CRYSTAL GROWTH & DESIGN 2002 VOL. 2, NO. 5 321-324
Communications Significant Decrease in the Solubility of Glucose Isomerase Crystals under High Pressure Yoshihisa Suzuki,*,† Gen Sazaki,‡,§ Kalevi Visuri,# Katsuhiro Tamura,† Kazuo Nakajima,‡ and Shin-ichiro Yanagiya$ Department of Chemical Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjima, Tokushima 770-8506, Japan, Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan, Center for Interdisciplinary Research, Tohoku University, Sendai 980-8578, Japan, Macrocrystal Oy, Ruukintie 20 F, 02,320 Espoo, Finland, and Department of Optical Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan Received February 28, 2002;
Revised Manuscript Received May 12, 2002
ABSTRACT: Solubility of glucose isomerase (from Streptomyces rubiginosus) crystals was measured in situ at 0.1 and 100 MPa. An equilibrium temperature of the crystal with the solution of a given concentration was measured using a two-beam interferometer. The solubility of the crystal decreased to about one-ninth with increasing pressure from 0.1 to 100 MPa at 30 °C. This means that the supersaturation, σ () ln C/Ce, C ) protein concentration, Ce ) solubility), increases significantly with increasing pressure at the same temperature. This strongly suggests that the substantial acceleration of the crystallization of glucose isomerase with increasing pressure reported by Visuri et al. is due to the significant decrease in the solubility. The enthalpy and entropy of the dissolution were estimated from the van’t Hoff plots. The volume change accompanying the dissolution took a large positive value as ∆V ) 54 ( 31 cm3 mol-1 at 30 °C. X-ray crystallography is indispensable to analyze the three-dimensional structure of protein molecule at an atomic level. However, protein crystallization is still a bottleneck of the crystallography. Many attempts have been so far carried out to find appropriate control parameters in the protein crystallization. Among those, pressure can be a good candidate, since hydrostatic pressure affects the whole system uniformly and can be changed very rapidly. Thus far, a lot of studies on the protein crystallization under high pressure have been reported.1-17 Visuri et al. reported, for the first time, that the crystallization of glucose isomerase crystals was significantly enhanced with increasing pressure.1 After their report, effects of pressure on the protein crystallization began to receive much attention. In the early studies, effects of pressure on the tetragonal crystal of hen egg-white lysozyme were often investigated,2-10 since extensive studies on the growth of the tetragonal lysozyme crystal had been already reported at atmospheric pressure.18 Most of these studies on the tetragonal lysozyme crystal revealed that the solubility increases,2,5,7-8,10,14 and the growth rate of the crystal2,4-8,12 * To whom correspondence should be sent. Dr. Yoshihisa Suzuki, 2-1 Minamijosanjima, Tokushima 770-8506, Japan; phone: +81-88-656-7415; fax: +81-88-655-7025; e-mail:
[email protected]. † Department of Chemical Science and Technology, The University of Tokushima. ‡ Institute for Materials Research, Tohoku University. § Center for Interdisciplinary Research, Tohoku University. # Macrocrystal Oy. $ Department of Optical Science and Technology, The University of Tokushima.
and the nucleation rate5 decrease with increasing pressure. From the relation between the growth rate and a supersaturation σ () ln(C/Ce), C: protein concentration, Ce: solubility), it was found that the pressure inhibited the growth kinetics.12 The increase in the average ledge surface energy of the two-dimensional nuclei with pressure explained the decrease in the growth rate.12,17 Effects of pressure on the lysozyme crystal of orthorhombic form has also been studied. In the case of the orthorhombic crystals, the solubility decreases with increasing pressure contrary to the case of the tetragonal form.10 Then, the supersaturation of the orthorhombic crystals increases with pressure; however, the pressure inhibits the growth kinetics of the crystals.19 In the case of another protein, recently, several studies have been reported on the crystallization of subtilisin.11,13,16 The solubility increases with increasing pressure,11,13 and the pressure inhibits the kinetics of the nucleation and the growth processes.13,16 Regardless of the pressure dependence of the solubilities, the growth kinetics of the tetragonal and orthorhombic lysozyme crystals and that of the subtilisin crystal were found to be inhibited by increase in pressure, as mentioned above. Then, we are strongly interested in whether such the negative pressure dependence of the growth kinetics is general or not. As far as we know, glucose isomerase is the only protein whose crystallization is significantly accelerated by an increase in pressure. To investigate whether such enhancement in the crystallization is due to the changes in the solubility or/and growth kinetics, in this
10.1021/cg025509f CCC: $22.00 © 2002 American Chemical Society Published on Web 06/29/2002
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study, the solubility of glucose isomerase crystals was measured at 0.1 and 100 MPa. In the present study, we used glucose isomerase (5 times recrystallization) from Streptomyces rubiginosus (D-xylose ketol-isomerase EC 5.3.1.5, molecular weight: 173 000. Glucose isomerase is a tetramer composed of four identical polypeptides of 43 000 Da.). Glucose isomerase was prepared by Macrocrystal Oy and supplied as a suspension of the small crystals (10-100 µm in size) by Hampton Research. We believe that the purity of the suspension is sufficiently high, since chromatography and electrophoresis with high load of the suspension give only one band.20 Then, we used the glucose isomerase without further purification. The glucose isomerase solution for the crystallization contains 0.91 M ammonium sulfate, 1 mM magnesium sulfate, and these are dissolved in 6 mM tris hydrochloride buffer (pH ) 7.0). The space group of the crystal is I222 and the unit cell dimensions are a ) 9.388 nm, b ) 9.964 nm, c ) 10.290 nm (Z ) 2).21 Solubility of the glucose isomerase crystal was measured using a two-beam interferometer.10,22 By this technique, the concentration distribution around the crystal can be visualized in situ. From the bending of the interference fringes around the crystals, we can determine the equilibrium temperature, Te, of the crystals in the solution of a given protein concentration within 3 h.10,22 The solubility curve was obtained by measuring Te at various protein concentration at 0.1 and 100 MPa. Further details of the measurements are described elsewhere.10 Seed crystals of 0.5-1.0 mm in size were used in this study. They were prepared as follows. The crystal suspension was heated to 40 °C to dissolve the crystals. To remove micro crystals or amorphous precipitates, the solution was filtered through a 0.45 µm filter (polypropylene filter media, Whatman Inc.). The concentration of the filtered solution was determined as 35.4 mg mL-1 using an absorption coefficient R280nm ) 1.04 mL mg-1 cm-1.23 The solution was poured into the cell for the in situ observation (the volume of the cell: 50 µL), and the temperature of the solution was kept at 4 °C. In a day, 2-3 crystals of 0.5-1.0 mm in size were obtained. These were used as the seed crystals. The glucose isomerase crystal and the surrounding interference fringes under high pressure are shown in Figure 1. The concentration of the solution was 35.4 mg mL-1, and the pressure was 100 MPa. When the temperature of the sample was set lower than its equilibrium temperature, the crystal grew (24.7 °C), and the fringes were bent in the vicinity of the crystal (Figure 1a), because of the decrease in the concentration around the crystal. On the other hand, when the temperature was raised higher than its equilibrium temperature, the crystals dissolved (44.1 °C), and the fringes were bent to the opposite direction (Figure 1b). From the observation of the fringes around the crystal, we determined the equilibrium temperature of the crystal and the solution of a given concentration. Details of this technique were reported by Sazaki et al.10,22 Figure 2 shows the solubility curves at 0.1 and 100 MPa. The numerical data are also summarized in Table 1. We have measured all Te using only one seed crystal. For each concentration of protein solution, after Te was measured at 0.1 and 100 MPa, Te was measured again at both the pressures to check the validity of the equilibrium temperatures. The errors in Te are not the standard deviation of multiple trials. The size of the error comes from the accuracy in measuring the straightness of the interference fringes.10,22 We determine the highest and lowest temperatures at which we can judge the bending of the interference fringes. These highest and lowest temperatures correspond to the left (growth) and right (dissolution) sides
Communications
Figure 1. Interferograms around the glucose isomerase crystal under 100 MPa. Concentration of glucose isomerase in bulk solution is 35.4 mg mL-1. (a) Growth (24.7 °C), (b) dissolution (44.1 °C). The scale bar represents 1 mm.
Figure 2. Solubility curves of the glucose isomerase crystals measured by our technique. Pressure: 0.1 MPa (O), 100 MPa (0). Solid curves are the exponential fits, and they are obtained by weighted least mean square method.
of the temperature values of the error bar, and the equilibrium temperature is defined as an average value of these two temperatures. The size of the error in the lower concentration range is larger than that in the higher concentration range. The errors of Te depend on the temperature derivative of the solubility of a target protein. Details of the error are described elsewhere.22 The solubilities less than 295 K under atmospheric pressure agree closely with the previous data (albeit a different stain) taken by Chayen et al.24 They have measured the dependence of the solubility of glucose isomerase (from Arthrobacter strain B3728) on the concentration of ammonium sulfate at 291.2 K under atmospheric pressure. The solubility at 0.9 M ammonium sulfate in 50 mM tris-acetate buffer (pH ) 5.0 to 5.8) is about 1 mg mL-1. In addition, Visuri et al. reported the dependence of the solubility of glucose isomerase (from Streptomyces rubiginosus) on the
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Table 1. Equilibrium Temperature (Te) of Glucose Isomerase at 0.1 and 100 MPaa protein conc (mg mL-1)
Te (K)
error (K)
35.4 17.7 11.8 8.9 5.9 4.4 3.0
(0.1 MPa) 307.5 304.5 302.9 301.0 299.2 294.4 291.4
1.1 1.0 1.5 1.9 5.5 5.0 8.4
35.4 17.7 11.8 8.9 5.9 4.4 3.0
(100 MPa) 314.8 312.4 311.5 308.0 306.3 302.6 302.0
1.3 0.4 0.8 2.7 4.1 6.1 8.2
a Conditions: 0.91 M ammonium sulfate, 1 mM magnesium sulfate in 6 mM Tris-HCl buffer (pH ) 7.0).
temperature at 0.1 MPa.25 They reported the solubility of glucose isomerase at 10 wt % (≈ 0.84 M) of ammonium sulfate. The values are 1, 3, 6, and 16 mg mL-1 at 290, 295, 300, and 305 K, respectively. These values also agree closely with our data shown in Figure 2. The solubility increased with temperature at each pressure (Figure 2). From the figure, it is clear that Te of a certain concentration increased with increasing pressure. In other words, the solubility at 100 MPa was much less than that at 0.1 MPa at the same temperature. This means that the supersaturation, σ, increases with increasing pressure at the same temperature and concentration. This increase in σ enhances the crystallization. In practice, the crystal in equilibrium with the solution at 0.1 MPa began to grow only by applying pressure up to 100 MPa. From these results, it was found out that the substantial acceleration of the crystallization reported by Visuri et al. is due to the significant decrease in the solubility with pressure shown in Figure 2. However, at present, we cannot distinguish whether the growth kinetics is also responsible for the remarkable acceleration of the crystallization with increasing pressure or not. The investigation about the effect of the pressure on the growth kinetics of glucose isomerase crystals is now in progress. From the solubility data, thermodynamic parameters can be calculated using the van’t Hoff plots. If the activity coefficient is assumed to be unity, we can estimate the partial molar enthalpy of dissolution, ∆H, from eq 1 and the partial molar entropy of dissolution, ∆S, from eq 2.
∂ ln Ce
∆H ) -R
∂(1/T)
∂ ln Ce ∆S ) R ln Xe + RT ∂T
Figure 3. van’t Hoff plots of the solubility shown in Figure 2. Pressure: 0.1 MPa (O), 100 MPa (0). Solid lines are obtained by weighted least mean square method. Table 2. List of the Volume Change Accompanying the Dissolution (∆V), the Enthalpy (∆H) and Entropy (∆S) of the Dissolutiona pressure ∆H/kJ mol-1: ∆S/J mol-1 K-1: ∆V/cm3 mol-1:
0.1 MPa
100 MPa
160 ( 40 420 ( 100 54 ( 31b
210 ( 60 580 ( 180
a ∆V is estimated at T ) 30 °C. b Calculated from the eq 4 at P ) 100 MPa.
60 kJ mol-1 at 100 MPa from the slope of each fitting line (Table 2). In a similar way, ∆S was also estimated as ∆S ) 420 ( 100 J mol-1 K-1 at 0.1 MPa and ∆S ) 580 ( 180 J mol-1 K-1 at 100 MPa (Table 2). All errors shown in Table 2 are estimated from ∆Te (Figure 2) with a law of propagation of errors, and they are rather large at the present stage. Thus, the increase in the enthalpy and entropy with the pressure is not significant taking the errors into consideration. More accurate measurement of the equilibrium temperature is necessary for further thermodynamic analysis. From the dependence of the solubility on pressure, the volume change accompanying the dissolution of 1 mol of glucose isomerase molecules, ∆V, is expressed as,
∆V ) -RT (1) (2)
where Ce ) solubility (mg mL-1); Xe ) solubility (mole fraction, Xe ) Ce/173000/55.51 as an approximation); and R ) the gas constant. To estimate ∆H, ln Ce is plotted against 1/T as shown in Figure 3. If we assume that ∆H does not depend on temperature within the experimental temperature range, ∆H is estimated by the linear fitting of the plot. In the present study, weighted fitting was carried out, since the error in Te (∆Te) was larger in lower concentration region. Here w ) 1/∆Te2 was used as the weight for the fitting. Fitted lines are also shown in Figure 3. ∆H was estimated as ∆H ) 160 ( 40 kJ mol-1 at 0.1 MPa and ∆H ) 210 (
∂ ln Ce . ∂p
(3)
Since the solubility of the other proteins is approximately proportional to pressure up to 100 MPa,7,8,10,14 we assumed that the solubility of glucose isomerase is also proportional to pressure and then ∆V is constant up to 100 MPa. If ∆V does not depend on pressure up to a prescribed pressure P MPa, ∆V is expressed as,
∆V ) -RT
ln Ce,P - ln Ce,0.1 P - 0.1
(4)
where Ce,P and Ce,0.1 indicate the solubility at P MPa and 0.1 MPa, respectively. At 30 °C, ∆V was estimated as ∆V ) 54 ( 31 cm3 mol-1 (Table 2). Thus, the volume of the solution expands when the crystal dissolves, and this positive ∆V value gives the negative pressure dependence of the solubility. ∆V generally include the change in (i) the
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constitutive volume of the protein, (ii) the void volume in the molecule, and (iii) the structure of water around the protein, and so on.26 However, the error in ∆V is too large to discuss further at this stage. Glucose isomerase is a tetramer composed of four identical subunits.23 Then, if the dissociation of the tetramer occurs, it probably causes the change in the partial molar volume of the protein, hence ∆V. The dissociation of the tetramer to the dimers in 0.1-0.15 N NaCl at atmospheric pressure was also reported,27 while there are no data under high pressure. To study the dissociation in our system in detail, we should measure the size distribution of the oligomers in the supersaturated solution under high pressure by dynamic light scattering method. High pressure also can change the molar volume of the crystal, hence ∆V. Thus, a high-pressure X-ray crystallography is also indispensable to study ∆V in detail. We conclude that the solubility of the glucose isomerase crystal decreased significantly with increasing pressure. This strongly suggests that the substantial acceleration of the crystallization with increasing pressure reported by Visuri et al. is due to this significant decrease in the solubility. To study an acceleration mechanism of the crystallization with increasing pressure in detail, it is indispensable to study the effects of the pressure on the growth kinetics of the crystals. The enthalpy and entropy of the dissolution were estimated from the van’t Hoff plots. The volume change accompanying the dissolution took a large positive value as ∆V ) 54 ( 31 cm3 mol-1 at 30 °C. Acknowledgment. The authors wish to thank Prof. Tetsuo Inoue of the University of Tokushima for his help in the preparation of the cell for the interferometric measurements and also thank MSc. Viveka Ehrnsten for giving the chromatography and electrophoresis data of glucose isomerase. This work was partially supported by Grant-in-Aid (No. 12750006) of Scientific Research of the Ministry of Education, Culture, Sports, Science, and Technology Japan. This work was performed under the interuniversity cooperative research program of the Institute for Materials Research, Tohoku University.
References (1) Visuri, K.; Kaipainen, E.; Kivimaki, J.; Niemi, H.; Leisola, M.; Palosaari, S. Bio/Technology 1990, 8, 547-549. (2) Gross, M.; Jaenicke, R. FEBS Lett. 1991, 284, 87-90. (3) Gross, M.; Jaenicke, R. Biophys. Chem. 1993, 45, 245-252. (4) Schall, C. A.; Wiencek, J. M.; Yarmush, M.; Arnold, E. J. Cryst. Growth 1994, 135, 548-554.
Communications (5) Suzuki, Y.; Miyashita, S.; Komatsu, H.; Sato, K.; Yagi, T. Jpn. J. Appl. Phys. 1994, 33, L1568-L1570. (6) Saikumar, M. V.; Glatz, C. E.; Larson, M. A. J. Cryst. Growth 1995, 151, 173-179. (7) Lorber, B.; Jenner, G.; Giege, R. J. Cryst. Growth 1996, 158, 103-117. (8) Takano, K. J.; Harigae, H.; Kawamura, Y.; Ataka, M. J. Cryst. Growth 1997, 171, 554-558. (9) Saikumar, M. V.; Glatz, C. E.; Larson, M. A. J. Cryst. Growth 1998, 187, 277-288. (10) Sazaki, G.; Nagatoshi, Y.; Suzuki, Y.; Durbin, S. D.; Miyashita, S.; Nakada, T.; Komatsu, H. J. Cryst. Growth 1999, 196, 204-209. (11) Webb, J. N.; Waghmare, R. Y.; Carpenter, J. F.; Glatz, C. E.; Randolph, T. W. J. Cryst. Growth 1999, 205, 563-574. (12) Suzuki, Y.; Miyashita, S.; Sazaki, G.; Nakada, T.; Sawada, T.; Komatsu, H. J. Cryst. Growth 2000, 208, 638-644. (13) Waghmare, R. Y.; Webb, J. N.; Randolph, T. W.; Larson, M. A.; Glatz, C. E. J. Cryst. Growth 2000, 208, 678-686. (14) Suzuki, Y.; Sawada, T.; Miyashita, S.; Komatsu, H.; Sazaki, G.; Nakada, T. J. Cryst. Growth 2000, 209, 1018-1022. (15) Logtenberg, E. H. P.; Meersman, F.; Rubens, P.; Heremans, K.; Frank J. High-Pressure Res. 2000, 19, 675-680. (16) Waghmare, R. Y.; Pan, X. J.; Glatz, C. E. J. Cryst. Growth 2000, 210, 746-752. (17) Suzuki, Y.; Sazaki, G.; Miyashita, S.; Sawada, T.; Tamura, K.; Komatsu, H. Biochim. Biophys. Acta 2002, 1595, 345356. (18) For a discussion, see for example, Rosenberger, F.; Vekilov, P. G.; Muschol, M.; Thomas, B. R. J. Cryst. Growth 1996, 168, 1-27. (19) (a) Miyashita, S.; Sazaki, G.; Nagatoshi, Y.; Suzuki, Y.; Sawada, T.; Nakada, T.; Komatsu, H.; Nakajima, K.; J. Jpn. Assoc. Cryst. Growth 1999, 26, 192-202 [in Japanese]. (b) Nagatoshi, Y.; Master thesis at Tohoku University, 1999 [in Japanese]. (20) Personal data from a study by Ehrnsten, V., and Visuri, K. (21) Carrell, H. L.; Glusker, J. P.; Burger, V.; Manfre, F.; Tritsch, D.; Biellmann, J.-F. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 4440-4444. (22) Sazaki, G.; Kurihara, K.; Nakada, T.; Miyashita, S.; Komatsu, H. J. Cryst. Growth 1996, 169, 355-360. (23) Products data of Hampton Research. (24) Chayen, N.; Akins, J.; Campbell-Smith, S.; Blow, D. M. J. Cryst. Growth 1988, 90, 112-116. (25) Visuri, K.; Uotila, S. In Principles of Large Scale Crystallization of Proteins, Recent Advances in Macromolecular Crystallization (International Conference), June 2-4, 1996, Le Bischenberg, France. (26) Gekko, K.; Noguchi, H. J. Biol. Chem. 1979, 83, 2706-2714. (27) Carrell, H. L.; Rubin, B. H.; Hurley, T. J.; Glusker, J. P. J. Biol. Chem. 1984, 259, 3230-3236.
CG025509F
