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Biomaterial crystallization.pptx
- 1. (CRYSTALLIZATION AND STABILITY OF DIFFERENT PROTEIN CRYSTAL MODIFICATIONS : A CASE STUDY OF LYSOZYME) SUBMITTED BY : SAWAN BHAT CB21M15
- 2. INTRODUCTION • Crystallization from solution is a separation technique where a solid phase is separated from a mother liquor. The process consists of two major steps, nucleation and crystal growth. • Nucleation is the step where the solute molecules dispersed in the solvent start to gather into clusters. • Crystal growth is the rate of displacement of a given crystal surface In the direction perpendicular to the face. • In this case study we will see, the crystallization, including both the phase diagram and the phase transition of hen egg white lysozyme (HEWL)
- 3. • Egg white lysozyme of chicken eggs belongs to the c-type lysozymes ,which are one of the major types identified in the animal kingdom, along with g-type (goose-type and bacterial lysozyme) and i-type (invertebrate lysozyme). • Lysozyme is endowed with a number of properties suitable for food application. It is a heat-stable protein and is not inactivated by solvents and does not lose its activity when redissolved in water. • Applications of Lysozyme include , cheese preservation , preservative in either pasteurized or sterile filtered beer, used in extension of shelf life of various food items such as vegetables.
- 4. CASE STUDY • A tetragonal modification and a needle modification were obtained during crystallization. • The phase diagram and stability of two modifications in both acid and basic pH solutions (pH 4.5, 8.0 and 9.0) were obtained. • Based on the phase diagram, phase transfer behavior was found to exist between the two modifications. • Change of temperature and pH were studied on the crystal structure.
- 5. • A model system for investigations into protein crystallization is the enzyme hen egg white lysozyme (HEWL). This protein can crystallize in different crystal forms by using different precipitants and by varying crystallization conditions like temperature, pH, supersaturation level and with the presence of small amounts of heavy atoms and traces of other materials. • Different morphologies that have been discovered till now , some of them are, using sodium chloride as the precipitant, the well-known tetragonal structure P43212 can be obtained from a sodium acetate buffer in a temperature ranging from −4 to 25 °C. • An orthorhombic structure P212121 (HTO) crystallizes from the acid buffer solution between 25 and 60 °C. This kind of HTO
- 6. • In addition, studies have also found that phase transformation can happen between different modifications of protein crystals. At temperatures higher than 25 °C, the tetragonal morphology will transfer to the orthorhombic modification (HTO- high temperature orthorhombic) with the existence of acid buffer solutions. • This case study is concerned with investigations of both phase diagram and the phase transition of different protein modifications. • Hen egg white lysozyme (HEWL) was chosen as the model protein. Tetragonal morphology and needle shaped crystals were obtained from the crystallization progress. Both kinds of crystals were identified by morphology and X-ray powder diffraction (XRPD) determination. • Phase diagrams were made in both acid and basic mediums and
- 7. EXPERIMENTAL • Lysozyme crystallization : Hen egg white lysozyme (HEWL) was purchased from Fluka , and used without further purification. Sodium chloride (NaCl) purchased from Carl Roth was chosen as the precipitant. All the protein and salt solutions were treated with buffer solutions. Lysozyme was dissolved in buffer and the precipitate solution was prepared by dissolving sodium chloride in the same buffer. The tetragonal crystals were prepared from a solution containing 8 mg/mL protein and 4% (w/v) NaCl. The crystallization process of the tetragonal modification was carried out in both 0.1 M sodium acetate buffer (pH 4.5) and 0.5 M Tris-HCl buffer (pH 8.5).
- 8. The needle shaped crystals were prepared from a solution with a final concentration of 12 mg/mL protein and 4% (w/v) NaCl in 0.5 M Tris-HCl buffer (pH 8.5). All the crystallization solutions were stored at 7 °C for crystallization. When the crystallization process finished, 100 μL of the crystallization solution was taken out for the microscope analysis. The mother liquor and crystals were separated by centrifugation.
- 9. X-RAY POWDER DIFFRACTION (XRPD) • X-ray powder diffraction was carried out at room temperature on a Brucker D8 Discover diffractometer equipped with a Cu Kα source. • X-ray diffraction analysis (XRD) is a technique used in materials science to determine the crystallographic structure of a material • The measurement is used to compare the crystal structure of different crystal forms. • Diffraction data were collected under 40 kV, 40 mA. Typical
- 10. ACTIVITY TEST • The activity test was carried out by the use of a Varian Cary 50 UV/Vis spectrophotometer. • The bacteria, M. lysodeikticus, of 8.4 mg were suspended in 50 mL 66mM phosphate buffer, pH 6.2 and used as the substrate. • The crystalline enzyme solutions (1 mg/mL) of tetragonal and needle forms were prepared with the same buffer. • The biological reaction was carried out by adding 100 μL enzyme solution to 2.5 mL bacteria solution at 25 °C. • The decreasing in the concentration of the bacteria in 2 min was monitored by a photometer at the wavelength of 450 nm.
- 11. SOLUBILITY MEASUREMENT • The solubility measurements were carried out in a STEM Integrity Parallel Synthesis Station. • To determine the solubility points, lysozyme crystal suspensions were heated up to a certain temperature. A heating rate of 0.1 K/min was involved here. The solubility point is defined as the point when transmission reaches a stable plateau. • The solubility of different lysozyme modifications was measured at three different pH values (pH = 4.5, 8.0 and 9.0). • The concentration of NaCl remained 4% (w/v) for all measurements. The protein concentration of the clear solution was verified by UV/Vis spectrophotometer at a wavelength of 280 nm after each experiment
- 12. PHASE TRANSITION EXPERIMENT • The phase transition between different modifications was observed by a microscope. • A cell was filled with 2 mL of protein solution by a pipette, and seed crystals were added to the solution. • The temperature of the cell was controlled by a thermostat. • Phase transition between tetragonal and needle modifications were observed under different pH and different temperature. • The progress was recorded by a microscope equipped with a digital camera.
- 13. RESULTS AND DISCUSSION • Lysozyme crystallization and crystal morphology : (a) Tetragonal crystals from 0.1M sodium acetate buffer pH 4.5 (8 mg/mL) (b) Tetragonal crystals from 0.5M Tris-HCl buffer pH 8.5 (8 mg/mL). (c) Needle shaped crystals from 0.5M Tris-HCl buffer pH 8.5 (12 mg/mL).
- 14. XPRD MEASUREMENT • The XRPD pattern of tetragonal and needle forms obtained from the experiment are shown in fig. below. • Two intense peaks in the needle pattern, at about 5 degree and 7 degree, are missing in the tetragonal pattern. • The pattern for the buffer and salt were also measured and the results were quite different from protein crystals, since the peaks of buffer and salt appear at higher degrees than most of the peaks for protein crystals. • It can be a proof that the two lysozyme morphologies have different crystal structures
- 15. SOLUBILITY AND PHASE DIAGRAM • The solubility was checked by the turbidity measurement and the method has been successfully used for measuring the solubility of protein crystals. • By measuring the solubility of different morphologies of lysozyme, it is possible to determine the stability of the different crystal forms. • The solubility of both forms increases with the temperature as normal behavior. However, the effect of pH is not as clear as that of temperature. • The solubility of needle shaped crystals decreases with an increase of the pH. But the relationship between solubility and pH for tetragonal crystals is not that obvious.
- 16. PHASE TRANSITION • Phase transformations of different lysozyme morphologies have been observed in a microscope cell. • It was found in the literature, the transition temperature from tetragonal to HTO lysozyme crystals was around 25 °C and LTO modification can transfer to HTO form when the temperature is higher than 35 °C. • The phase transition from needle shape too tetragonal morphology was carried out in acid conditions. • For the transformation experiment, a lysozyme solution with a concentration of 5 mg/mL was prepared in 0.1 M sodium acetate buffer. • Seed crystals of the needle morphology, without special treatment, were added to the solution. The solution was kept at 20 °C to avoid transformation to the HTO modification and no change in the pH, ionic strength and protein concentration were involved. Fig. Phase transformation of needle shaped to tetragonal crystals. The process was carried out in 0.1 M sodium acetated buffer at 20 °C
- 17. • The phase transition process can be more complicated in basic buffer solutions. When pH increases to 9, needle shaped crystals are more stable according to the phase diagram. • The phase transition process in pH 9 buffer solutions was observed in a microscope cell. • Tetragonal seed crystals were added to a protein solution of 8 mg/mL in 0.5 M Tris-HCl buffer at 20 °C.
- 18. • The effect of pH was studied on these sample. A lysozyme solution of 8 mg/mL was prepared with pH 8, pH 8.5 and pH 9 buffer, respectively and all the experiments were carried out at 20 °C. Fig. Effect of pH on the phase transition behavior. All experiments were carried out at 20 °C. A: pH 9, B: pH 8.5, and C: pH 8.
- 19. • Experiments found that tetragonal crystals can still be obtained at low temperature with the existence of needle shaped crystals. Effect of temperature on the phase transition behavior. A: 35 °C, B: 30 °C, C: room temperature, D: 7 °C.
- 20. CONCLUSION • The phase diagram and phase transition of hen egg white lysozyme were studied. • Tetragonal and needle modifications were investigated and the stable regions of both modifications were determined. • It is more likely to obtain the tetragonal crystal form in an acid solution, and in solutions with a pH 9, needle shaped crystal forms nucleate more likely. • The tetragonal modification can be obtained in a basic solution as well, but is only stable at low temperature. • In addition to the transition behaviors already discovered, phase transformation between tetragonal modification and needle shaped crystals was found. • In acid buffer solutions, the needle shaped crystals can, transfer to