Dissolution Behavior of Lysozyme Crystals - Crystal Growth & Design

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The dissolution behavior of lysozyme crystals Ronny Oswald, and Joachim Ulrich Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.5b00821 • Publication Date (Web): 05 Aug 2015 Downloaded from http://pubs.acs.org on August 13, 2015

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Crystal Growth & Design

The dissolution behavior of lysozyme crystals R. Oswald∗ and J. Ulrich∗ Martin-Luther-Universität Halle-Wittenberg, Zentrum für Ingenieurwissenschaften, Verfahrenstechnik / TVT, D-06099 Halle (Saale), Germany E-mail: [email protected]; [email protected] Phone: +049 (0)345 55 - 28 400. Fax: +049 (0)345 55 - 27 358

Abstract Protein crystal dissolution was investigated. Solutions with different compositions (distilled water, buffer solutions, different pH values, different salt concentrations and salts, different precipitates and solvents) were used to dissolve different lysozyme crystal modifications. The results show, that two distinctly different types of dissolution mechanisms could be found. Crystals dissolved at the same pH value as used in their crystallization process, show to a conventually known dissolution mechanism. That means the crystals get rounded and shrink with time. In case the pH value in dissolution differs significantly from that in crystallization process, the crystals are “falling apart” into many small particles which subsequently dissolve separately and due to the large surface area the overall dissolution shows a faster rate. In case of different precipitants it is also possible to induce the second dissolution mechanism. A first explanation for these mechanisms are given.

∗

To whom correspondence should be addressed

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Introduction Crystallization of proteins was in past decades mostly used to prepare large and high pure crystals to determine the structure of the protein crystals by x-ray measurements. With the increase of known industrial applications of proteins, the desired amount of high pure proteins is increasing, too. The most important advantage of protein purification by crystallization is the low cost in industrial mass production of high pure protein crystals. Crystallized proteins, furthermore, offer an improved shelf life with a very high degree of preservation of the protein activity. Many studies on the crystallization of proteins with the aim to optimize purity, yield, shape or activity were performed, e.g. by Durbin and Feher 1 or McPherson 2 . In research papers on protein crystallization, lysozyme is the most often described protein because of it‘s good availability and it‘s fairly low costs. In most cases, however, only the crystallization processes are investigated, the dissolution is neglected even though dissolution of crystals is known to be the contrary of crystal growth process (see e.g. Ulrich and Stelzer 3 ). Müller and Ulrich 4 described the first time an unusual dissolution behavior for protein crystals which is different from a conventional dissolution behavior and cannot be explained as the known dissolution processes. For the future and a better controlled use of crystallized proteins it is necessary to observe and understand the dissolution mechanisms and kinetics. One reason for this importance is the prediction of the bioavailability for pharmaceuticals.

Material and methods Crystallization of lysozyme crystals Crystals for dissolution experiments were prepared by a salting out crystallization. Two solutions were mixed at a given constant temperature. Both solutions include water as solvent and acetate or glycine as buffer and have a given pH and a given concentration

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(see Table 1). One solution contains the lysozyme, hen egg white (Sigma Aldrich product No. 62971, Fluka) as protein, the other solution contains the sodium chloride (>99.5 %, Carl Roth) with a concentration of 8-12 wt% as precipitant, which is known to reduce the solubility of lysozyme drastically (see e.g. Wiencek 5 ). By mixing both solutions, the lysozyme solubility is lowered and thereby the nucleation is initiated and crystals growth took place within 2 days. This method was already discribed by Müller 6 and Aldabaibeh 7 and delivers fairly large (∼200 µm in terms of tetragonal modification) crystals of high quality. Lysozyme crystals exist in different modifications (monoclinic, triclinic, tetragonal, high temperature orthorhombic, low temperature orthorhombic) which can be crystallized in accordance with the crystallization conditions. Here only three modifications: the tetragonal, high temperature orthorhombic (HTO) and the low temperature orthorhombic (LTO) modification were examined. The crystallization conditions (mixed solutions) for the different modifications are shown in Table 1. Furthermore, it can be seen that a change of only one parameter in the crystallization process can already change the crystal modification. Table 1: Lysozyme crystallization conditions Tetragonal 50 mg/ mL lysozyme acetate buffer 0.1 M - pH 5.0 - 4 ◦C 4 wt% sodium chloride

HTO 100 mg/ mL lysozyme acetate buffer 0.1 M - pH 5.0 - 40 ◦C 6 wt% sodium chloride

LTO 50 mg/ mL lysozyme glycine buffer 0.05 M - pH 9.8 - 20 ◦C 4 wt% sodium chloride

Dissolution experiments The used microscope cell is temperature controlled by a thermostatic bath (Julabo F32ME). The experimental set-up is the same as described by Müller 4 and is shown in Figure 1. The cell was filled with 2 mL of solution. The crystals to be dissolved will be harvested from a crystal growth process and the dissolution is observed via a microscope (Olympus BM2) with an attached camera system (Olympus SC-30). Depending on the dissolution kinetics, time steps for recording between 15 s and 300 s were used. 3



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Figure 1: microscope cell

Results of dissolution experiments In distilled water The dissolution experiments started with distilled water as solvent. The crystals positioned in the solution are “falling apart” into smaller fragments. Those fragments were in the dissolution process outwardly moved away from the crystals like in an explosion or a firework. An example is shown in an image in Figure 2. All three protein crystal modifications used here show this dissolution behavior. The use of distilled water causes problems, because the solution is then totally unbuffered. Low amounts of third substances, like carbon dioxide from the air, will result in a high modification in the pH value. The dissolution in pure water is the fastest out of all the different solvents (different pH of buffer, different amount of precipitant, etc.) used. The fast dissolution causes problems in observation since the

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handling (e.g. positioning of the crystals in the cell, bringing the microscope in focus) needs time.

Figure 2: explosion like dissolution

In different concentrations of precipitant The concentration of the precipitant opens options for a control of the dissolution kinetics since it controls in some degree the solubility of the protein. The precipitant needs to be actively working for the desired protein. It should be able to lower the solubility of the used protein. It has been recognized, that the concentration of sodium chloride inside the solution changes the rate of the dissolution in a range between minutes (no salt) and weeks (with 8 wt% salt).

In different concentrations of lysozyme The lysozyme concentration, however, shows only a low influence on the dissolution rate since the solubility of lysozyme without salt is extremely high. Due to the high costs of the

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pure protein, experiments without salt as precipitant were here not possible to be carried out.

At different pH values To maintain the pH value of a solution constant, a buffer solution is needed. To control the dissolution kinetics, the undersaturation level must be controlled. The dissolution conditions were first observed at a constant temperature 20 ◦C and the same pH value which were used for the crystallization of the observed crystal modification. The images of these results are shown in Figures 3a-3c for the HTO, Figures 4p-4r for the LTO and Figures 5a-5c for the teragonal lysozyme crystals. The crystals dissolve just as expected which is known, e.g. from inorganic salts. That means the edges will be rounded and the crystals will get smaller in size by time (see Figures 5a-5c concerning the tetragonal lysozyme crystals). It looks like the HTO lysozyme crystals falling apart (see Figures 3a-3c), but this is only due to the non perfect generation of these crystals, from agglomerates of different crystals. If agglomerated crystals dissolve, at one point they fall apart into separate crystals. The LTO lysozyme crystals shown in Figures 4p-4r shrink over the complete size. On the elongated ends the LTO crystals look like a comb with its tooth’s aligned in the direction away from the elongated crystal ends during dissolution (see window in Figure 4r). Some seconds after the start of the dissolution experiment with a change of the pH value, the transparent HTO and tetragonal crystals have changed and become opaque as to be seen in Figures 3h and 5k. The microscope shows black shades and the time until these shady state is reached depends on the rate of dissolution and change of the pH value. When the crystals are falling apart into small particles, those dissolve separately. Then due to the created high surface area, the overall rate of dissolution will be increased. The LTO crystals show fasciated bars after a few seconds (see Figure 4g), thereafter those crystals are falling apart (Figures 4i and 4r). The amount of fasciated bars will be increased if the pH value is changed more strongly. The gap between these bars will decrease. A number of the 6


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gaps related to the pH of the solution is shown in Table 2. The dissolution mechanism will suddenly change at a certain pH value. For crystals grown at pH value of 5.0 (HTO and tetragonal lysozyme crystals), a change in pH to approximately 7.0 is required to see that change in the dissolution mechanism from “conventional” to “falling apart”. LTO crystals crystallized at pH 9.8 need a lower pH value than approximately pH 7.8 in order to observe a change of the mechanism. Table 2: Measurement gap of fasciated bars abs. pH pH change 9.80 0.00 8.40 1.40 8.05 1.75 7.70 2.10 7.20 2.60 5.00 4.80

minimal gap maximal gap average gap 0.00 µm 0.00 µm 0.00 µm 0.00 µm 0.00 µm 0.00 µm 29.72 µm 230.98 µm 94.49 µm 16.10 µm 102.93 µm 43.83 µm 3.42 µm 12.68 µm 6.41 µm 1.66 µm 6.68 µm 4.32 µm

At different temperature levels The temperature level is another parameter enabling the control of the dissolution kinetics of protein crystals. With increasing temperature the kinetics of dissolution will increase. With a lowered temperature the kinetics will be slowed down. Due to the existence of the dissolution rate dispersion (see e.g. Fabian and Ulrich 8 ), it is not possible to give a mean value for the dissolution rate, since the number of examined crystals is not high enough to achieve a reliable main value.

At different type of precipitants In literature (e.g. Wiencek 5 , Lim et al. 9 , Harata and Akiba 10 and Forsythe et al. 11 ) there are different precipitants for the crystallization of lysozyme crystals described. In this work sodium bromide, sodium nitrate, ammonium sulfate, polyethylene glycol (PEG) 6000, ethanol and acetone where used. All of these precipitants are used in crystallization and purification of protein crystals of the different modifications. 7



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Sodium bromide is used for the crystallization of tetragonal lysozyme crystals. 9 The dissolution of protein crystals in a sodium bromide solution is not anymore as conventionally known. The crystals are “falling apart” when dissolving at the pH values where they were crystallized. Sodium nitrate and ammonium sulfate will be used for the crystallization of lysozyme to achieve the modifications trigonal and monoclinic. 10, 11 Both substances show again a dissolution mechanism of the kind of “falling apart” of the tetragonal crystals. PEG 6000, ethanol and acetone are generally used for protein crystallization and again the tetragonal crystals show a “falling apart” dissolution mechanism for the three precipitants. The HTO crystals show for all the 6 precipitation agents the same dissolution behavior as the tetragonal crystals, they “falling apart”. The low temperature orthorhombic modification (LTO) show a different behavior. Except for the ammonium sulfate, the crystals dissolve in a conventional way, they are rounding and shrinking in size. In case of ammonium sulfate, the LTO crystals show lengthways stripes, but not in the extent of the fascinated bars where found by the change of the pH value as in the case of the other precipitants.

Discussion To have a clear idea of the nature of a protein crystal, is important at this stage of the discussion. Many authors, especially, Jones 12 as well as Ulrich and Pietzsch 13 clarified that protein crystals contain besides the protein itself, water, buffer and salt. This is different from conventionally known crystals which are when pure most of the time a 100 % of the material of which they carry its name. Matthews 14 described an amount between 27 and 65 % of water inside the protein crystals. The so called “water”, however, is not only pure water! There are two kinds of “water”, a bounded water as we know from hydrates (this is real water) and the so called “free water” which is actually a buffer and it contains dissolved precipitant agents (often salts). That means buffer and also the precipitant is included inside

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the protein crystal. Some experiments with coloration support these statements as shown earlier by Müller. 6 The colorant will be an impurity, but it will not disturbe crystal growth. In this work a pH sensitive colorant was used to colorize the protein crystals. It was shown by this experiments, that the color inside of the protein crystals is changing due to a pH change in the solution. Inside the protein crystals is buffer (the “free water”) and it has clearly a diffusial exchange with the solution in which the crystals are dispersed. In literature 15, 16, 17 the “falling apart” mechanism is only described in similar way in case of a binary system at conditions when only one of the components will dissolved or will partially be molten. We do not face this situation here. A protein is a macromolecule, built of different amino acids by polypeptide bonds. Distributed over these molecule chains, some functional groups from different amino acids exist. These functional groups can hold charges. 5 By protonation or deprotonation this charges which depend on the pH value, can be neutralized. From those charges also the protein molecules have an overall charge distribution on its total surface 18, which results in attractive and repulsive forces between the protein molecules in a crystal lattice. The crystallization step incorporates some counter ions and aligns of the molecules to reach the lowest repulsion forces within the crystal. Due to the large solvent content (the “free water”) and the low binding energy, protein crystals are soft and sensitive to small changes in external conditions. 19 If the pH value will be changed by more than 2 units, the repulsion forces between all molecules in the crystal will be increased enough, to push the protein molecules apart. This changes also (speeds up) the dissolution kinetics, because of the small amount of specific sites, with which an ordered array (a crystal) can be formed. 19 Only low forces are necessary to destroy such a crystal lattice of a protein crystal. Supported by the experimentally found results the hypothesis is therefore that the repulsion forces generated by a pH value difference of more than 2 units compared to the growth conditions of the crystals delivers enough energy to disrupt the lattice so that the crystals can fall into pieces. This holds for the case of the same precipitation agent.

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The effect of the precipitant substances looks more complex. The dissolution mechanism of the HTO and tetragonal modification gives a different result than crystals of the LTO modification. For all dissolution experiments here only the precipitant is changed. The pH values of the undersaturated solution is kept constant by the buffer. The pH value is kept on the level of the solution used to crystallize the crystals which are thereafter examined in the dissolution experiments. The first result is, that the salts sodium bromide, sodium nitrate and ammonium sulfate change (substitute) the counter ions inside (the “free water”) and on the outer surface of the protein crystals. The counter ions are necessary to neutralize the protein molecules inside the crystal. The counter ions can be changed for example from Cl– to Br– or NO3– . The counter ions do not have the same size. In case the new counter ions are sterical bigger then the exchange will result in a breakage by the “falling apart” dissolution mechanism in case of the lysozyme crystals. The exchange of Cl– by SO42– will result additionaly in a different charge of the single ion, which results in different amounts of needed counter ions. If NO3– and SO42– will be used for the crystallization of lysozyme, it will result in a different crystal morphology, the triclinic and monoclinic form. 10, 11 The usage of organic compounds or solvents show the same dissolution mechanism, the “falling apart” of the crystals. These substances change the dielectric constant and react as anti solvent which lowers the solubility of proteins. These substances are not able to exchange the counter ion, but the use of all these substances will result in an undersaturation against the counter ion, since the new solvent in its composition contains no chloride anymore. It seems the extraction of the counter ion by undersaturation generates again the “falling apart” dissolution mechanism. The LTO modification shows except ammonium sulfate no “falling apart” dissolution behavior. An explaination is the position of the system with respect to the pI, the isoelectric point. At this point the charge of the protein molecule in its total, is neutral. At the pI, theoretically no counter ions are needed. As an example, in solutions with a pH value more acid than the pI, the molecules get a positiv overall charge. In the liquid phase there are

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several ions which result in a neutral charged solution. For the solid phase, the protein crystal, it is necessary to incorporate some counter ions inside the crystal lattice to reach a neutral crystal. The gap between the crystallization pH and the pH equivalent to the amount of counter ions needed, make the difference between the different modifications. The pI for lysozyme is located between pH values of 9.3 and 11.3 units. 20, 21 That means, for the LTO modification less counter ions are needed to create a neutral charge of the crystals. If the protein crystal needs only a reasonable small amount of included counter ions for the neutralization, the dissolution mechanism does not change and the dissolution mechanism for HTO and tetragonal crystals on the one hand and LTO lysozyme crystals on the other hand differs from each other. It is the same effect which is to be observed for different pH values which need a difference of about 2 pH units to change the dissolution mechanism.

Conclusions The dissolution behavior of three different modifications of lysozyme protein crystals is observed. The protein crystals show two different dissolution mechanisms. The first is the already known mechanism where, the edges of the crystals will be rounded and the crystals shrink with time. The second dissolution mechanism is more complex. The crystals when having the appropriate conditions are “falling apart” and then the little pieces dissolve. Due to the so created high surface area the dissolution is then faster under the conditions compared to the first mechanism. The second mechanism (the “falling apart”) is always in operation when the pH value (more than 2 units), or the used precipitant in the solution in which the crystals should dissolve differs from the conditions used to grow the crystals. This knowledge helps to enhance a proper use of protein crystals. The choise of a faster or a slower dissolution mechanism is possible by choosing the appropriate condition for dissolution. A door for tayloring the dissolution rate of protein contain drugs seems to be opened.

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a) pH 5.0 - ∆t 0 min

b) pH 5.0 - ∆t 120 min

c) pH 5.0 - ∆t 240 min

d) pH 5.9 - ∆t 0 min

e) pH 5.9 - ∆t 120 min

f) pH 5.9 - ∆t 240 min

g) pH 7.2 - ∆t 0 min

h) pH 7.2 - ∆t 120 min

i) pH 7.2 - ∆t 240 min

j) pH 7.7 - ∆t 0 min

k) pH 7.7 - ∆t 120 min

l) pH 7.7 - ∆t 240 min

m) pH 8.4 - ∆t 0 min

n) pH 8.4 - ∆t 120 min

o) pH 9.8 - ∆t 240 min

p) pH 9.8 - ∆t 0 min

q) pH 9.8 - ∆t 120 min

r) pH 9.8 - ∆t 240 min

Figure 3: dissolving of HTO modification of lysozyme (scale-bar in Figure 3r represents 200 µm und is applicable for all images) 12


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a) pH 5.0 - ∆t 0 min

b) pH 5.0 - ∆t 60 min

c) pH 5.0 - ∆t 120 min

d) pH 7.2 - ∆t 0 min

e) pH 7.2 - ∆t 60 min

f) pH 7.2 - ∆t 120 min

g) pH 7.7 - ∆t 0 min

h) pH 7.7 - ∆t 120 min

i) pH 7.7 - ∆t 240 min

j) pH 8.05 - ∆t 0 min

k) pH 8.05 - ∆t 120 min

l) pH 8.05 - ∆t 240 min

m) pH 8.4 - ∆t 0 min

n) pH 8.4 - ∆t 120 min

o) pH 9.8 - ∆t 240 min

p) pH 9.8 - ∆t 0 min

q) pH 9.8 - ∆t 100 min

r) pH 9.8 - ∆t 200 min

Figure 4: dissolving of LTO modification of lysozyme (scale-bar in Figure 4r represents 200 µm und is applicable for all images) 13



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a) pH 5.0 - ∆t 0 min

b) pH 5.0 - ∆t 120 min

c) pH 5.0 - ∆t 240 min

d) pH 5.9 - ∆t 0 min

e) pH 5.9 - ∆t 120 min

f) pH 5.9 - ∆t 240 min

g) pH 7.2 - ∆t 0 min

h) pH 7.2 - ∆t 120 min

i) pH 7.2 - ∆t 240 min

j) pH 7.7 - ∆t 0 min

k) pH 7.7 - ∆t 120 min

l) pH 7.7 - ∆t 240 min

m) pH 9.8 - ∆t 0 min

n) pH 9.8 - ∆t 120 min

o) pH 9.8 - ∆t 240 min

Figure 5: dissolving of tetragonal modification of lysozyme (scale-bar in Figure 5o represents 200 µm und is applicable for all images)

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Acknowledgement The authors are gratefully for the support of Chinese-German Center to Promote Science, GZ: 935

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http://digital.bibliothek.uni-halle.de/hs/content/titleinfo/1177647, 2012. (7) Aldabaibeh, N. Towards an improved understanding of the variables controlling crystal modification and phase transition in protein crystals. Ph.D. thesis, Illinois Institute of Technology, http://gradworks.umi.com/34/17/3417924.html, 2009. (8) Fabian, J.; Ulrich, J. In proceeding 12th Symposium on Industrial Crystallization; Rojkowski, Z. H., Ed.; 1993; Vol. 2; pp 41–46.

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(9) Lim, K.; Nadarajah, A.; Forsythe, E. L.; Pusey, M. L. Locations of bromide ions in tetragonal lysozyme crystals. Acta Cryst D 1998, 54, 899–904. (10) Harata, K.; Akiba, T. Structural phase transition of monoclinic crystals of hen eggwhite lysozyme. Acta Cryst D 2006, 62, 375–382. (11) Forsythe, E. L.; H. Snell, E.; Malone, C. C.; Pusey, M. L. Crystallization of chicken egg white lysozyme from assorted sulfate salts. Journal of Crystal Growth 1999, 196, 332–343. (12) Jones, M. On the Industrial Crystallization of Proteins; Berichte aus der Verfahrenstechnik; Shaker Aachen, 2014. (13) Ulrich, J.; Pietzsch, M. What is a protein crystal? Can we apply the terminology of classical industrial crystallization to them? Cryst. Res. Techn. 2015, 50, 560–565. (14) Matthews, B. W. Solvent content of protein crystals. J Mol Biol 1968, 33, 491–497. (15) Matsuoka, M.; Sumitani, A. Rate of composition changes of organic solid solution crystals in sweating operations. J. Chem. Eng. Japan 1988, 21, 6–10. (16) Beilles, S.; Cardinael, P.; Ndzié, E.; Petit, S.; Coquerel, G. Preferential crystallisation and comparative crystal growth study between pure enantiomer and racemic mixture of a chiral molecule: 5-ethyl-5-methylhydantoin. Chemical Engineering Science 2001, 56, 2281–2294. (17) Pauchet, M.; Coquerel, G. Polar Dissolution and Regrowth of (±)-Modafinil Twins Grown in Gels. Crystal Growth & Design 2007, 7, 1612–1614. (18) Pusey, M. L.; Nadarajah, A. A Model for Tetragonal Lysozyme Crystal Nucleation and Growth. Crystal Growth & Design 2002, 2, 475–483. (19) Kam, Z.; Shore, H.; Feher, G. On the crystallization of proteins. Journal of Molecular Biology 1978, 123, 539–555. 16


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(20) Moritz, R. L.; Simpson, R. J. Liquid-based free-flow electrophoresis-reversed-phase HPLC: a proteomic tool. Nature Methods 2005, 2, 863–873. (21) Tanford, C.; Roxby, R. Interpretation of protein titration curves. Application to lysozyme. Biochemistry 1972, 11, 2192–2198.

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Graphical TOC Entry Lysozyme protein crystals can be dissolved at different conditions. The crystals will show two different dissolution mechanisms. The parameters which influences the dissolution mechanisms where systematically investigated and an explanation for these different mechanisms are given.

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Dissolution Behavior of Lysozyme Crystals - Crystal Growth & Design

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