/ . Biochem., TI, 343-351 (1975)
Crystallization and Preliminary X-ray Investigation of Soybean /3-Amylase Yuhei MORITA,* Shigeo AIBARA,* Honami YAMASHITA,* Fumio YAGI,* Toshihiko SUGANUMA,** and Keitaro HIROMI** * Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, and ** Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, June 22, 1974
J9-Amylase [1,4-a-D-glucan maltohydrolase, EC 3.2.1.2] has been purified from defatted soybean meal by fractional precipitation with ammonium sulfate, ionexchange chromatography on CM- and DEAE-Sephadex and gel filtration chromatography on Sephadex G-100. Two different components of ^-amylase were crystallized from ammonium sulfate solutions, and the homogeneity of each preparation was confirmed by sedimentation and disc electrophoretic analyses. Both components of soybean /3-amylase formed large single crystals (trigonal crystal system) from 40—50% saturated ammonium sulfate solution buffered at pH 5.4 on dialyzing concentrated protein solution in the apparatus of Zeppezauer et al. Preliminary X-ray diffraction data gave a hexagonal lattice with unit cell dimensions a=86.1 A and c=144.4 A. The space group corresponds to P3i21 or P3j21, and one asymmetric unit contains one molecule of /3-amylase, assuming a crystal density of 1.25 g/ml and a molecular weight of the enzyme of 60,000 daltons. In this case, the crystal has a volume of 2.53 A8 per atomic mass unit, and the percentage of protein in the crystal is about 52.
"Crystalline 0-amylases [EC 3.2.1.2] have been prepared from sweet potato, barley, wheat, soybean, and Japanese-radish (1—5). The first X-ray investigation was done by Colman and Matthews (6) using tetragonal crystals of the sweet potato enzyme, prepared according to the method of Balls et al. (7). However, it seems difficult to expect a structural analysis at high resolution from these crystals because of the large dimensions of the unit cell, due to the high molecular weight of sweet potato Vol. 77, No. 2, 1975
/3-amylase (196,000 daltons) and the highly symmetric packing of many molecules in a unit cell (P4i22). The object of the present study was to prepare large single crystals, suitable for Xray analysis, of /9-amylase from soybean. This is a monomeric protein and has a lower molecular weight than the sweet potato enzyme (5). Crystallization of soybean /5-amylase was first achieved by Fukumoto and Tsujisaka (4), who demonstrated an apparent hexagonal crys-
343
344
Y. MORITA, S. AIBARA, H. YAMASHITA, F. YAGI, T. SUGANUMA, and K. HIROMI
tal system, though this was not confirmed. However, attempts at purification according to their method were unsuccessful due to the instability of the enzyme. In the present investigation, /3-amylase was purified by ammonium sulfate precipitation, heat treatment, ion-exchange chromatography with CM- and DEAE-Sephadex, and gel filtration with Sephadex G-100. High recovery of enzymatic activity was obtained in the presence of /3-mercaptoethanol and EDTA, as in the case of barley /S-amylase (9). Finally, the purified enzyme was crystallized with ammonium sulfate, and preliminary X-ray crystallographic data were obtained. EXPERIMENTAL Assay Methods—/)-Amylase activity was measured by the method of Bernfeld (10) with some modifications. One gram of amylopectin, Schoch's B fraction (11), was dissolved in 100 ml of 0.02 M acetate buffer, pH 5.0, instead of 0.016 M buffer, pH 4.8, and the reaction was performed at 37° instead of 20°. The absorbance of the reduction product of 3, 5-dinitrosalicylate was determined at 540 nm. Some difficulty was encountered with /!amylase due to its instability. Full activity was retained in concentrated solutions of the enzyme in the presence of EDTA and jSmercaptoethanol, as in the case of barley /3amylase (9), but these substances did not stabilize the enzyme in highly diluted solutions (less than 10 fig protein per ml). Therefore, the activity was determined immediately after the enzyme had been appropriately diluted at low temperature (in an ice bath). One amylase unit (A.U.) was defined as the amount of enzyme which liberates 1 //mole maltose per min in the reaction mixture at 37°. Specific activity was expressed as A.U. per mg of protein. Protein content was determined according to Lowry et al. (12) using bovine serum albumin as a standard. Purification Procedure—Defatted soybean meal was generously supplied by Honen Seiyu Co., Tokyo. The meal was milled and sieved through 40 mesh. Step 1. Extraction: One kirogram of soy-
bean flour was suspended in 10 liters of 10 mM acetate buffer, pH 5.4. The suspension was stirred for 2 hr and centrifuged. The pH of the supernatant was adjusted to 5.2 by the addition of 1M acetic acid. The precipitate was filtered in a cold room to obtain a clear filtrate. A total of 28 kg of flour was treated and 227 liters of extract was obtained. Step 2. Ammonium sulfate precipitation : The enzyme was precipitated by ammonium sulfate (80% saturation) and collected by filtration. The precipitate was dissolved in 4 volumes of distilled water containing 3.5 mM /3-mercaptoethanol and 1 mM EDTA, and ammonium sulfate was added to make 25% saturation. After the precipitate had been removed, a further amount of ammonium sulfate was added to make 60% saturation. The precipitate was collected by filtration. Step 3. Dialysis and heat treatment: The protein was dialyzed against 10 mM acetate buffer, pH 5.0, containing 10 mM calcium acetate and 18 mM /3-mercaptoethanol at 5° for 4 days. The precipitate formed was removed by centrifugation. The clear solution was divided into 1 liter aliquots, and the aliquots were heated at 60° for 20 min with stirring in a water bath and then chilled with icewater. The precipitate formed was removed by filtration, and a total of 8 liters of clear filtrate was obtained. To this solution was added 10 ml of /3-mercaptoethanol (18 mM final concentration), 14 g of EDTA (5 mM), and then solid ammonium sulfate to give 70% saturation. The precipitate was collected by centrifugation. Step 4. Ammonium sulfate fractionation: The precipitate was dissolved in 0.05 M acetate buffer, pH 5.4, containing 18 mM /3-mercaptoethanol and 5 mM EDTA. To this solution, saturated ammonium sulfate solution was added to give 40% saturation, and the precipitate was spun off. Saturated ammonium sulfate solution was added to the supernatant to give 55% saturation, and the precipitate was collected by centrifugation. The precipitate was dialyzed against 50 mM phosphate buffer, pH 7.0, containing 18 mM /3-mercaptoethanol and 1 mM EDTA. The dialyzate was then fractionated with ammonium sulfate. Two fractions, / . Biochem.
X-RAY CRYSTALLOGRAPHY OF SOYBEAN 0-AMYLASE
40—55% saturated and 55-70% saturated fractions, were obtained. Each fraction was dialyzed against 50 mM acetate buffer, pH 5.0, containing 3.5 mM /3-mercaptoethanol at 5° for 5 days, and the precipitate was spun off. Both fractions exhibited similar values of total and specific activities. However, as will be described later, the fractions contained different relative amounts of two /9-amylase components. Step 5. CM-Sephadex chromatography: Cation-exchange chromatography of the above two fractions was carried out on a CM-Sephadex C-50 column, 2.5x93 cm, equilibrated with 50 mM acetate buffer, pH 5.0, containing 3.5 mM /)-mercaptoethanol. Elution was performed by increasing the pH of buffer as follows: 1, 50 mM acetate buffer, pH 5.0, to pH 5.5 (500 ml each); 2, 50 mM acetate buffer, pH 5.5, to 50 mM phosphate buffer, pH 6.4, (200 ml each); 3, 50 mM phosphate buffer, pH 6.4, to 50 mM phosphate buffer, pH 7.0, (500 ml each). Two active fractions, Components I and II, were obtained. The corresponding fractions of each component in the two chromatographic runs were combined separately, and ammonium sulfate was added to give 70% saturation. Each precipitate was collected by centrifugation, and dialyzed against 50 mM phosphate buffer, pH 7.0, containing 3.5 mM fimercaptoethanol at 5° for 4 days. Step 6. DEAE-Sephadex chromatography: Anion-exchange chromatography was carried out for the two components on a DEAE-Sephadex column, 2.5x92 cm, equilibrated with 50 mM phosphate buffer, pH 7.0, containing 3.5 mM j9-mercaptoethanol. After the enzyme solution had been put onto the column, the column was ^vashed with 1 liter of the above buffer solution. Elution was performed by increasing NaCl concentration linearly from 0 to 0.5 M in the same buffer. The appropriate fractions of the eluates for the two components were collected separately and dialyzed against 80% saturated ammonium sulfate solution containing 18 mM /3-mercaptoethanol and 5 mM EDTA. Step 7. Gel filtration: The two components were finally purified by gel filtration on a Sephadex G-100 column equilibrated with 0.1 Vol. 77, No, 2, 1975
345
M phosphate buffer, pH 6.0, containing 0.2 M NaCl and 7 mM /S-mercaptoethanol at 4°. The enzyme precipitate described above was collected by centrifugation and dissolved in the equilibration buffer (protein concentration 50 mg/ml). A 10 ml aliquot was applied to the column and chromatography was performed with the same buffer solution. The fractions with highest activities in repeated runs for both components were collected. The collected solutions were dialyzed against 80% saturated ammonium sulfate solution containing 50 mM acetate buffer, pH 5.4, 18 mM /9-mercaptoethanol, and 5 mM EDTA. Step 8. Crystallization: Crystallization of Component I was achieved as follows. A suspension of the enzyme in 80% saturated ammonium sulfate solution was centrifuged. The precipitate -was dissolved at 0° in a minimum amount of 0.2 M acetate buffer, pH 5.4, containing 18 mM /)-mercaptoethanol and 5 mM EDTA, and the solution was left to stand at 4°. In this solution, the protein concentration was 140 mg/ml, and the ammonium sulfate was nearly at 50% saturation. After 4 days crystals appeared, and more than 98% of the enzyme had crystallized after 3 weeks. For the crystallization of Component II, a suspension of the enzyme in 80% saturated ammonium sulfate solution was centrifuged, and the precipitate was dissolved in 0.2 M acetate buffer, pH 5.4, containing 18 mM /S-mercaptoethanoI and 5 mM EDTA. In this solution the protein concentration was 170 mg/ml and the ammonium sulfate was at 44% saturation. To this solution was added saturated ammonium sulfate solution at room temperature until the enzyme solution became slightly turbid. The saturation of ammonium sulfate was found to be near 50% at this point. The solution was chilled in ice-water, where the turbidity disappeared, and was left to stand at 4°. Crystals appeared after'3 days and the crystallization was completed after 4 weeks. Disc Eledrophoresis—Disc electrophoresis was performed according to Davis (13). Polyacrylamide gels were prepared with 6.0% monomer concentration. The proteins were located by staining with Amide Black 10B. Sedimentation—Sedimentation experiments
346
Y. MORITA, S. AIBARA, H. YAMASHITA, F. YAGI, T. SUGANUMA, and K. HIROMI
were carried out at 20° in a Spinco model E analytical ultracentrifuge equipped with Schlieren optics. The sedimentation velocity was determined at 59,780 rpm, and the values were corrected to SJO Calculation of the sedimentation coefficient was performed by using the partial specific volume, v, of 0.73 cm*g"1, as derived from the amino acid composition of the enzyme protein (8). X-ray Diffraction—Crystals were mounted in thin-walled glass capillaries, produced by Paul Raebiger, Berlin-Spandau, Germany, in the conventional manner (14). X-ray photographs were taken at room temperature with a Buerger precession camera, Rigaku Denki Co., with Ni-filtered CuKa radiation from a Philips fine-focusing tube. Sakura X-ray film, Industrial type N, was used.
RESULTS AND DISCUSSION
Purification of Soybean p-Amy lose—The elution patterns obtained in the purification procedure for soybean /?-amylase by successive column chromatographies are shown in Figs. 1—3. Figure 1A shows the elution profile from CM-Sephadex for the fraction obtained by 55— 70% saturation with ammonium sulfate; two active components, I and II, are seen. The activity ratio of the two components was found to be about 1 : 10. The 40-55% saturation fraction exhibited a similar elution pattern, but the activity ratio of the two components was about 6 : 10 (Fig. IB). Thus Component I was precipitated preferentially at the lower concentration of ammonium sulfate. Components I and II were collected separately, as shown by horizontal bars in the figure, and
(
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- 20
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2
3
I
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I
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-
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rocedure •.. Preparation of Large Single Crystals— Large crystals were formed when Component I solution (30 mg/ml) in cellophane tubing was dialyzed at 4° against 40% saturated ammonium sulfate solution buffered with 0.1 M acetate, pH 5.4, containing 0.1 M NaCI, 18 mM £-mercaptoethanol, and 5 mM EDTA. Crystals appeared after 4 days and grew slowly to a size suitable for X-ray analysis (0.4x0.4x0.4 mm). The mother liquor, still containing the enzyme at high concentration, was removed carefully,
350
Y. MORITA, S. AIBARA, H. YAMASHITA, F. YAGI, T. SUGANUMA, and K. HIROMI
and the crystals were washed with 45 and 50% saturated ammonium sulfate solutions successively and finally transferred to 55% saturated ammonium sulfate solution. Alternatively, the preparation of large crystals was performed with the dialysis apparatus of Zeppezauer et al. (15). Thus, 0.2 ml of the enzyme solution, 50 mg/ml, was poured into the apparatus, 3 mm inner diameter, and dialyzed at 4° against 55% saturated ammonium sulfate solution containing
0.1 M acetate buffer, pH 5.4, 0.1 M NaCl, 18 mM /3-mercaptoethanol, and 5 mM EDTA. Crystals appeared after 2 days and grew large (0.5x0.5x0.5 mm) within 2 weeks. A microphotograph of crystals produced by this method is shown in Fig. 7. On the other hand, Component II was crystallized at higher ammonium sulfate concentrations. Thus, the crystals appeared and grew slowly when the enzyme solution (30 mg/ml) in cellophane tubing was dialyzed against 45% saturated ammonium sulfate solution. The crystals were washed with 50 and 55% saturated ammonium sulfate solutions successively and finally transferred to 60% saturated ammonium sulfate solution. Crystallization was alternatively achieved with the apparatus of Zeppezauer et al. as in the case of Component I. The crystal form of Component II was found to be identical with that of Component I by X-ray diffraction. It was noted that the crystallization of the enzyme by dialysis against 40% saturated ammonium sulfate solution buffered at pH 4.0 in the apparatus of Zeppezauer et al. gave another type of crystal, which may be identical with that reported by Fukumoto and Tsujisaka (4). These crystals seemed to belong to the hexagonal system in appearance, but curiously they did not give a characteristic X-ray diffraction pattern, probably because of very rapid deterioration under X-ray irradiation under the conditions used.
Fig. 7. Microphotograph of single crystals of soybean ^-amylase (Component I). The crystals were formed in the apparatus of Zeppezauer et al. (see the text).
b Fig. 8. Precession photographs (8°) of soybean 0-amylase (Component I), (a) Photograph with beam along the rhombohedral axis, (b) hkO zone, and (c) hOl zone. The exposure time was 5 hr for each photograph. / . Biochem.
3C-RAY CRYSTALLOGRAPHY OF SOYBEAN 0-AMYLASE
Crystallographic Data—Figure 8 (a) shows an 8° precession photograph taken with the X-ray beam parallel to the rhombohedral axis of the crystal. The photographs with the beam parallel to the other two axes were of the same pattern, showing that the crystal belongs to the trigonal crystal system. Figure 8 (b) and (c) shows precession photographs (8°) with the X-ray beam parallel to the hexagonal c and a axes, giving hkO and hOI, respectively. These and upper-level precession photographs •of principal reciprocal lattice planes indicated that the space group was P3i21 or P3j21 and unit-cell parameters were a=86.1 A and c— 144.4 A. The volume of the unit cell was •927,000 A*. Assumption of a crystal density of 1.25 g/cm* yields the asymmetric unit weight of 116,000 daltons. Therefore, it can be concluded that one asymmetric unit contains one molecule of ^-amylase and that the percentage of protein in the crystal is about 52, assuming a molecular weight of 60,000 daltons (8). The •crystal has a volume of 2.58 A* per atomic .mass unit. The X-ray data show that the present •crystals of soybean /3-amylase will be suitable for further structural analysis in preference to various other crystals of a- and /9-amylases hitherto reported in preliminary X-ray studies {6, 16, 17). As structural analysis requires isomorphous replacement of heavy atoms, a search for heavy atom derivatives is now under way. Already £-chloromercuriben2enesulfonate has produced visible changes in the intensities, probably by combination with reactive sulfhydryl groups in the enzyme molecule (
Crystallization and Preliminary X-ray Investigation of Soybean /3-Amylase Yuhei MORITA,* Shigeo AIBARA,* Honami YAMASHITA,* Fumio YAGI,* Toshihiko SUGANUMA,** and Keitaro HIROMI** * Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, and ** Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, June 22, 1974
J9-Amylase [1,4-a-D-glucan maltohydrolase, EC 3.2.1.2] has been purified from defatted soybean meal by fractional precipitation with ammonium sulfate, ionexchange chromatography on CM- and DEAE-Sephadex and gel filtration chromatography on Sephadex G-100. Two different components of ^-amylase were crystallized from ammonium sulfate solutions, and the homogeneity of each preparation was confirmed by sedimentation and disc electrophoretic analyses. Both components of soybean /3-amylase formed large single crystals (trigonal crystal system) from 40—50% saturated ammonium sulfate solution buffered at pH 5.4 on dialyzing concentrated protein solution in the apparatus of Zeppezauer et al. Preliminary X-ray diffraction data gave a hexagonal lattice with unit cell dimensions a=86.1 A and c=144.4 A. The space group corresponds to P3i21 or P3j21, and one asymmetric unit contains one molecule of /3-amylase, assuming a crystal density of 1.25 g/ml and a molecular weight of the enzyme of 60,000 daltons. In this case, the crystal has a volume of 2.53 A8 per atomic mass unit, and the percentage of protein in the crystal is about 52.
"Crystalline 0-amylases [EC 3.2.1.2] have been prepared from sweet potato, barley, wheat, soybean, and Japanese-radish (1—5). The first X-ray investigation was done by Colman and Matthews (6) using tetragonal crystals of the sweet potato enzyme, prepared according to the method of Balls et al. (7). However, it seems difficult to expect a structural analysis at high resolution from these crystals because of the large dimensions of the unit cell, due to the high molecular weight of sweet potato Vol. 77, No. 2, 1975
/3-amylase (196,000 daltons) and the highly symmetric packing of many molecules in a unit cell (P4i22). The object of the present study was to prepare large single crystals, suitable for Xray analysis, of /9-amylase from soybean. This is a monomeric protein and has a lower molecular weight than the sweet potato enzyme (5). Crystallization of soybean /5-amylase was first achieved by Fukumoto and Tsujisaka (4), who demonstrated an apparent hexagonal crys-
343
344
Y. MORITA, S. AIBARA, H. YAMASHITA, F. YAGI, T. SUGANUMA, and K. HIROMI
tal system, though this was not confirmed. However, attempts at purification according to their method were unsuccessful due to the instability of the enzyme. In the present investigation, /3-amylase was purified by ammonium sulfate precipitation, heat treatment, ion-exchange chromatography with CM- and DEAE-Sephadex, and gel filtration with Sephadex G-100. High recovery of enzymatic activity was obtained in the presence of /3-mercaptoethanol and EDTA, as in the case of barley /S-amylase (9). Finally, the purified enzyme was crystallized with ammonium sulfate, and preliminary X-ray crystallographic data were obtained. EXPERIMENTAL Assay Methods—/)-Amylase activity was measured by the method of Bernfeld (10) with some modifications. One gram of amylopectin, Schoch's B fraction (11), was dissolved in 100 ml of 0.02 M acetate buffer, pH 5.0, instead of 0.016 M buffer, pH 4.8, and the reaction was performed at 37° instead of 20°. The absorbance of the reduction product of 3, 5-dinitrosalicylate was determined at 540 nm. Some difficulty was encountered with /!amylase due to its instability. Full activity was retained in concentrated solutions of the enzyme in the presence of EDTA and jSmercaptoethanol, as in the case of barley /3amylase (9), but these substances did not stabilize the enzyme in highly diluted solutions (less than 10 fig protein per ml). Therefore, the activity was determined immediately after the enzyme had been appropriately diluted at low temperature (in an ice bath). One amylase unit (A.U.) was defined as the amount of enzyme which liberates 1 //mole maltose per min in the reaction mixture at 37°. Specific activity was expressed as A.U. per mg of protein. Protein content was determined according to Lowry et al. (12) using bovine serum albumin as a standard. Purification Procedure—Defatted soybean meal was generously supplied by Honen Seiyu Co., Tokyo. The meal was milled and sieved through 40 mesh. Step 1. Extraction: One kirogram of soy-
bean flour was suspended in 10 liters of 10 mM acetate buffer, pH 5.4. The suspension was stirred for 2 hr and centrifuged. The pH of the supernatant was adjusted to 5.2 by the addition of 1M acetic acid. The precipitate was filtered in a cold room to obtain a clear filtrate. A total of 28 kg of flour was treated and 227 liters of extract was obtained. Step 2. Ammonium sulfate precipitation : The enzyme was precipitated by ammonium sulfate (80% saturation) and collected by filtration. The precipitate was dissolved in 4 volumes of distilled water containing 3.5 mM /3-mercaptoethanol and 1 mM EDTA, and ammonium sulfate was added to make 25% saturation. After the precipitate had been removed, a further amount of ammonium sulfate was added to make 60% saturation. The precipitate was collected by filtration. Step 3. Dialysis and heat treatment: The protein was dialyzed against 10 mM acetate buffer, pH 5.0, containing 10 mM calcium acetate and 18 mM /3-mercaptoethanol at 5° for 4 days. The precipitate formed was removed by centrifugation. The clear solution was divided into 1 liter aliquots, and the aliquots were heated at 60° for 20 min with stirring in a water bath and then chilled with icewater. The precipitate formed was removed by filtration, and a total of 8 liters of clear filtrate was obtained. To this solution was added 10 ml of /3-mercaptoethanol (18 mM final concentration), 14 g of EDTA (5 mM), and then solid ammonium sulfate to give 70% saturation. The precipitate was collected by centrifugation. Step 4. Ammonium sulfate fractionation: The precipitate was dissolved in 0.05 M acetate buffer, pH 5.4, containing 18 mM /3-mercaptoethanol and 5 mM EDTA. To this solution, saturated ammonium sulfate solution was added to give 40% saturation, and the precipitate was spun off. Saturated ammonium sulfate solution was added to the supernatant to give 55% saturation, and the precipitate was collected by centrifugation. The precipitate was dialyzed against 50 mM phosphate buffer, pH 7.0, containing 18 mM /3-mercaptoethanol and 1 mM EDTA. The dialyzate was then fractionated with ammonium sulfate. Two fractions, / . Biochem.
X-RAY CRYSTALLOGRAPHY OF SOYBEAN 0-AMYLASE
40—55% saturated and 55-70% saturated fractions, were obtained. Each fraction was dialyzed against 50 mM acetate buffer, pH 5.0, containing 3.5 mM /3-mercaptoethanol at 5° for 5 days, and the precipitate was spun off. Both fractions exhibited similar values of total and specific activities. However, as will be described later, the fractions contained different relative amounts of two /9-amylase components. Step 5. CM-Sephadex chromatography: Cation-exchange chromatography of the above two fractions was carried out on a CM-Sephadex C-50 column, 2.5x93 cm, equilibrated with 50 mM acetate buffer, pH 5.0, containing 3.5 mM /)-mercaptoethanol. Elution was performed by increasing the pH of buffer as follows: 1, 50 mM acetate buffer, pH 5.0, to pH 5.5 (500 ml each); 2, 50 mM acetate buffer, pH 5.5, to 50 mM phosphate buffer, pH 6.4, (200 ml each); 3, 50 mM phosphate buffer, pH 6.4, to 50 mM phosphate buffer, pH 7.0, (500 ml each). Two active fractions, Components I and II, were obtained. The corresponding fractions of each component in the two chromatographic runs were combined separately, and ammonium sulfate was added to give 70% saturation. Each precipitate was collected by centrifugation, and dialyzed against 50 mM phosphate buffer, pH 7.0, containing 3.5 mM fimercaptoethanol at 5° for 4 days. Step 6. DEAE-Sephadex chromatography: Anion-exchange chromatography was carried out for the two components on a DEAE-Sephadex column, 2.5x92 cm, equilibrated with 50 mM phosphate buffer, pH 7.0, containing 3.5 mM j9-mercaptoethanol. After the enzyme solution had been put onto the column, the column was ^vashed with 1 liter of the above buffer solution. Elution was performed by increasing NaCl concentration linearly from 0 to 0.5 M in the same buffer. The appropriate fractions of the eluates for the two components were collected separately and dialyzed against 80% saturated ammonium sulfate solution containing 18 mM /3-mercaptoethanol and 5 mM EDTA. Step 7. Gel filtration: The two components were finally purified by gel filtration on a Sephadex G-100 column equilibrated with 0.1 Vol. 77, No, 2, 1975
345
M phosphate buffer, pH 6.0, containing 0.2 M NaCl and 7 mM /S-mercaptoethanol at 4°. The enzyme precipitate described above was collected by centrifugation and dissolved in the equilibration buffer (protein concentration 50 mg/ml). A 10 ml aliquot was applied to the column and chromatography was performed with the same buffer solution. The fractions with highest activities in repeated runs for both components were collected. The collected solutions were dialyzed against 80% saturated ammonium sulfate solution containing 50 mM acetate buffer, pH 5.4, 18 mM /9-mercaptoethanol, and 5 mM EDTA. Step 8. Crystallization: Crystallization of Component I was achieved as follows. A suspension of the enzyme in 80% saturated ammonium sulfate solution was centrifuged. The precipitate -was dissolved at 0° in a minimum amount of 0.2 M acetate buffer, pH 5.4, containing 18 mM /)-mercaptoethanol and 5 mM EDTA, and the solution was left to stand at 4°. In this solution, the protein concentration was 140 mg/ml, and the ammonium sulfate was nearly at 50% saturation. After 4 days crystals appeared, and more than 98% of the enzyme had crystallized after 3 weeks. For the crystallization of Component II, a suspension of the enzyme in 80% saturated ammonium sulfate solution was centrifuged, and the precipitate was dissolved in 0.2 M acetate buffer, pH 5.4, containing 18 mM /S-mercaptoethanoI and 5 mM EDTA. In this solution the protein concentration was 170 mg/ml and the ammonium sulfate was at 44% saturation. To this solution was added saturated ammonium sulfate solution at room temperature until the enzyme solution became slightly turbid. The saturation of ammonium sulfate was found to be near 50% at this point. The solution was chilled in ice-water, where the turbidity disappeared, and was left to stand at 4°. Crystals appeared after'3 days and the crystallization was completed after 4 weeks. Disc Eledrophoresis—Disc electrophoresis was performed according to Davis (13). Polyacrylamide gels were prepared with 6.0% monomer concentration. The proteins were located by staining with Amide Black 10B. Sedimentation—Sedimentation experiments
346
Y. MORITA, S. AIBARA, H. YAMASHITA, F. YAGI, T. SUGANUMA, and K. HIROMI
were carried out at 20° in a Spinco model E analytical ultracentrifuge equipped with Schlieren optics. The sedimentation velocity was determined at 59,780 rpm, and the values were corrected to SJO Calculation of the sedimentation coefficient was performed by using the partial specific volume, v, of 0.73 cm*g"1, as derived from the amino acid composition of the enzyme protein (8). X-ray Diffraction—Crystals were mounted in thin-walled glass capillaries, produced by Paul Raebiger, Berlin-Spandau, Germany, in the conventional manner (14). X-ray photographs were taken at room temperature with a Buerger precession camera, Rigaku Denki Co., with Ni-filtered CuKa radiation from a Philips fine-focusing tube. Sakura X-ray film, Industrial type N, was used.
RESULTS AND DISCUSSION
Purification of Soybean p-Amy lose—The elution patterns obtained in the purification procedure for soybean /?-amylase by successive column chromatographies are shown in Figs. 1—3. Figure 1A shows the elution profile from CM-Sephadex for the fraction obtained by 55— 70% saturation with ammonium sulfate; two active components, I and II, are seen. The activity ratio of the two components was found to be about 1 : 10. The 40-55% saturation fraction exhibited a similar elution pattern, but the activity ratio of the two components was about 6 : 10 (Fig. IB). Thus Component I was precipitated preferentially at the lower concentration of ammonium sulfate. Components I and II were collected separately, as shown by horizontal bars in the figure, and
(
A
100 -
i
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1
2
3
I
I
I
k
a
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-
r
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\
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rocedure •.. Preparation of Large Single Crystals— Large crystals were formed when Component I solution (30 mg/ml) in cellophane tubing was dialyzed at 4° against 40% saturated ammonium sulfate solution buffered with 0.1 M acetate, pH 5.4, containing 0.1 M NaCI, 18 mM £-mercaptoethanol, and 5 mM EDTA. Crystals appeared after 4 days and grew slowly to a size suitable for X-ray analysis (0.4x0.4x0.4 mm). The mother liquor, still containing the enzyme at high concentration, was removed carefully,
350
Y. MORITA, S. AIBARA, H. YAMASHITA, F. YAGI, T. SUGANUMA, and K. HIROMI
and the crystals were washed with 45 and 50% saturated ammonium sulfate solutions successively and finally transferred to 55% saturated ammonium sulfate solution. Alternatively, the preparation of large crystals was performed with the dialysis apparatus of Zeppezauer et al. (15). Thus, 0.2 ml of the enzyme solution, 50 mg/ml, was poured into the apparatus, 3 mm inner diameter, and dialyzed at 4° against 55% saturated ammonium sulfate solution containing
0.1 M acetate buffer, pH 5.4, 0.1 M NaCl, 18 mM /3-mercaptoethanol, and 5 mM EDTA. Crystals appeared after 2 days and grew large (0.5x0.5x0.5 mm) within 2 weeks. A microphotograph of crystals produced by this method is shown in Fig. 7. On the other hand, Component II was crystallized at higher ammonium sulfate concentrations. Thus, the crystals appeared and grew slowly when the enzyme solution (30 mg/ml) in cellophane tubing was dialyzed against 45% saturated ammonium sulfate solution. The crystals were washed with 50 and 55% saturated ammonium sulfate solutions successively and finally transferred to 60% saturated ammonium sulfate solution. Crystallization was alternatively achieved with the apparatus of Zeppezauer et al. as in the case of Component I. The crystal form of Component II was found to be identical with that of Component I by X-ray diffraction. It was noted that the crystallization of the enzyme by dialysis against 40% saturated ammonium sulfate solution buffered at pH 4.0 in the apparatus of Zeppezauer et al. gave another type of crystal, which may be identical with that reported by Fukumoto and Tsujisaka (4). These crystals seemed to belong to the hexagonal system in appearance, but curiously they did not give a characteristic X-ray diffraction pattern, probably because of very rapid deterioration under X-ray irradiation under the conditions used.
Fig. 7. Microphotograph of single crystals of soybean ^-amylase (Component I). The crystals were formed in the apparatus of Zeppezauer et al. (see the text).
b Fig. 8. Precession photographs (8°) of soybean 0-amylase (Component I), (a) Photograph with beam along the rhombohedral axis, (b) hkO zone, and (c) hOl zone. The exposure time was 5 hr for each photograph. / . Biochem.
3C-RAY CRYSTALLOGRAPHY OF SOYBEAN 0-AMYLASE
Crystallographic Data—Figure 8 (a) shows an 8° precession photograph taken with the X-ray beam parallel to the rhombohedral axis of the crystal. The photographs with the beam parallel to the other two axes were of the same pattern, showing that the crystal belongs to the trigonal crystal system. Figure 8 (b) and (c) shows precession photographs (8°) with the X-ray beam parallel to the hexagonal c and a axes, giving hkO and hOI, respectively. These and upper-level precession photographs •of principal reciprocal lattice planes indicated that the space group was P3i21 or P3j21 and unit-cell parameters were a=86.1 A and c— 144.4 A. The volume of the unit cell was •927,000 A*. Assumption of a crystal density of 1.25 g/cm* yields the asymmetric unit weight of 116,000 daltons. Therefore, it can be concluded that one asymmetric unit contains one molecule of ^-amylase and that the percentage of protein in the crystal is about 52, assuming a molecular weight of 60,000 daltons (8). The •crystal has a volume of 2.58 A* per atomic .mass unit. The X-ray data show that the present •crystals of soybean /3-amylase will be suitable for further structural analysis in preference to various other crystals of a- and /9-amylases hitherto reported in preliminary X-ray studies {6, 16, 17). As structural analysis requires isomorphous replacement of heavy atoms, a search for heavy atom derivatives is now under way. Already £-chloromercuriben2enesulfonate has produced visible changes in the intensities, probably by combination with reactive sulfhydryl groups in the enzyme molecule (
