- Email: [email protected]
A novel spin filter method for the measurement of solubility
Journal of Crystal Growth 232 (2001) 17–20
A novel spin filter method for the measurement of solubility Lesley F. Hairea,*, David M. Blowb a
Division of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK b Biophysics Section, Physics Department, Imperial College of Science Technology & Medicine, London, SW1 2BZ, UK
Abstract A spin filter method for the measurement of the solubility of protein crystals is described. A crystal slurry is equilibrated with protein solution and then centrifuged through a filter to assay the protein concentration. The technique is rapid and easy to perform and does not require any specialised or expensive equipment. Data for hen eggwhite lysozyme and carboxypeptidase G2 are presented. # 2001 Elsevier Science B.V. All rights reserved. Keywords: A1. Biocrystallization; A1. Solubility
1. Introduction The first studies on protein solubility were carried out by Green who established empirical solubility laws [1,2]. More recent investigations on protein solubilities and crystal/solution phase diagrams have been done mainly with the model hen egg-white lysozyme e.g. Refs. [3–6]. Other proteins studied have included canavalin [7], concanavalin A [8], g crystallin [9], cytochrome-c oxidase [10], the photoreaction centre from Rhodobacter sphaeroides [11], carboxypeptidase G2 [12] and a collagenase from Hypoderma lineatum [13]. It is clear that there is a scarcity of solubility data for proteins and this can be explained by the relatively large amounts of protein, typically gram quantities, and the excessively long equilibration times required for the large-scale batch crystal-
*Corresponding author. Tel.: +44-20-8959-3666; fax: +4420-8906-4477. E-mail address: [email protected] (L.F. Haire).
lization experiments traditionally used for such measurements. In more recent years, several laboratories have reported solubility measurements made on 10 ml drops using vapour diffusion techniques instead of batch crystallization e.g. [14,8,15]. Crystallization droplets are setup under defined conditions and as crystals grow the change in the protein concentration in the drops is monitored with time. When the protein concentration becomes constant, this concentration can be regarded as being in equilibrium with the crystals, that is, the solubility of the crystals. A similar concentration change can be measured when crystals are added to an undersaturated solution. Equilibrium is reached when crystal dissolution no longer occurs. A more accurate solubility determination can be made by performing both crystallization and dissolution experiments in parallel. If the two solubility measurements agree, it can be reasonably assumed that the result represents the true equilibrium saturation concentration at the given temperature. Although these microscale methods reduced the quantity of protein required for the solubility
0022-0248/01/$ - see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 2 4 8 ( 0 1 ) 0 1 1 4 1 - 1
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L.F. Haire, D.M. Blow / Journal of Crystal Growth 232 (2001) 17–20
measurements, the experiments still remained time-consuming (approximately eight weeks required for equilibration). Other techniques for measuring solubility include an optical scintillation method [16,17] and Michelson laser interferometry [18]. These methods have the drawback of requiring specialized equipment, which in the latter case is very expensive. Pusey and Gernert [19] introduced an alternative method for measurement of protein solubilities in which crystals were packed in a pair of columns through which protein solutions, under- and oversaturated respectively, were forced to flow. The method was based on the maximization of the available crystalline surface and the minimization of the free solution to be brought into equilibrium. Thus, the time required for equilibrium was decreased from months to days. The method was found to be rapid and accurate (one solubility/day/temperature), but required gram amounts of protein. A semi-micromethod was later described [20] requiring 15– 20 mg protein per column. Using this method, equilibrium was reached in 1–5 days. This report describes a technique which also uses a bed of crystals for the solubility measurements, but which avoids the problems associated with packing and eluting miniature columns, by using spin filter units. A description of the method and results are presented with lysozyme and carboxypeptidase G2.
0.1 mM zinc chloride), 13% PEG 4000, 0.1 M sodium cacodylate pH 6.3, 0.2 M zinc acetate at 108C. 2.3. Solubility measurement The crystal slurry, prepared as described, was suspended in an appropriate stabilizing solution, and an aliquot pipetted into the insert of a 0.45 mm microcentrifuge tube filter (Whatman Ltd. UK) and sedimented by spinning at 2000 g. Duplicate filters were prepared. The crystals were resuspended in the stabilizing solution and centrifuged again. This procedure was repeated twice more. Then the crystals were resuspended in either 400 ml (a) oversaturated solution (crystallization) or (b) undersaturated solution (dissolution) and rotated end over end and equilibrated at 188C (or other chosen temperature) for 2–12 h. The filters were then spun (at the appropriate temperature) to sediment the crystals, and an aliquot (10 ml) of the filtrate removed for dilution and measurement of protein concentration by the absorbance at 280 nm. The crystals were resuspended in the filtrate, equilibrated again and the protein concentration of the filtrate measured after centrifugation. The solubility value was reached when the protein concentration of the crystallization and dissolution filtrates was within a range of 10–15%.
3. Results 2. Experimental procedure 2.1. Preparation of lysozyme crystals Lysozyme (Sigma L-6876) crystals were grown by batch crystallization from 5% NaCl, 27–30 mg/ ml protein, 50 mM sodium acetate buffer pH 4.5 at 188C. The protein was used as supplied and dissolved in high quality water. 2.2. Preparation of carboxypeptidase G2 crystals Crystals were grown from 20 mg/ml protein solution (in 0.05 M Tris-HCl pH 7.7, 0.1 M NaCl,
Lysozyme solubility was found to be most sensitive to temperature variations at low salt concentration (55% NaCl). The solubility curve for lysozyme is represented in Fig. 1 and these results are compared with those obtained by published methods in Table 1. The results of the spin filter solubility measurements for carboxypeptidase G2 are shown in Fig. 2. The solubility of carboxypeptidase G2 decreased by a factor of two over a 2% change in PEG concentration (10–12%) at 188C. The solubility was also very sensitive to temperature over this range of PEG concentration and
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L.F. Haire, D.M. Blow / Journal of Crystal Growth 232 (2001) 17–20
decreased with lower temperatures. The results of the spin filter solubility assay were compared to those estimated from the phase diagram established by the batch dilution method [12] and are shown in Table 2. The solubility values of the batch dilution method are approximate values because the solubility line was estimated by eye from the experimental phase diagram. The solubilities for the spin filter method tend to be lower than the batch dilution method, which is consistent with the results obtained for lysozyme in Table 1 comparing solubilities obtained by the batch method and column technique.
4. Discussion The modified spin filter method used in this study requires approximately 20 mg of protein per filter. The method is used because of its simplicity
Fig. 2. Solubility determination for Carboxypeptidase G2 using the spin filter method.
Table 2 Solubility of carboxypeptidase G2 determined by the batch dilution and the spin filter methods
Fig. 1. Solubility determination for lysozyme using the spin filter method.
% PEG w/v
Solubility (mg/ml) by batch dilution at 188C
Solubility (mg/ml) by spin filter method at 188C
10 11 12
12.5 10.7 8.3
11.9 8.9 5.8
Table 1 Solubilities of lysozyme obtained by different experimental techniques %NaCl
Hanging drop LAa
Hanging drop LBb
Batch methodc
Column methodd
Spin filter methode
3 4 5
7.9 0.9 4.4 0.3 2.8 0.3
8.9 0.5 5.7 0.5 3.0 0.3
18 11 6
8.6 0.5 3.2 0.2 2.2 0.3
7.7 0.2 3.9 0.1 2.5 0.2
a Measurements were performed on 10 ml drops which were equilibrated at 208C, pH 4.5, against 1 ml reservoirs in Linbro trays for 60 days. LA signifies Sigma lysozyme L2879. Data from Skouri et al. [15]. b Same experiment as (1), but using lysozyme LB which represents Sigma batch L 6876 contaminated with 2% ovalbumin. Data from Skouri et al. [15]. c Literature data from Howard et al. [6]. The solubility was measured at 208C with 40 mM sodium acetate buffer pH 4.5, using lysozyme 3 recrystallized. No experimental error was given. d Solubility was determined at 208C with 100 mM sodium acetate buffer pH 4.5, using recrystallized and dialyzed lysozyme, data from Cacioppo et al. [20]. e Solubility was measured at 188C with 50 mM sodium acetate buffer pH 4.5 using Sigma lysozyme L 2879.
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L.F. Haire, D.M. Blow / Journal of Crystal Growth 232 (2001) 17–20
and the avoidance of problems associated with packing and eluting miniature columns such as: minimizing dead volume; leaks or blockages; maintaining the column and tubing at the required temperature. Continual mixing of the crystalline slurry during equilibration by rotating the filter end over end increases the efficiency of exchange at the crystal surface. Crystallization conditions were selected so that small crystals (550 mm) were used in the experiments to optimize this exchange. The filters used for the under- and oversaturated solutions were reversed in each experiment to avoid excessive crystal dissolution in the undersaturated condition. The solubility measurements from the present work were combined with experimental data from microbatch experiments, where the protein and precipitant concentrations were varied with temperature (4, 10 and 188C), and used to construct phase diagrams from which the approximate position of the metastable zone could be estimated [21]. This information was then used in the design of experiments to uncouple nucleation and growth by temperature change [21,22].
kinetics. It is clear from these considerations that the determination of protein solubility is not a trivial task and that measurements must be made under clearly defined conditions. The spin filter method described here makes the measurement of solubility more practicable for crystal growers who wish to characterize novel proteins and design crystallization strategies or correlate the solubility with other parameters such as the second virial coefficient.
5. Conclusions
[10]
Despite the waning popularity of lysozyme as a model protein, its usefulness in testing new methodologies in protein crystallization is undiminished owing to its cost effectiveness, and the huge body of data available from previous studies which make it invaluable as a benchmark protein. Significant differences in lysozyme solubilities have reported by various authors. This has been attributed to different experimental methods. Also, preparation of the lysozyme solution is important, as the presence of the sodium chloride in variable amounts in commercial samples may affect the results. Experiments by [15] comparing solubilities obtained with pure and contaminated lysozyme (Table 1) showed that differences in solubility are only shown at low salt concentrations. They concluded that impurities in the lysozyme preparation mainly affected nucleation and/or crystal
References [1] [2] [3] [4] [5] [6] [7] [8] [9]
[11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
A.A. Green, J. Biol. Chem. 93 (1931) 495. A.A. Green, J. Biol. Chem. 95 (1932) 47. M. Ataka, S. Tanaka, Biopolymers 25 (1986) 337. M. Ataka, M. Asai, J. Crystal Growth 90 (1988) 86. M. Ataka, Phase Transitions 45 (1993) 205. S. Howard, P.J. Twigg, J.K. Baird, E.J. Meehan Jr., J. Crystal Growth 90 (1988) 94. R.C. DeMattei, R.S. Feigelson, J. Crystal Growth 110 (1991) 34. V. Mikol, R. Giegee, J. Crystal Growth 97 (1989) 324. C.R. Berland, G.M. Thurston, M. Kondo, M.L. Broide, J. Pande, O. Ogun, G.B. Benedek, Proc. Natl. Acad. Sci. USA 89 (1992) 1214. M. Ataka, K. Shinzawaitoh, S. Yoshikawa, J. Crystal Growth 122 (1992) 60. T. Odahara, M. Ataka, T. Katsura, Acta Crystallogr. D 50 (1994) 639. E.S. Saridakis, P.D. Shaw Stewart, L.F. Lloyd, D.M. Blow, Acta Crystallogr. D50 (1994) 293. C. Carbonnaux, M. Riees-Kautt, A. Ducruix, Prot. Sci. 4 (1995) 2123. N.E. Chayen, L.F. Lloyd, C.A. Collyer, D.M. Blow, J. Crystal Growth 97 (1988) 367. M. Skouri, B. Lorber, R. Giegee, J.-P. Munch, J.S. Candau, J. Crystal Growth 152 (1995) 209. F. Rosenberger, S.B. Howard, J.W. Sowers, T.A. Nyce, J. Crystal Growth 122 (1993) 1. A.R. FeelingTaylor, R.M. Banish, R.E. Hirsch, P.G. Vekilov, Rev. Sci. Instr. 70 (1999) 2845. G. Sazaki, E. Yoshida, T. Nakada, S. Miyashita, H. Komatsu, J. Crystal Growth 169 (1996) 355. M.L. Pusey, K. Gernert, J. Crystal Growth 88 (1988) 419. E. Cacioppo, S. Munson, M.L. Pusey, J. Crystal Growth 110 (1991) 66. L.F. Haire, Ph.D. Thesis, University of London, 1996. D.M. Blow, N.E. Chayen, L.F. Lloyd, E. Saridakis, Prot. Sci. 3 (1994) 1638.
A novel spin filter method for the measurement of solubility Lesley F. Hairea,*, David M. Blowb a
Division of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK b Biophysics Section, Physics Department, Imperial College of Science Technology & Medicine, London, SW1 2BZ, UK
Abstract A spin filter method for the measurement of the solubility of protein crystals is described. A crystal slurry is equilibrated with protein solution and then centrifuged through a filter to assay the protein concentration. The technique is rapid and easy to perform and does not require any specialised or expensive equipment. Data for hen eggwhite lysozyme and carboxypeptidase G2 are presented. # 2001 Elsevier Science B.V. All rights reserved. Keywords: A1. Biocrystallization; A1. Solubility
1. Introduction The first studies on protein solubility were carried out by Green who established empirical solubility laws [1,2]. More recent investigations on protein solubilities and crystal/solution phase diagrams have been done mainly with the model hen egg-white lysozyme e.g. Refs. [3–6]. Other proteins studied have included canavalin [7], concanavalin A [8], g crystallin [9], cytochrome-c oxidase [10], the photoreaction centre from Rhodobacter sphaeroides [11], carboxypeptidase G2 [12] and a collagenase from Hypoderma lineatum [13]. It is clear that there is a scarcity of solubility data for proteins and this can be explained by the relatively large amounts of protein, typically gram quantities, and the excessively long equilibration times required for the large-scale batch crystal-
*Corresponding author. Tel.: +44-20-8959-3666; fax: +4420-8906-4477. E-mail address: [email protected] (L.F. Haire).
lization experiments traditionally used for such measurements. In more recent years, several laboratories have reported solubility measurements made on 10 ml drops using vapour diffusion techniques instead of batch crystallization e.g. [14,8,15]. Crystallization droplets are setup under defined conditions and as crystals grow the change in the protein concentration in the drops is monitored with time. When the protein concentration becomes constant, this concentration can be regarded as being in equilibrium with the crystals, that is, the solubility of the crystals. A similar concentration change can be measured when crystals are added to an undersaturated solution. Equilibrium is reached when crystal dissolution no longer occurs. A more accurate solubility determination can be made by performing both crystallization and dissolution experiments in parallel. If the two solubility measurements agree, it can be reasonably assumed that the result represents the true equilibrium saturation concentration at the given temperature. Although these microscale methods reduced the quantity of protein required for the solubility
0022-0248/01/$ - see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 2 4 8 ( 0 1 ) 0 1 1 4 1 - 1
18
L.F. Haire, D.M. Blow / Journal of Crystal Growth 232 (2001) 17–20
measurements, the experiments still remained time-consuming (approximately eight weeks required for equilibration). Other techniques for measuring solubility include an optical scintillation method [16,17] and Michelson laser interferometry [18]. These methods have the drawback of requiring specialized equipment, which in the latter case is very expensive. Pusey and Gernert [19] introduced an alternative method for measurement of protein solubilities in which crystals were packed in a pair of columns through which protein solutions, under- and oversaturated respectively, were forced to flow. The method was based on the maximization of the available crystalline surface and the minimization of the free solution to be brought into equilibrium. Thus, the time required for equilibrium was decreased from months to days. The method was found to be rapid and accurate (one solubility/day/temperature), but required gram amounts of protein. A semi-micromethod was later described [20] requiring 15– 20 mg protein per column. Using this method, equilibrium was reached in 1–5 days. This report describes a technique which also uses a bed of crystals for the solubility measurements, but which avoids the problems associated with packing and eluting miniature columns, by using spin filter units. A description of the method and results are presented with lysozyme and carboxypeptidase G2.
0.1 mM zinc chloride), 13% PEG 4000, 0.1 M sodium cacodylate pH 6.3, 0.2 M zinc acetate at 108C. 2.3. Solubility measurement The crystal slurry, prepared as described, was suspended in an appropriate stabilizing solution, and an aliquot pipetted into the insert of a 0.45 mm microcentrifuge tube filter (Whatman Ltd. UK) and sedimented by spinning at 2000 g. Duplicate filters were prepared. The crystals were resuspended in the stabilizing solution and centrifuged again. This procedure was repeated twice more. Then the crystals were resuspended in either 400 ml (a) oversaturated solution (crystallization) or (b) undersaturated solution (dissolution) and rotated end over end and equilibrated at 188C (or other chosen temperature) for 2–12 h. The filters were then spun (at the appropriate temperature) to sediment the crystals, and an aliquot (10 ml) of the filtrate removed for dilution and measurement of protein concentration by the absorbance at 280 nm. The crystals were resuspended in the filtrate, equilibrated again and the protein concentration of the filtrate measured after centrifugation. The solubility value was reached when the protein concentration of the crystallization and dissolution filtrates was within a range of 10–15%.
3. Results 2. Experimental procedure 2.1. Preparation of lysozyme crystals Lysozyme (Sigma L-6876) crystals were grown by batch crystallization from 5% NaCl, 27–30 mg/ ml protein, 50 mM sodium acetate buffer pH 4.5 at 188C. The protein was used as supplied and dissolved in high quality water. 2.2. Preparation of carboxypeptidase G2 crystals Crystals were grown from 20 mg/ml protein solution (in 0.05 M Tris-HCl pH 7.7, 0.1 M NaCl,
Lysozyme solubility was found to be most sensitive to temperature variations at low salt concentration (55% NaCl). The solubility curve for lysozyme is represented in Fig. 1 and these results are compared with those obtained by published methods in Table 1. The results of the spin filter solubility measurements for carboxypeptidase G2 are shown in Fig. 2. The solubility of carboxypeptidase G2 decreased by a factor of two over a 2% change in PEG concentration (10–12%) at 188C. The solubility was also very sensitive to temperature over this range of PEG concentration and
19
L.F. Haire, D.M. Blow / Journal of Crystal Growth 232 (2001) 17–20
decreased with lower temperatures. The results of the spin filter solubility assay were compared to those estimated from the phase diagram established by the batch dilution method [12] and are shown in Table 2. The solubility values of the batch dilution method are approximate values because the solubility line was estimated by eye from the experimental phase diagram. The solubilities for the spin filter method tend to be lower than the batch dilution method, which is consistent with the results obtained for lysozyme in Table 1 comparing solubilities obtained by the batch method and column technique.
4. Discussion The modified spin filter method used in this study requires approximately 20 mg of protein per filter. The method is used because of its simplicity
Fig. 2. Solubility determination for Carboxypeptidase G2 using the spin filter method.
Table 2 Solubility of carboxypeptidase G2 determined by the batch dilution and the spin filter methods
Fig. 1. Solubility determination for lysozyme using the spin filter method.
% PEG w/v
Solubility (mg/ml) by batch dilution at 188C
Solubility (mg/ml) by spin filter method at 188C
10 11 12
12.5 10.7 8.3
11.9 8.9 5.8
Table 1 Solubilities of lysozyme obtained by different experimental techniques %NaCl
Hanging drop LAa
Hanging drop LBb
Batch methodc
Column methodd
Spin filter methode
3 4 5
7.9 0.9 4.4 0.3 2.8 0.3
8.9 0.5 5.7 0.5 3.0 0.3
18 11 6
8.6 0.5 3.2 0.2 2.2 0.3
7.7 0.2 3.9 0.1 2.5 0.2
a Measurements were performed on 10 ml drops which were equilibrated at 208C, pH 4.5, against 1 ml reservoirs in Linbro trays for 60 days. LA signifies Sigma lysozyme L2879. Data from Skouri et al. [15]. b Same experiment as (1), but using lysozyme LB which represents Sigma batch L 6876 contaminated with 2% ovalbumin. Data from Skouri et al. [15]. c Literature data from Howard et al. [6]. The solubility was measured at 208C with 40 mM sodium acetate buffer pH 4.5, using lysozyme 3 recrystallized. No experimental error was given. d Solubility was determined at 208C with 100 mM sodium acetate buffer pH 4.5, using recrystallized and dialyzed lysozyme, data from Cacioppo et al. [20]. e Solubility was measured at 188C with 50 mM sodium acetate buffer pH 4.5 using Sigma lysozyme L 2879.
20
L.F. Haire, D.M. Blow / Journal of Crystal Growth 232 (2001) 17–20
and the avoidance of problems associated with packing and eluting miniature columns such as: minimizing dead volume; leaks or blockages; maintaining the column and tubing at the required temperature. Continual mixing of the crystalline slurry during equilibration by rotating the filter end over end increases the efficiency of exchange at the crystal surface. Crystallization conditions were selected so that small crystals (550 mm) were used in the experiments to optimize this exchange. The filters used for the under- and oversaturated solutions were reversed in each experiment to avoid excessive crystal dissolution in the undersaturated condition. The solubility measurements from the present work were combined with experimental data from microbatch experiments, where the protein and precipitant concentrations were varied with temperature (4, 10 and 188C), and used to construct phase diagrams from which the approximate position of the metastable zone could be estimated [21]. This information was then used in the design of experiments to uncouple nucleation and growth by temperature change [21,22].
kinetics. It is clear from these considerations that the determination of protein solubility is not a trivial task and that measurements must be made under clearly defined conditions. The spin filter method described here makes the measurement of solubility more practicable for crystal growers who wish to characterize novel proteins and design crystallization strategies or correlate the solubility with other parameters such as the second virial coefficient.
5. Conclusions
[10]
Despite the waning popularity of lysozyme as a model protein, its usefulness in testing new methodologies in protein crystallization is undiminished owing to its cost effectiveness, and the huge body of data available from previous studies which make it invaluable as a benchmark protein. Significant differences in lysozyme solubilities have reported by various authors. This has been attributed to different experimental methods. Also, preparation of the lysozyme solution is important, as the presence of the sodium chloride in variable amounts in commercial samples may affect the results. Experiments by [15] comparing solubilities obtained with pure and contaminated lysozyme (Table 1) showed that differences in solubility are only shown at low salt concentrations. They concluded that impurities in the lysozyme preparation mainly affected nucleation and/or crystal
References [1] [2] [3] [4] [5] [6] [7] [8] [9]
[11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
A.A. Green, J. Biol. Chem. 93 (1931) 495. A.A. Green, J. Biol. Chem. 95 (1932) 47. M. Ataka, S. Tanaka, Biopolymers 25 (1986) 337. M. Ataka, M. Asai, J. Crystal Growth 90 (1988) 86. M. Ataka, Phase Transitions 45 (1993) 205. S. Howard, P.J. Twigg, J.K. Baird, E.J. Meehan Jr., J. Crystal Growth 90 (1988) 94. R.C. DeMattei, R.S. Feigelson, J. Crystal Growth 110 (1991) 34. V. Mikol, R. Giegee, J. Crystal Growth 97 (1989) 324. C.R. Berland, G.M. Thurston, M. Kondo, M.L. Broide, J. Pande, O. Ogun, G.B. Benedek, Proc. Natl. Acad. Sci. USA 89 (1992) 1214. M. Ataka, K. Shinzawaitoh, S. Yoshikawa, J. Crystal Growth 122 (1992) 60. T. Odahara, M. Ataka, T. Katsura, Acta Crystallogr. D 50 (1994) 639. E.S. Saridakis, P.D. Shaw Stewart, L.F. Lloyd, D.M. Blow, Acta Crystallogr. D50 (1994) 293. C. Carbonnaux, M. Riees-Kautt, A. Ducruix, Prot. Sci. 4 (1995) 2123. N.E. Chayen, L.F. Lloyd, C.A. Collyer, D.M. Blow, J. Crystal Growth 97 (1988) 367. M. Skouri, B. Lorber, R. Giegee, J.-P. Munch, J.S. Candau, J. Crystal Growth 152 (1995) 209. F. Rosenberger, S.B. Howard, J.W. Sowers, T.A. Nyce, J. Crystal Growth 122 (1993) 1. A.R. FeelingTaylor, R.M. Banish, R.E. Hirsch, P.G. Vekilov, Rev. Sci. Instr. 70 (1999) 2845. G. Sazaki, E. Yoshida, T. Nakada, S. Miyashita, H. Komatsu, J. Crystal Growth 169 (1996) 355. M.L. Pusey, K. Gernert, J. Crystal Growth 88 (1988) 419. E. Cacioppo, S. Munson, M.L. Pusey, J. Crystal Growth 110 (1991) 66. L.F. Haire, Ph.D. Thesis, University of London, 1996. D.M. Blow, N.E. Chayen, L.F. Lloyd, E. Saridakis, Prot. Sci. 3 (1994) 1638.