overnight at 4 “ C in saline (2.5 1). The mixture was centrifuged at 10000 x g for 20 min at 4 ’C, the precipitate discarded, and the supernatant (1575 ml) brought to 70 ”/, ammonium sulfate saturation by gradual addition of the salt at room temperature. The precipitate was isolated by centrifugation as above, redissolved in a minimal volume of distilled water. dialyzed exhaustively against distilled water at room temperature, and the ammonium sulfate precipitation was repeated. After dissolving the precipitate in distilled water, the pH was lowered to 4.0 by the dropwise addition of saturated citric at 0 ’C. The insoluble residue was removed by centrifugation at 15000 x g for 20 min, and the supernatant dialyzed against four changes of ‘chromatography’ buffer (McIlvaine buffer, pH 4.0, containing 0.1 niM NazEDTA and 0.01 NaN3).
Ajfin ity Chromat ogrup hy The solution obtained above (465 ml) was passed through a column (8.5 x 2.6) of N-&-aminocaproyl a-D-galactopyranosylamine-Sepharoseconjugate preequilibrated with chromatography buffer and the column washed until the absorbance of the effluent at 280 nm was less than 0.01. Elution of the a-D-galactosidase was achieved with 50 ml of 0.2 M D-galactose in chromatography buffer. Fractions absorbing at 280 nm were pooled, dialyzed thoroughly against chromatography buffer, and stored at 4 C in polyethylene containers. The results of a typical experiment in which the enzyme-containing fractions were dialyzed individually, is shown in Fig. 1. As indicated, the specific activity across the enzyme peak was constant. The purification scheme described above is summarized in Table 1. As can be seen, the ammonium
42 1
N. Harpaz, H. M. Flowers, and N. Sharon
sulfate precipitation step removed material (presumably oligosaccharides and polysaccharides) which inhibited a-D-galactosidase activity. When affinity chromatography was performed directly on the crude extract, even after prolonged dialysis, less than 50% of the enzyme was retained on the column. Retention was improved significantly after one ammonium sulfate precipitation but was complete only after the second precipitation step. The final yield of the enzyme was high (over 90 based on the first ammonium sulfate precipitation step), and the product was highly pure, as shown below. Soybean agglutinin [21] was not bound to the affinity adsorbent, presumably because of its low galactose-binding activity at room temperature and low pH (H. Lis and N. Sharon, unpublished results), and the final enzyme preparation was free of the lec-
Fraction number
Fig. 1. A , f f i / ~ i t jchroniutogrciph~ < oj cc-u-guluc./o.FidcisL.. Partially purified r-o-galactosidase (4200 mg protein in 230 ml of McIlvaine buffer, pH 4.0, containing 0.1 m M Na2EDTA and 0.01 'I;, NaN3) was passed through a column (8.5 x 2.6 cm) of N-6-aminocaproyl a-D-galactopyranosylamine-Sepharoseconjugate pre-equilibratcd with the same buffer. Arrow indicates elution with buffer containing 0.2 M o-galactose. Fractions (6 ml) were monitored at 280 nm and assayed for r-o-galactosidase activity. Fractions containing D-gdlaCtoSe were dialyzed individually against the chromatography buffer and the specific activity of the enzyme fractions determined by assay of protein 1151 and of enzyme activity. (-) Absorbance at 280 n m ; (0-- --0) enzyme activity; ( x ) spccific activity
tin as judged by the failure of a 175 pg/ml solution to agglutinate trypsinized rabbit erythrocytes under microscopic inspection.
Cutalytic Properties of Soybean a-D-Guluctosidase The purified enzyme preparation showed no activity towards the p-nitrophenyl glycopyranosides of fl-D-galactoSe, a-D-glucose, fl-D-glucose, a-L-fucose, a-D-mannose, N-acetyl b-D-glucosamine, N-acetyl a-D-galactosamine and N-acetyl/l-D-galactosamine. In these tests, the a-D-galactosidase (0.2 U, 2 pg) was incubated with 1.25 mM solutions of substrate in McIlvaine buffers of pH 5.0 and 6.0 for 1 h at 37 "C. The enzyme showed a broad pH dependence towards p-nitrophenyl a-D-galactopyranoside, as shown in Fig.2, with an optimum at pH 5.0. At this pH, the K,, determined by a Lineweaver-Burk plot, was 0.57 mM, and the corresponding V was 141 pmol x min-' x mg of protein-' (Fig. 3). Coffee bean a - ~ galactosidase, in comparison, has a pH optimum at pH 6.0; its K , is 0.3 mM and corresponding V 124 pmol x min-l x mg-' (authors' unpublished results). As shown in Fig. 4, concentrations ofp-nitrophenyl a-D-galactopyranoside above 1.25 mM partially inhibited the enzyme. Substrate inhibition by aryl a-Dgalactopyranosides is a feature common to coffee bean 2-D-galactosidase (authors' unpublished results) as well as other a-D-galactosidases [I]. The pH dependence of the enzymic destruction of blood-type-B specificity in ovarian cyst B substance is shown in Fig. 5 ; as indicated, maximal loss occurred at pH 4.0. Blood-type-H specificity, as determined with U . europeus lectin (Fig.6) and with eel anti-H agglutinin, was increased 16-fold when B specificity was completely abolished. Of the various sugars tested, only D-galactose and methyl a-D-galactopyranoside inhibited this reaction (Fig. 7). Treatment of intact human type-B erythrocytes with 50 U (0.5 mg) of soybean a-D-galactosidase for 2.5 h resulted in the loss of their B specificity and in the enhancement of their H specificity to a level identical to that of normal 0 erythrocytes (Fig. 8).
Table 3 . Purifi'cation of soybean % - D - g ~ h C t o s i C h S P For details of the purification procedures and definition of enzyme units, see text. Yields and degrees ofpurification are given relative to the first ammonium sulfate precipitation, since activity in the crude extract is inhibited by galactose-containing compounds Procedure
Crude extract from 500 g soybean meal 1st ammonium sulfate precipitate 2nd ammonium sulfate precipitate Acidification Affinity chromatography
Volume
Protein
Enzyme activity
ml
1%
U
1575 650 520 465 40
91 000 45 500 38000 8 500 10
800 1030 1005 965 945
Yield
Specific activity
Purification
0,
/
U/mg
-fold
78 100 98 94 92
0.009 0.023 0.026 0.114 94.5
~
1
1.1 5.0 4100
422
Soybean r-Galactosidase I
I
I
I
7
I
i
0.60
0.50
5
Fl--
LD .;f 0
0.40
%
em
ss: 0.20 I
0
I
5O .
4.0
6.0
7.0
PH
Fig.2. Egret of p H on the activity of a-D-galactosidase towards p-nitrophenyl cc-D-~alactopyranoside. 50 pl of purified enzyme (0.60 U/ml) was incubated with 50 pl of substrate (5.0 mM) in the presence of 0.1 ml of McIlvaine buffers ol‘ various pH values, at 37 “C. Reactions were terminated after 0.5, 1.0 and 1.5 min by the addition of 0.8 ml of Na2C03(0.5 M) and thc increase per min of absorbance at 405 nm plotted as a function of pH
Fig. 4. Suhsrrate inhihition of n-n-~alactosidu.rc. Reactions were performed as described in the legend to Fig. 3, and reaction rates plotted as a function of substrate concentration
i
. .
c ._
E
0 c
64
-
._ r ._ c
._ 0
80-
. -
16
2 ._
-
3
-
b
m 0)
40
3
-
,
4 g
I ,
,’ , ’
I
,, ‘
/,
g
tl
4.0
4.5
5.0
5.5
6.0
6.5
7.0
0
1 / [ S ] ( mM”)
Fig. 3. Lineweavrr-Burk plot of rn;.vmic’ 1 i y d r . 0 1 ~oj ~ 1p-nitrophenyl ~ x-a-galrctopyranoside. 50 p1 of purified enzyme (0.44 U m l ) was incubated with 0.1 ml of Mcllvaine buffer, pH 5.0, and 50 pl of substrate at various concentrations, at 37 ’C. Reactions were terminated as in the legend to Fig.2, and the reciprocals of the reaction rates plotted as a function of the reciprocal of substrate concentrations
I
1
I
I
Fig. 5. F;/fiJcfcc.r of p H on the activitj. of a-n-jicrloc,to.sidusr activit.c toward~?ovarian cyst B-suhstance. Reaction mixtures containing purified x-n-galactosidase (2.5 U/ml). B substance. 50 pg:ml, and Mcllvaine buffers of various pH values. final volume 50 pl, were incubated at 37 “C for 24 h. After the addition of 50 pl of phosphatebuffered saline, pH 7.0, serial dilutions (50 pl) were treated with an equal volume of blood-grouping anti-B serum (four hemdgglutinating doses) at room temperature. After 30 min, 50 p1 of a 2“,, suspension of saline-washed, type-B erythrocytes was added, and the mixtures inspected for complete agglutination after 3 h [8]
I
I
-a-Galactosidase
I
I
I
1 -
+a-Galactosidase
0
t
I
I
I
I
2
8
32
I
I 1 1 I I 128 512 2 8 Hemagglutination inhibition titer
I
I 32
I 128
I
I I 512
Fig. 6. Destruction of B .specificity and enharzcrmmt of H specificity in ovarian c ~ . . s rB-suhstuiicc~by x-a-jial(ic,ro,sidase. Reaction mixtures containing purified x-D-galactosidase (2.5 U/ml),H substance, 440 pgjml, and Mcllvaine buffer, pH 4.0, final volume 0.1 ml, were incubated at 37 ”C for 48 h. After the addition of 0.1 ml of plio~phate-bufferedsaline, pH 7.0, one half of each mixture was tested for B activity and one half for H activity, using blood-grouping anti-B serum and B erythrocytes, and U. ruroprus lectin and 0 erythrocytes, respectively, as described in the legend to Fig. 5
423
N Harpaz, H. M. Flowers, and N. Sharon
1 512
-g L
a,
._ -32
._ c c m
._
I
3
-8
2 - 2.
I
-0 B
I
I
I
H
Fig. 8. Destruction of B specificity and enhancement of H .specificity of human type-B erythrocytes by a-o-guluctosidase. 40 p1 of freshlydrawn, packed, type-B erythrocytes was incubated with 90 p1 of purified x-D-galactosidase, 50 U (0.5 mg), in 0.1 M citric acid/0.2 M Na2HP04,p H 5.0. containing 3 ( w h ) glycerol. A parallel control was incubated without the enzyme. After 2.5 h, samples were brought to about 1 ml by the gradual addition of phosphate-buffered saline, pH 7.0, and washed twice with buffered saline by centrifugation. 2 :d suspensions of erythrocytes in buffered saline were tested for complete agglutination by serial dilutions of blood-grouping anti-R serum and of U . europeus lectin
Fig. I . Inhibition by succliurides of a-D-guluctosiduse activity ton.urc/s ovarian cyst B-substance. Reaction mixtures containing purified x-D-galactosidase (4.0 U/ml), ovarian cyst B-substance, 50 pgjml, McIlvaine buffer, p H 4.0, and various saccharides, 0.1 M, final volume, 50 pl, were incubated at 37 'T for 36 h. Hemagglutination inhibition activities were determined as described in the legend to Fig. 5
1
B
H
I
I
se
Fraction number
Fig. 9. Gelfiltration ofpurified x-u-galuctosiduse un S e p h u d a G-200 ut p H 4.0 ( A ) undpt-l 6.0 ( B ) . Approximately 2 ml of cc-D-galactosidase (0.3 mg/ml) in McIlvaine buffer, p H 4.0, containing 0.1 m M Na2EDTA, 0.01 NaN3 and 10% sucrose, was applied to a column (53.5 x 2.3 cm) of Sephadex G-200 (fine) and eluted at room temperature with McIlvaine buffer, p H 4.0 or 6.0, containing 0.1 mM Na2EDTA and 0.01 yo NaN3, at a constant hydrostatic pressure of 15 cm. Fractions (2.5 ml) were collected and monitored for protein at 280 nm (-) and for a-D-galactosidase activity at 405 nm ( - - ~ ). The peak maxima of dextran blue 2000 and of sucrose are indicated ~~
Molecular Weight and Subunit Composition Gel filtration of the purified enzyme on a column (53.5 x 2.3 cm) of Sephadex G-200 at pH 4.0 resulted in a symmetrical enzyme peak (Fig.9A), whose elution volume corresponded to a molecular weight of 150 000 i 5000 (Fig. 10) [22]. Chromatography at pH 6.0 re-
sulted in an additional enzyme peak (Fig. 9 B) of molecular weight 40000 -t 3000 (Fig. 10). Upon velocity sedimentation the enzyme was shown to dissociate reversibly from a predominantly (about 90 %) high-molecular-weight form ( s , , ~ o = 7.45 S) to a low-molecular-weight form (sw, z0 = 4.15 S) as the pH was increased from 4.0-8.0
424
Soybean a-Galactosidase
I
7.45
sl
0.35
I'
o
I 0.2
I 0.6
I 0.4
I
0.8
1.0
K
Fig. 10. Molwulur weight determinutions (?Jsc-n-gnlucto.Fiduse oligonier und monomer by gel fi'lfration. The column of Sephadex G-200
described in the legend to Fig. 9 was calibrated with the following proteins at p H 4.0 (molecular weights in parentheses): (1) rabbit immunoglobulin G (160000); (2) lactate dehydrogenase (144000); (3) soybean agglutinin (120000); (4) bovine serum albumin (68000); (5) ovalbumin (43000); (6) chymotrypsinogen (25700); (7) cytochrome c (12300). The logarithms of the molecular weights were plotted as a function of K , defined as (V,- Vo)/(V,- Vo),where Ve = elution volume of protein; V , = elution volume of dextran blue 2000; V , = elution volume of sucrose, and are indicated by circles. K values of soybean a-D-galactosidase oligomer and monomer were obtained from the data shown in Fig.8. A second column (119.0 x 1.0 cm) of Sephadex G-200 (fine) was equilibrated with saline containing 0.1 2, sodium dodecyl sulfate and calibrated with the following proteins (subunit molecular weights in parentheses): ( I ) ovalbumin (43000); (2) alcohol dehydrogenase (41 000); (3) lactate dehydrogenase (36000); (4) soybean trypsin inhibitor (20500). Proteins were boiled for 3 min in the presence of 1 sodium dodecyl sulfate and 10 mM dithiothrcitol prior to chromatography. The data. indicated by triangles, are plotted as above
/, polyacrylamide gels, pH 7.2 [lo]. Gels stained with Cooniassie blue were photographed as transparencies and scanned spectrophotometrically at 580 nm. The relative migration of bromphenol blue is taken as 1.0
* Determined as cysteic acid.
Corrected value, based on yield of 79Y0 obtained with lysozyme.
amino acid analysis of 0.5 mg of enzyme protein. By the NaB3H4 procedure, less than 2 ng of sugar was observed in 4 pg of the protein. The enzyme is thus not a glycoprotein. Concluding Remarks
, 1
o
0.2
0.4 0.6 Relative migration
0.8
1.0
Fig. 13. Molecular tcvighl determiriation ofx-n-guluctosidu.,e 12). polyacrylutnide gel electrophoresis in dadecyl .sn&re. Electrophoresis of about 10 pg of purified enzyme was performed for 5 h at 90 mA in parallel with the following protein standards (subunit molecular weights in parentheses): (1j P-gdhctosidase (1 30000); (2) bovine serumalbumiti(68000); (3)catalase(58000); (4) ovalbumin(43000): (5) alcohol dehydrogenase (41 000); (6) lactate dehydrogenase (36000); (7) soybean agglutinin (30000); (8) chymotrypsinogen (25 700). Logarithms of molecular weights are plotted as a function of migrations relative to bromphenol blue. Arrow indicates relative migration of ?-D-galactosidase
The presence of multiple forms of a-11-galactosidase in soybeans germinated for 3 days has been previously reported [23]. One enzyme had a molecular weight of 130000- 150000, and two others molecular weights of 40000, as determined by gel filtration. Since the pH conditions under which these results were obtained were not specified, it is possible that one or both of the lower-molecular-weight species might have been related to the high-molecular-weight enzyme by a monomer-oligomer relationship. The redistribution of 1-D-galactosidases from high to low-molecularweight forms during germination has been reported in a variety of seeds [24], and it would be interesting to compare the chemical properties of purified enzyme species as a function of germination. Affinity chromatography may provide a convenient method for purifying such enzymes. Soybean a-11-galactosidase, whose facile purification from a cheap and readily available source has been described, may be useful in studies of blood-group substances or other cell surface glycoconjugates. This work was supported by grant number 548 from the United States - Israel Binational Foundation.
426
N . Harpaz, H. M. Flowers. and N. Sharon: Soybean cc-Galactosidase
REFERENCES 1. Dey, P. M. & Pridham, J. B. (1972) Adv. Enzymol. 36, 91 - 129. 2. Watkins, W. M. (1956) Biochem. J . 64, 21P. 3. Iseki, S., Furukawa, K. & Yamamoto, S. (1959) Proc. Jap. Acad. 35, 507-512. 4. Hakomori, S. I., Siddiqui, B., Li, Y.-T., Li, S.-C. & Hellerqvist, C. G. (1971) J . Bid. Chem. 246, 2271 -2271. 5. Oishi, K. & Aida, K. (1972) Agric. Biol. Chem. 36, 578 - 587. 6. Zarnitz, M. L. & Kabat, E. A. (1960) J . Am. Chenz. Soc. 82, 3953 3957. 7. Harpaz, N., Flowers, H. M. & Sharon, N. (1974) Biochim. Biophys. Acta, 341, 213-221. 8. Harpaz, N., Flowers, H. M. & Sharon, N. (1975) Arch. Biochem. Biophys. 170,676-683. 9. Mcllvaine, T. C. (1921) J . Biol. Chem. 49, 183- 186. 10. Weber, K. & Osborn, M. (1969) J . Biol. Chem. 244,4406-4412. 11. Zacharius, R. M., Zell, T. E., Morrison, J. H. & Woodlock, J. J. (1969) Anal. Biochem. 30, 148- 152. 12. Spackman, D. H., Stein, W. H. & Moore, S. (1958) Anal. Chem. 30,1190- 1206. -
13. Spencer, R. L. &Wold, F. (3969) Anal. Biochem. 32, 185-189. 14. Matsubara, H. & Sasaki, R. M. (1969) Biochem. Biophys. Res. Commun. 35, 175-181. 15. Lowry, 0. J., Rosebrough, N . J., Farr, A. L. & Randall. R. J. (1951) J . Bid. Chem. 193, 265-275. 16. Gray, W. R. (1967) Methods Enzymol. 11, 139-151. 17. Woods, K. R. & Wang, K . T. (1967) Biochim. Biophys. Acto, 133, 369-370. 18. Takasaki, S. & Kobaia, A. (1974) J . Biochem. 76, 783-789. 19. Eshdat, Y. & Mirelman, D. (1972) .I. Chromatogr. 65,458-459. 20. Trevylan, W. E., Procter, D. P. & Harrison, J. S. (1950) Nature (Lond.) 166,444-445. 21. Gordon, J. A,, Blumberg, S., Lis, H. &Sharon, N. (1972) F E B S Lett. 24, 193-196. 22. Andrews. P. (1964) Biochem. J . 91, 222-233. 23. McCleary, B. V. & Matheson, N. K. (1974) Phytoclzernistry, 13, 1747- 1757. 24. Barham, D., Dey, P. M., Griffiths, D. & Pridham, J. B. (1971) Phytochemistry, 10, 1759 - 1763.
N Harpaz, H M Flowers, and N Sharon*, Department of Biophysics, The Weizmann Institute of Science, P 0 Box 26, Rehovot, Israel ~
* To whom correspondence should be addressed
