The crystallization of lysozyme in the system of ionic liquid [BMIm][BF4]‐water

Lysozyme crystallization was conducted in the ionic liquid (IL) 1‐butyl‐3‐methylimidizolium tetrafluoroborate ([BMIm][BF₄]) with different buffer/IL proportions. It was found that the addition of [BMIm][BF₄] could promote the crystallization process, during which more lager single crystals with controllable morphologies could be obtained due to the manageable crystal growth velocity. A probable explanation was proposed based on the influence of the ionic polarization and kinetics in the lysozyme crystallization. Moreover, the transform in coordination number and the relative growth rate of different crystal faces were discussed. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Second virial coefficient: variations with lysozyme crystallization conditions

Interparticle lysozyme interactions in solution have been studied by small angle X-ray scattering (SAXS) as a function of salt type (NaCl, NaNO₃, NaSCN and NaOAc), salt concentration, and as a function of temperature between 30°C and 10°C. The choice of conditions was made to cover variations from (undersaturated) solutions to (supersaturated) crystallization conditions. The second virial coefficients (A₂) were determined from the X-ray structure factors extrapolated to the origin, as a function of protein concentration. The A₂ values which correspond to lysozyme crystallization conditions were found to be in a range from about zero to −8.0×10⁻⁴ mol ml g⁻², in agreement with previous determinations by other groups. The variations of the second virial coefficient from positive (repulsive interactions) to negative (attractive interactions) were found to follow the efficiency of salts to induce crystallization. The choice of the second virial coefficient as a tool to predict crystallization conditions is discussed.     

Atomic force microscopy study of lysozyme crystallization

Crystallization of lysozyme from solutions has been studied by the atomic force microscopy method. The surface morphology and the growth kinetics of several faces of the orthorhombic and monoclinic modifications of lysozyme crystals are considered. The surface images are obtained at molecular resolution. For the (010) face of orthorhombic lysozyme, the phenomenon of the surface reconstruction is established—doubling of the unit-cell parameter along the a-axis. The main growth parameters of lysozyme are determined—the kink density at steps, probabilities of the attachment and detachment of building blocks, the kink and step velocities, and the dependence of the fluctuation in the step position on time.     

Effects of purification on the crystallization of lysozyme

We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20°C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal ↔ orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.     

In situ monitoring and control of lysozyme concentration during crystallization in a hanging drop

Fiber optic Raman spectroscopy combined with a partial least-squares regression model was demonstrated as a monitor of lysozyme concentration during crystallization in a hanging drop experiment in real time. Raman spectral features of the buffer and protein were employed to build the regression model. The use of fiber optic technology coupled with Raman spectroscopy, which is ideal for use with aqueous solutions, results in a powerful noninvasive probe of the changing environment within the solution. Lysozyme concentrations were monitored in experiments at a constant reservoir ionic strength. Data from these uncontrolled experiments were used to determine rates of supersaturation, induction times, and the number and size of the resultant lysozyme crystals. Control experiments were performed by introducing step changes in the reservoir ionic strength. The step changes were initiated by comparing in situ rates of supersaturation with the rates of supersaturation calculated from the uncontrolled data. Monitoring the concentration changes of the lysozyme within the hanging drop permits a measurement of the level of supersaturation of the system and enhances the possibility of dynamic control of the crystallization process.     

Solvent freeze out crystallization of lysozyme from a lysozyme‐ovalbumin mixture

Hen egg white lysozyme (HEWL) crystallization conditions from an ovalbumin‐lysozyme mixture were found by screening tests and further located in pseudo‐phase diagrams. This information was used to set up the initial conditions for the solvent freeze out (SFO) process. The process uses the freezing of ice to create the supersaturation for the proteins to crystallize out of the solution. The crystallization of HEWL (15 mg/mL) out of a lysozyme‐ovalbumin mixture (1.7 mg/mL) is carried out by SFO. Under the reported conditions, a crystallization yield of 69 % was obtained. A mean crystal size of 77.8 µm was enhanced in a crystallization time of 15.1 h. The lysozyme nature of the crystals is proven by SDS PAGE and enzymatic activity tests. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Uncertainties in crystallization of hen‐egg white lysozyme: reproducibility issue

The reproducibility of biomacromolecular crystallization (tetragonal and orthorhombic lysozyme crystals) was studied by monitoring the evolution of protein concentration during the crystallization process using Mach‐Zehnder interferometer. It was found that formation of both tetragonal and orthorhombic crystals exhibited poor reproducibility. When the crystallization occurred under isothermal conditions, the protein concentration in the solution varied differently in different experiments under identical conditions (for both types of crystals). Moreover, in the case of orthorhombic lysozyme crystallization (under either isothermal or thermal gradient conditions), it is clear that the crystals could not be always readily formed. When formation of tetragonal lysozyme crystals was conducted at a temperature gradient condition, however, the evolution of concentration was reproducible. The phenomena found in this study revealed that biomacromolecular crystallization can be uncertain, which is probably caused by the process of nucleation. Such uncertainties will be harmful for the efforts of screening crystallization conditions for biomacromolecules. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Spherulitic branching in the crystallization of liquid selenium

Liquid selenium is a spherulite-forming liquid. In a previous study [G. Ryschenkow and G. Faivre, J. Crystal Growth 87 (1988) 221], several spherulitic modes of crystallization have been observed to coexist, at a given undercooling of the liquid, with the growth of single crystals. The spherulitic modes were thought to be basically due to a mechanism inducing a regular polygonization of crystal during growth (small-angle branching). We present a morphological investigation of the spherulites of selenium, by optical and electron microscopy, which substantiate this conjecture. At medium undercooling of the liquid, the small-angle branching periodically triggers a homoepitaxial large-angle branching. This gives rise to nonringed spherulites. At higher undercooling the spherulites are ringed, which we attribute to the ineffectiveness of the large-angle branching. The existence of several spherulitic modes signifies that several stable regimes of the small-angle branching exist.     

Influence of various factors on the liquid/liquid diffusion crystallization of proteins

From a numerical simulation of pre-nucleation solute transport in the liquid/liquid diffusion crystallization of proteins, it is derived that there are various factors influencing the spatial and temporal distributions of supersaturation. They include the ratio of the length of the protein solution to that of the salt solution in a tube crystallizer, the initial concentrations of protein and salt, and the initial salt concentration in the protein solution. Their influences on the protein crystallization have been demonstrated by the corresponding experiments of gel crystal growth. It may be deduced that the experimental conditions for the liquid/liquid diffusion growth of protein crystals could be optimized under given conditions in order to develop the advantages of this method.     

Exploration on nano-composite fumed silica-based composite polymer electrolytes with doping of ionic liquid

Fumed silica (SiO₂)-based composite polymer electrolytes were prepared by means of solution casing technique. Horizontal attenuated total reflectance-Fourier Transform Infrared (HATR-FTIR) study shows the complexation between polymer matrix and SiO₂. The highest ionic conductivity of (4.11 ± 0.01) × 10^? 3 Scm^? 1 is achieved upon inclusion of 8 wt.% of SiO₂. Three different regions have been observed in the frequency dependence–ionic conductivity study. The conductivity rises sharply with frequency at low frequency regime. It is followed by a frequency independent plateau region and sharp decrease in the conductivity at high frequency range. The dielectric permittivity (ε^') and dielectric loss (ε^") are decreased with increasing the frequency. This phenomenon is mainly attributed to the electrode polarization effect. The formation of electrical double layer has been proven in these dielectric permittivity studies. This indicates the non-Debye properties of the nano-composite polymer electrolytes.     

Structural order and crystallization of an iron-rich aluminosilicate liquid under oxidizing condition

An iron-rich aluminosilicate liquid is studied in different atmospheres by using differential scanning calorimetry. The results show that the oxidation state of iron and the maximum temperature of the scan have an effect on the liquid structure and on the crystallization behavior of the liquid during both cooling and reheating processes. Increase in Fe²⁺/Fe³⁺ enhances crystallization and favors formation of a stable crystal structure. The structural order conserved in the liquid is an intrinsic phenomenon, which is not related to the oxidation state of iron.     

Molecular dynamics simulation of the crystallization of a liquid gold nanoparticle

Molecular dynamics simulation of the crystallization behavior of a liquid gold (Au) nanoparticle, about 4 nm in diameter, on cooling has been carried out based on the modified embedded-atom-method potential. With decreasing cooling rate, the final structure of the particle changes from amorphous to crystalline via icosahedron-like structure. While the outer shell of the icosahedron-like particle shows crystalline feature with {1 1 1}-like facets, the inner core remains amorphous. It is found that the structure of the fully crystallized particle is polycrystalline face-centered cubic (fcc). The fcc structure of the gold nanoparticle is proved energetically the most stable form.     

Relationship between the nucleation and precrystallization liquid phase during homogeneous crystallization of metals

A comparative estimation of the size parameters of clusters of precrystallization liquid phase (PLP) of metals and critical crystalline nuclei has revealed the sizes of critical crystalline nuclei to be smaller than those of PLP clusters. Based on the results of estimation of the radii of critical crystalline nucleus by the methods of classical theory and molecular dynamics, it has been found for the first time that the radius of critical crystalline nucleus is about 0.2–0.4 of the PLP cluster radius. This fact confirmed the hypothesis of Ubellode et al. about the formation of a transition layer with a partially ordered structure between the solid and liquid phases. A scheme of the nucleation and growth of a crystalline nucleus under conditions of transition layer formation is proposed.

     

The dissolution phenomenon of lysozyme crystals

Dissolution studies on lysozyme crystals were carried out since the observed dissolution pattern look different from non‐protein dissolved crystals. The Tetragonal, High Temperature and Low Temperature Orthorhombic morphologies, crystallized using sodium chloride, were chosen and the influence of different pH, salt and protein concentration on their dissolution was investigated. An increase in pH and/or salt concentration can modify the dissolution behaviour. The pattern of the crystals during the dissolution process will, therefore, develop differently. Frequently a skeleton like crystal pattern followed by a falling apart of the crystals is observed. The multi‐component character of the lysozyme crystal (protein, water, buffer, salt) as well as “solvatomorphism” gives first insights in the phenomena happening in the dissolution process. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Ion conducting corn starch biopolymer electrolytes doped with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate

Biodegradable corn starch-lithium hexafluorophosphate (LiPF₆) based biopolymer electrolytes were prepared by solution casting technique. Ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate (BmImPF₆) was doped into the polymer matrix. Upon addition of 50 wt.% BmImPF₆, the maximum ionic conductivity of (1.47 ± 0.02) × 10^− 4 Scm^− 1 was achieved due to its higher amorphous region. This result had been further proven in ATR-FTIR study. Frequency dependence of conductivity and dielectric studies reveal the occurrence of polarization at the electrolyte-electrode interface and thus form the electrical double layer, asserting the non-Debye characteristic of polymer electrolytes. This result is in good agreement with dielectric loss tangent study. Based on the changes in shift, changes in intensity, changes in shape and existence of some new peaks, attenuated total reflectance-Fourier Transform Infrared (ATR-FTIR) divulged the complexation between corn starch, LiPF₆ and BmImPF₆, as shown in the spectra.     

Continuous compositional changes of crystal and liquid during crystallization of a sodium calcium silicate glass

This paper deals with a systematic study of crystal nucleation and growth kinetics in a 14.6Na₂O–34.0CaO–51.4SiO₂ mol% glass, which is close to the CaO · SiO₂–Na₂O · SiO₂ pseudo-binary section, just left of the stoichiometric Na₂O · 2CaO · 3SiO₂ (N₁C₂S₃) compound. We show that crystallization begins with nucleation of a Na_4+2xCa_4−x[Si₆O₁₈] (0 < x < 1) solid solution that is enriched in sodium as compared with both parent glass and the N₁C₂S₃ compound; while a fully crystallized sample is composed only by a solid solution that is stable at very high temperatures, but is metastable in the temperatures under investigation. We thus confirm a continuous compositional change of the crystals during the course of crystallization.     

The distribution of impurities in lysozyme crystals

The effective distribution coefficient, K_Ieff, of turkey egg white lysozyme into host hen egg white lysozyme crystals was measured for varying levels of relative supersaturation, impurity concentrations and temperatures. Turkey egg white lysozyme served as a test impurity of high homology to hen egg white lysozyme. K_Ieff increased with increasing host protein concentration and temperature, consistent with a kinetically controlled incorporation process. The principles of slow crystal growth rate to limit impurity incorporation were applied to a commercial source of lysozyme with concomitant improvement in purity of re-crystallized protein.     

The crystallization of amorphous germanium films

Germanium thin films were prepared in an amorphous form by vacuum deposition onto room temperature fused silica substrates. The amorphous—crystalline transition was studied as a function of time and temperature by measuring the optical transmission near 0.65 μm, where the absorption constant is most sensitive to the phase transformation. At a fixed temperature, the time for half the volume of the sample to become crystallized was found to be consistent with the relation t_c = τ exp(E₀/kT), with an activation energy E₀ = 2.96 eV.     

The effect of diluting crystallization droplets on protein crystallization in vapor diffusion method

In this study, effects of diluting either protein or crystallization agents in the droplets on the success rate of protein crystallization was investigated. Diluting the crystallization agent was found to increase the success rate of protein crystallization. Theoretical analysis showed that, concentration ranges of both protein and crystallization agent that can be scanned during the vapor diffusion process are wider with diluting the crystallization agent than that without dilution, resulting in more opportunities for the crystallization solution to be in the nucleation zone. On the other hand, diluting protein could lead to controversial results depending on the location of the initial concentration relative to that of the nucleation zone in the phase diagram. The method of diluting the crystallization agent is therefore proposed as an alternative modification to the conventional vapor diffusion method for obtaining more crystallization conditions in protein crystallization screening. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)