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Impurity effect on defect formation of protein crystals
ARTICLE IN PRESS
Journal of Crystal Growth 275 (2005) e1423–e1429 www.elsevier.com/locate/jcrysgro
Impurity effect on defect formation of protein crystals H. Hondoh, T. Nakada Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan Available online 15 December 2004
Abstract Etch pit formation on lysozyme crystals was observed in situ by using atomic force microscopy. No etch pit was nucleated at low undersaturation (so0:08). Flat-bottomed etch pits (F-pits) were observed to form at undersaturation of 0.08–0.14, and their number was increased with undersaturation. In addition to F-pits, deep flat-bottomed etch pits (DF-pits) appeared when understauration was increased over 0.30. It was found that the locations of F- and DF-pits were independent and randomly nucleated. In the case of pure lysozyme crystals, the bottoms of F-pits were flat at a molecular level. In contrast, streaks consisted of several cords with a length of more than 100 nm were often found at the bottoms of the F-pits on contaminated lysozyme crystals. These streaks would be the one of the origins of F-pits on the contaminated crystals. r 2004 Elsevier B.V. All rights reserved. PACS: 61.72.Ff; 61.72.Ji; 81.10.Dn; 87.14.Ee Keywords: A1. Atomic force microscopy; A1. Defects; A1. Etching; A1. Impurities; B1. Biological macromolecules; B1. Lysozyme
1. Introduction In protein crystal growth, influence of impurity must be considered because target samples are consistently contaminated by other proteins. Even in commercially available proteins, such as lysozyme, several kinds of proteins are contained [1]. These impurities could be incorporated into protein crystals to form lattice defects. In the last decades, therefore, numerous studies about impurity effects on protein crystal growth have been Corresponding author. Fax: +81 77 561 2657.
E-mail address: [email protected] (H. Hondoh).
carried out [2–6]. Vekilov et al. [7] reported the impurity effect on the growth step kinetics. A step velocity significantly increased with increasing purity of samples. Nakada et al. [8] directly observed the incorporation of dimers into crystals at molecular level by atomic force microscopy (AFM). Effects of impurity on crystal quality have also been revealed by AFM and X-ray diffraction experiments [9]. They reported that crystal quality tended to degrade as the impurity concentration increased. On the other hand, however, defects of protein crystals have not been studied well because of a lack of suitable techniques. With developing
0022-0248/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jcrysgro.2004.11.236
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H. Hondoh, T. Nakada / Journal of Crystal Growth 275 (2005) e1423–e1429
synchrotron facilities, X-ray topographic studies have been performed in recent years [10–16], but point defects in the protein crystals could not be observed because of the limit of spatial resolution of this method. Very recently, etch figure method has been successfully applied to investigate defects of protein crystals [17]. In this study, three types of etch pits were observed on the (1 1 0) face of tetragonal lysozyme crystals; a flat-bottomed pit (F-pit), a point-bottomed pit (P-pit) and a deep flat-bottomed pit (DF-pit). DF-pits have the depth of more than 100 nm, while F-pits are of 6 nm depth. It is also revealed that the etch pit density of contaminated lysozyme crystals was greater than that of pure lysozyme crystals. However, the mechanism of defect formation in protein crystals is still unclear. In this paper, we report the in situ observation of etching lysozyme crystals by AFM. Direct measurement of the developing etch pits determined the undersaturations for the pit formation. The behavior of etch pits was investigated to understand the etch pit formation mechanism. In addition, the possible source of the defect is also described.
2. Experimental procedure 2.1. Preparation of seed crystals We employed two varieties of hen egg white lysozyme samples of different purity. The Seikagaku Corporation product was used as a contaminated sample without further purification. A pure sample, which was purified from the Seikagaku product, was purchased from Maruwa Foods Industries. Inc. The purity of these samples was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as 98% and X99.99%, respectively. Seed crystals of lysozyme tetragonal form were obtained by a batch method at 20 1C. The crystallization solution was composed of 100 mM sodium acetate buffer, pH 4.5, 25 mg/ml NaCl and 80 mg/ml lysozyme. Floating crystals, nucleated at a surface of the solution, were applied for further studies as seed crystals
because they were not influenced by containers. All the solution in these studies was filtered with Millipore 0.22-mm membranes before use. 2.2. Polyurethane substrate We applied polyurethane-coated cover glasses as substrates to prevent detachment of crystals from substrates during etching. Polyurethane Desmolac 4340, which was shared from Sumika Bayer Urethane Co. Ltd., was diluted by 20% with benzene, and then coated cover glasses with itself. The glasses were dried overnight. Because Desmolac 4340 does not elite into the crystallization solution after drying up, it will have no effect on crystal growth. The polyurethane substrates fixed protein crystals so tightly that the seed crystals did not move even in the solution containing no protein. 2.3. AFM observation of etching crystal Atomic force microscopy was used for in situ observation of etching lysozyme crystals. The AFM was a NanoScope E from Digital Instruments operating in the contact mode with Si3N4 cantilevers (the spring constant was 0.06 N/m). To increase a chance of DF-pit formation, the contaminated sample was mainly applied for the seed crystal preparation and the etch figure observation. The etching and the observation were performed in a fluid-cell on polyurethane substrates at 20 1C. The composition of the solution was 100 mM sodium acetate buffer, pH 4.5, 25 mg/ ml NaCl and lysozyme. Undersaturation sðs ¼ jC Cej=CeÞ was controlled by varying the concentration of lysozyme from 16 to 7 mg/ml ðs ¼ 0:0020:56Þ: The equilibrium concentration at 20 1C in this solution is 16.1 mg/ml. The solutions were kept to flow at 40 ml/min by a peristaltic pump to neglect the temperature vibration in the fluid-cell. Since the volume of the fluid-cell is less than 100 ml, all the solution in the cell would be replaced within 3 min. To replace the solution in the fluid-cell completely, the solutions are maintained to flow more than 30 min after they reached the cell. The solubility of the lysozyme crystals was based on the results of Sazaki et al. [18].
ARTICLE IN PRESS H. Hondoh, T. Nakada / Journal of Crystal Growth 275 (2005) e1423–e1429
3. Results and discussion 3.1. In situ observation of etching crystals Etch pit formation on lysozyme crystals was observed in situ by AFM. There were only two types of etch pits, F- and DF-pits, on the (1 1 0) faces in the series of this study. No spiral growth hillock and P-pit was found. The F-pits appeared when the undersaturation was in the range of 0.08–0.14 (Fig. 1(a)). The depth of these F-pits was 6 or 12 nm. Considering that the unit height of growing 2D islands is 6 nm [19], the unit depth of dissolving pits is the same as that during growth. The number of F-pits was increased with understauration (Fig. 1(b)). When the undersaturation was raised over 0.30, DF-pits were developed in
e1425
addition to F-pits (Fig. 1(c)). The F-pits tended to nucleate at a fresh surface on other developing Fpits as shown in Fig. 1(c). This indicates that the origin of the F-pits appeared at the surface by dissolving. Because the depth of the nucleated Fpits was also 6 nm, the strains from the source of the pit would distribute only within 6 nm. The independence of locations of F- and DF-pits are also suggested in the assumption. Furthermore, because F-pits were formed at the undersaturation lower than that for DF-pits formation, the nucleation of F-pits is much easier than that of DF-pits. Thus, the strains from the sources of Fpits should be greater than that at DF-pits. We also investigated the behavior of pits during a repetition of dissolution and growth of crystals (Figs. 2(a–c)). DF-pits were completely buried
Fig. 1. A series of AFM images of etching on the (1 1 0) face of lysozome crystals at different values of undersaturation: (a) s ¼ 0:14; (b) s ¼ 0:30 and (c) s ¼ 0:42: The F-pits generated at the fresh surface on other F-pits are enclosed with circles. The DF-pits were indicated by arrow-heads. The size of these images is 20 20 mm2.
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after growth for 30 min at the supersaturation of 0.9 (Fig. 2(b)). The surface became flat and no specific feature of DF-pits was observed. After reetching, most of the DF-pits developed again at the same position, as illustrated in Fig. 2(c). However, some of the DF-pits became shallower despite the fact that the time of the re-etching was longer than the first one. This suggests that the origin of DF-pits is not moved but partially removed by etching.
3.2. The origin of F-pit To reveal the origin of F-pits, detailed observations were carried out at the center of the pits. On pure lysozyme crystals, almost all the F-pits was flat at a molecular level, but some pits contained a particle at the center of them (Fig. 3). The pit was nucleated at the particle and developed during etching. When the surface of the crystal was etched more, this particle
Fig. 2. Topographic images of the (a) etched, (b) grown and (c) re-etched surface. Sectional plots are shown along the lines. The DFpits which became shallower were indicated by triangles. The size of these images is 50 50 mm2.
ARTICLE IN PRESS H. Hondoh, T. Nakada / Journal of Crystal Growth 275 (2005) e1423–e1429
Fig. 3. A topographic image of the extension at the center of the F-pit on the pure lysozyme crystal. The size of this image is 1 1 mm2.
vanished. Because the purity of lysozyme was high and the solution was filtered, the particle could be a microcrystal of lysozyme, which was nucleated in the solution and included into the seed crystal during growth. In the case of contaminated lysozyme crystals, a streak was often found at the bottom of F-pits (Figs. 4(a) and (b)). The position of the streaks was shifted depending on the fast scan direction of AFM. When the scan was from left to right, the streaks were found on the right side of the images, and vice versa. The streaks had approximately 2 nm height and the length was more than 100 nm. The magnified view revealed that the streaks were consisted of several cords. The streaks were not removed during the observation, even though the surface was slightly dissolved. During dissolving, the surface sometimes dug out the buried parts of the streaks and made them longer. Therefore, we conclude that the streaks were bunches of cords of which both ends were buried in crystals. The schematic image of the streaks was shown in Fig. 4(c). The streaks would be one of the origins of the F-pits because they were found in approximately 50% at the
e1427
bottom of the F-pits. Therefore, it is most likely that lysozyme layers grown on the streaks have a strain and preferentially dissolve in undersaturated condition. Since DF-pit has never developed from the streaks, the streaks are not the origin of DF-pit. Taking into account that the streaks were not found on the pure lysozyme crystals, the streaks should be a contaminated protein. From the length and the height of the streaks, one single cord is considered to be a denatured stretched protein. If the length of the buried parts of the denatured protein in crystal was assumed to be the same as that on the surface, the total length of the denatured protein was more than 200 nm. This value is corresponding to 500 amino acid (AA) residues (this was calculated from the length of b-strand). Although egg white contains various kinds of proteins, there are few proteins with 500 or more residues, such as ovotransferrin (686 AA), ovomacroglobulin (1437 AA) and ovomucin (2087 AA) [20]. Because ovomucin has a lot of sugar-chains and affinity for lysozyme, ovomucin has a potential to contaminate a commercial lysozyme and be one of the origins of F-pit.
4. Conclusions By using AFM, in situ observation of etching was carried out on the (1 1 0) face of lysozyme crystals. F-pits were formed at the under saturation of 0.08–0.14. The number of F-pits was increased with undersaturation. DF-pits were observed to form when undersaturation was increased over 0.30. The locations of DF-pits were independent of the F-pit sites. The strains from the sources of F-pits seem to be greater than that at DF-pits. Because the repetition of etching and growth made DF-pits shallower, the source of DFpits could be removed by etching. At the bottom of F-pits on contaminated crystals, bunches of several cords with the length of more than 100 nm were often found. These bunches would be the one of the origin of F-pit in the contaminated crystals.
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Fig. 4. (a) Topographic and (b) phase contrast images of the streak at the bottom of the F-pit. The directions of the fast scan were indicated by arrows. The size of these images is 250 250 nm2. (c) A schematic image of the streak in the lysozome crystal. Gray lines show the layers parallel to the (1 1 0) face.
Acknowledgements We thank Sumika Bayer Urethane Co. Ltd. for kindly sharing of Desmolac 4030. This work was partially supported by Grant-in-Aids (no. 14655013) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.
References [1] B.R. Thomas, P.G. Vekilov, F. Rosenberger, Acta Crystallogr. D 52 (1996) 776. [2] F. Rosenberger, P.G. Vekilov, M. Muschol, B.R. Thomas, J. Crystal Growth 168 (1996) 1.
[3] B.R. Thomas, D. Carter, F. Rosenberger, J. Crystal Growth 187 (1998) 499. [4] B.R. Thomas, P.G. Vekilov, F. Rosenberger, Acta Crystallogr. D 54 (1998) 226. [5] D.C. Carter, K. Lim, J.X. Ho, B.S. Wright, P.D. Twigg, T.Y. Miller, J. Chapman, K. Keeling, J. Ruble, P.G. Vekilov, B.R. Thomas, F. Rosenberger, A.A. Chernov, J. Crystal Growth 196 (1999) 623. [6] B.R. Thomas, A.A. Chernov, P.G. Vekilov, D.C. Carter, J. Crystal Growth 211 (2000) 149. [7] P.G. Vekilov, F. Rosenberger, J. Crystal Growth 158 (1996) 540. [8] T. Nakada, G. Sazaki, S. Miyashita, S.D. Durbin, H. Komatsu, J. Crystal Growth 196 (1999) 503. [9] I. Yoshizaki, A. Kadowaki, Y. Iimura, N. Igarashi, S. Yoda, H. Komatsu, J. Synchrotron Radiat. 11 (2004) 30. [10] K. Izumi, S. Sawamura, M. Ataka, J. Crystal Growth 168 (1996) 106.
ARTICLE IN PRESS H. Hondoh, T. Nakada / Journal of Crystal Growth 275 (2005) e1423–e1429 [11] V. Stojanoff, D.P. Siddons, Acta Crystallogr. A 52 (1996) 498. [12] I. Dobrianov, K.D. Finkelstein, S.G. Lemay, R.E. Thorne, Acta Crystallogr. D 54 (1998) 922. [13] F. Otalora, J.M. Garcia-Ruiz, J.A. Gavira, B. Capelle, J. Crystal Growth 196 (1999) 546. [14] Z.W. Hu, B. Lai, Y.S. Chu, Z. Cai, D.C. Mancini, B.R. Thomas, A.A. Chernov, Phys. Rev. Lett. 87 (2001) 148101-1. [15] I. Dobrianov, S. Kriminski, C.L. Caylor, S.G. Lemay, C. Kimmer, A. Kisselev, K.D. Finkelstein, R. E Thorne, Acta Crystallogr. D 57 (2001) 61.
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[16] M. Tachibana, H. Koizumi, K. Izumi, K. Kajiwara, K. Kojima, J. Synchrotron Radiat. 10 (2003) 416. [17] H. Hondoh, T. Nakada, Jpn. J. Appl. Phys. 43 (2004) 4529. [18] G. Sazaki, K. Kurihara, T. Nakada, S. Miyashita, H. Komatsu, J. Crystal Growth 169 (1996) 355. [19] S.D. Durbin, W.C. Carlson, J. Crystal Growth 122 (1992) 71. [20] Swiss-Plot, http://kr.expasy.org/sprot/.
Journal of Crystal Growth 275 (2005) e1423–e1429 www.elsevier.com/locate/jcrysgro
Impurity effect on defect formation of protein crystals H. Hondoh, T. Nakada Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan Available online 15 December 2004
Abstract Etch pit formation on lysozyme crystals was observed in situ by using atomic force microscopy. No etch pit was nucleated at low undersaturation (so0:08). Flat-bottomed etch pits (F-pits) were observed to form at undersaturation of 0.08–0.14, and their number was increased with undersaturation. In addition to F-pits, deep flat-bottomed etch pits (DF-pits) appeared when understauration was increased over 0.30. It was found that the locations of F- and DF-pits were independent and randomly nucleated. In the case of pure lysozyme crystals, the bottoms of F-pits were flat at a molecular level. In contrast, streaks consisted of several cords with a length of more than 100 nm were often found at the bottoms of the F-pits on contaminated lysozyme crystals. These streaks would be the one of the origins of F-pits on the contaminated crystals. r 2004 Elsevier B.V. All rights reserved. PACS: 61.72.Ff; 61.72.Ji; 81.10.Dn; 87.14.Ee Keywords: A1. Atomic force microscopy; A1. Defects; A1. Etching; A1. Impurities; B1. Biological macromolecules; B1. Lysozyme
1. Introduction In protein crystal growth, influence of impurity must be considered because target samples are consistently contaminated by other proteins. Even in commercially available proteins, such as lysozyme, several kinds of proteins are contained [1]. These impurities could be incorporated into protein crystals to form lattice defects. In the last decades, therefore, numerous studies about impurity effects on protein crystal growth have been Corresponding author. Fax: +81 77 561 2657.
E-mail address: [email protected] (H. Hondoh).
carried out [2–6]. Vekilov et al. [7] reported the impurity effect on the growth step kinetics. A step velocity significantly increased with increasing purity of samples. Nakada et al. [8] directly observed the incorporation of dimers into crystals at molecular level by atomic force microscopy (AFM). Effects of impurity on crystal quality have also been revealed by AFM and X-ray diffraction experiments [9]. They reported that crystal quality tended to degrade as the impurity concentration increased. On the other hand, however, defects of protein crystals have not been studied well because of a lack of suitable techniques. With developing
0022-0248/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jcrysgro.2004.11.236
ARTICLE IN PRESS e1424
H. Hondoh, T. Nakada / Journal of Crystal Growth 275 (2005) e1423–e1429
synchrotron facilities, X-ray topographic studies have been performed in recent years [10–16], but point defects in the protein crystals could not be observed because of the limit of spatial resolution of this method. Very recently, etch figure method has been successfully applied to investigate defects of protein crystals [17]. In this study, three types of etch pits were observed on the (1 1 0) face of tetragonal lysozyme crystals; a flat-bottomed pit (F-pit), a point-bottomed pit (P-pit) and a deep flat-bottomed pit (DF-pit). DF-pits have the depth of more than 100 nm, while F-pits are of 6 nm depth. It is also revealed that the etch pit density of contaminated lysozyme crystals was greater than that of pure lysozyme crystals. However, the mechanism of defect formation in protein crystals is still unclear. In this paper, we report the in situ observation of etching lysozyme crystals by AFM. Direct measurement of the developing etch pits determined the undersaturations for the pit formation. The behavior of etch pits was investigated to understand the etch pit formation mechanism. In addition, the possible source of the defect is also described.
2. Experimental procedure 2.1. Preparation of seed crystals We employed two varieties of hen egg white lysozyme samples of different purity. The Seikagaku Corporation product was used as a contaminated sample without further purification. A pure sample, which was purified from the Seikagaku product, was purchased from Maruwa Foods Industries. Inc. The purity of these samples was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as 98% and X99.99%, respectively. Seed crystals of lysozyme tetragonal form were obtained by a batch method at 20 1C. The crystallization solution was composed of 100 mM sodium acetate buffer, pH 4.5, 25 mg/ml NaCl and 80 mg/ml lysozyme. Floating crystals, nucleated at a surface of the solution, were applied for further studies as seed crystals
because they were not influenced by containers. All the solution in these studies was filtered with Millipore 0.22-mm membranes before use. 2.2. Polyurethane substrate We applied polyurethane-coated cover glasses as substrates to prevent detachment of crystals from substrates during etching. Polyurethane Desmolac 4340, which was shared from Sumika Bayer Urethane Co. Ltd., was diluted by 20% with benzene, and then coated cover glasses with itself. The glasses were dried overnight. Because Desmolac 4340 does not elite into the crystallization solution after drying up, it will have no effect on crystal growth. The polyurethane substrates fixed protein crystals so tightly that the seed crystals did not move even in the solution containing no protein. 2.3. AFM observation of etching crystal Atomic force microscopy was used for in situ observation of etching lysozyme crystals. The AFM was a NanoScope E from Digital Instruments operating in the contact mode with Si3N4 cantilevers (the spring constant was 0.06 N/m). To increase a chance of DF-pit formation, the contaminated sample was mainly applied for the seed crystal preparation and the etch figure observation. The etching and the observation were performed in a fluid-cell on polyurethane substrates at 20 1C. The composition of the solution was 100 mM sodium acetate buffer, pH 4.5, 25 mg/ ml NaCl and lysozyme. Undersaturation sðs ¼ jC Cej=CeÞ was controlled by varying the concentration of lysozyme from 16 to 7 mg/ml ðs ¼ 0:0020:56Þ: The equilibrium concentration at 20 1C in this solution is 16.1 mg/ml. The solutions were kept to flow at 40 ml/min by a peristaltic pump to neglect the temperature vibration in the fluid-cell. Since the volume of the fluid-cell is less than 100 ml, all the solution in the cell would be replaced within 3 min. To replace the solution in the fluid-cell completely, the solutions are maintained to flow more than 30 min after they reached the cell. The solubility of the lysozyme crystals was based on the results of Sazaki et al. [18].
ARTICLE IN PRESS H. Hondoh, T. Nakada / Journal of Crystal Growth 275 (2005) e1423–e1429
3. Results and discussion 3.1. In situ observation of etching crystals Etch pit formation on lysozyme crystals was observed in situ by AFM. There were only two types of etch pits, F- and DF-pits, on the (1 1 0) faces in the series of this study. No spiral growth hillock and P-pit was found. The F-pits appeared when the undersaturation was in the range of 0.08–0.14 (Fig. 1(a)). The depth of these F-pits was 6 or 12 nm. Considering that the unit height of growing 2D islands is 6 nm [19], the unit depth of dissolving pits is the same as that during growth. The number of F-pits was increased with understauration (Fig. 1(b)). When the undersaturation was raised over 0.30, DF-pits were developed in
e1425
addition to F-pits (Fig. 1(c)). The F-pits tended to nucleate at a fresh surface on other developing Fpits as shown in Fig. 1(c). This indicates that the origin of the F-pits appeared at the surface by dissolving. Because the depth of the nucleated Fpits was also 6 nm, the strains from the source of the pit would distribute only within 6 nm. The independence of locations of F- and DF-pits are also suggested in the assumption. Furthermore, because F-pits were formed at the undersaturation lower than that for DF-pits formation, the nucleation of F-pits is much easier than that of DF-pits. Thus, the strains from the sources of Fpits should be greater than that at DF-pits. We also investigated the behavior of pits during a repetition of dissolution and growth of crystals (Figs. 2(a–c)). DF-pits were completely buried
Fig. 1. A series of AFM images of etching on the (1 1 0) face of lysozome crystals at different values of undersaturation: (a) s ¼ 0:14; (b) s ¼ 0:30 and (c) s ¼ 0:42: The F-pits generated at the fresh surface on other F-pits are enclosed with circles. The DF-pits were indicated by arrow-heads. The size of these images is 20 20 mm2.
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after growth for 30 min at the supersaturation of 0.9 (Fig. 2(b)). The surface became flat and no specific feature of DF-pits was observed. After reetching, most of the DF-pits developed again at the same position, as illustrated in Fig. 2(c). However, some of the DF-pits became shallower despite the fact that the time of the re-etching was longer than the first one. This suggests that the origin of DF-pits is not moved but partially removed by etching.
3.2. The origin of F-pit To reveal the origin of F-pits, detailed observations were carried out at the center of the pits. On pure lysozyme crystals, almost all the F-pits was flat at a molecular level, but some pits contained a particle at the center of them (Fig. 3). The pit was nucleated at the particle and developed during etching. When the surface of the crystal was etched more, this particle
Fig. 2. Topographic images of the (a) etched, (b) grown and (c) re-etched surface. Sectional plots are shown along the lines. The DFpits which became shallower were indicated by triangles. The size of these images is 50 50 mm2.
ARTICLE IN PRESS H. Hondoh, T. Nakada / Journal of Crystal Growth 275 (2005) e1423–e1429
Fig. 3. A topographic image of the extension at the center of the F-pit on the pure lysozyme crystal. The size of this image is 1 1 mm2.
vanished. Because the purity of lysozyme was high and the solution was filtered, the particle could be a microcrystal of lysozyme, which was nucleated in the solution and included into the seed crystal during growth. In the case of contaminated lysozyme crystals, a streak was often found at the bottom of F-pits (Figs. 4(a) and (b)). The position of the streaks was shifted depending on the fast scan direction of AFM. When the scan was from left to right, the streaks were found on the right side of the images, and vice versa. The streaks had approximately 2 nm height and the length was more than 100 nm. The magnified view revealed that the streaks were consisted of several cords. The streaks were not removed during the observation, even though the surface was slightly dissolved. During dissolving, the surface sometimes dug out the buried parts of the streaks and made them longer. Therefore, we conclude that the streaks were bunches of cords of which both ends were buried in crystals. The schematic image of the streaks was shown in Fig. 4(c). The streaks would be one of the origins of the F-pits because they were found in approximately 50% at the
e1427
bottom of the F-pits. Therefore, it is most likely that lysozyme layers grown on the streaks have a strain and preferentially dissolve in undersaturated condition. Since DF-pit has never developed from the streaks, the streaks are not the origin of DF-pit. Taking into account that the streaks were not found on the pure lysozyme crystals, the streaks should be a contaminated protein. From the length and the height of the streaks, one single cord is considered to be a denatured stretched protein. If the length of the buried parts of the denatured protein in crystal was assumed to be the same as that on the surface, the total length of the denatured protein was more than 200 nm. This value is corresponding to 500 amino acid (AA) residues (this was calculated from the length of b-strand). Although egg white contains various kinds of proteins, there are few proteins with 500 or more residues, such as ovotransferrin (686 AA), ovomacroglobulin (1437 AA) and ovomucin (2087 AA) [20]. Because ovomucin has a lot of sugar-chains and affinity for lysozyme, ovomucin has a potential to contaminate a commercial lysozyme and be one of the origins of F-pit.
4. Conclusions By using AFM, in situ observation of etching was carried out on the (1 1 0) face of lysozyme crystals. F-pits were formed at the under saturation of 0.08–0.14. The number of F-pits was increased with undersaturation. DF-pits were observed to form when undersaturation was increased over 0.30. The locations of DF-pits were independent of the F-pit sites. The strains from the sources of F-pits seem to be greater than that at DF-pits. Because the repetition of etching and growth made DF-pits shallower, the source of DFpits could be removed by etching. At the bottom of F-pits on contaminated crystals, bunches of several cords with the length of more than 100 nm were often found. These bunches would be the one of the origin of F-pit in the contaminated crystals.
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Fig. 4. (a) Topographic and (b) phase contrast images of the streak at the bottom of the F-pit. The directions of the fast scan were indicated by arrows. The size of these images is 250 250 nm2. (c) A schematic image of the streak in the lysozome crystal. Gray lines show the layers parallel to the (1 1 0) face.
Acknowledgements We thank Sumika Bayer Urethane Co. Ltd. for kindly sharing of Desmolac 4030. This work was partially supported by Grant-in-Aids (no. 14655013) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.
References [1] B.R. Thomas, P.G. Vekilov, F. Rosenberger, Acta Crystallogr. D 52 (1996) 776. [2] F. Rosenberger, P.G. Vekilov, M. Muschol, B.R. Thomas, J. Crystal Growth 168 (1996) 1.
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