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Inclusion of protein lysozyme molecules in the lipidic mono-olein cubic phase induces a transition from a Pn3m structure to an Im3m one. The small-angle x-ray scattering method with high-intensity synchrotron radiation enabled us to follow closely the transition depending on the conditions of lysozyme solutions. We show that concentrated lysozyme solutions induced the appearance of the Im3m structure coexisting with the Pn3m structure. From the relation between the lattice parameters of these two structures it is shown that they are related by the Bonnet transformation of the underlying triply periodic minimal surfaces. We found that the transition also occurred at lower lysozyme concentration when NaCl induced an attraction between lysozyme molecules. The origin of the transition was considered as a frustration in the cubic phase where lysozyme molecules were highly confined. A simple estimation of the frustration was given, which took into account the translational entropy of lysozyme molecules. At the highest concentration of lysozyme and NaCl the Im3m structure was found to disappear and left only the Pn3m structure. This was probably either due to the crystallization or phase separation of lysozyme solutions ongoing microscopically, which absorbed lysozyme molecules from channels of the cubic phase and thus removed the frustration.

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We applied dynamic light scattering technique on the model system of hen egg lysozyme in salt-free aqueous ethanol solution to study the mechanism of denaturation and aggregation of protein. At low ethanol concentration [0-63% (v/v)], the fast relaxation mode was observed, which was caused by lysozyme molecules in the solution interacting with each other with strong repulsive electrostatic force. At 45 and 63% (v/v) ethanol, the slow relaxation mode was also observed, which showed translational diffusive nature, similar to that observed in salt-free polyelectrolyte solution. At 72 or 81% (v/v) ethanol, the slow mode disappeared, leaving only the fast mode. However, the mutual diffusion coefficients obtained from the fast mode at 72 and 81% (v/v) ethanol decreased by about one order of magnitude compared with those from the fast mode at 0-63% (v/v). The reported alcohol-induced conformational transformation of lysozyme molecules at >60% (v/v) ethanol from their native structure to an alpha-helix-rich structure might cause such drastic decrease in the mutual diffusion coefficients. At the highest ethanol concentration of 90% (v/v), the slow mode reappeared, and its relaxation rate was decreasing with elapsed time, which is possibly due to the growth of aggregates of lysozyme molecules. X-ray diffraction results suggested that the intermolecular beta-sheet formation caused the aggregation. Thus, our results indicated that the change in molecular structure of lysozyme closely relates to the diffusion of molecules and their aggregation.

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Protein crystallization under micro gravity has been already tried many times in the United States and other countries, and it is reported that about 20% of proteins were better crystallized under microgravity than on earth. This verified that microgravity is sometimes effective in protein crystallization. However, if these procedures continued to be carried out without clarifying which processes are effective, improved development of protein crystallization cannot be expected. The most effective way to study the process is to carry out protein crystallization experiments, each elementary stage of which is clearly observed. To this end, the dissolution rate of a single crystal of hen egg-white lysozyme has been measured both under microgravity and on earth in order to study the mechanism of protein crystallization. In May 1997, we had an opportunity to have an experiment on protein crystallization with use of STS-84 space shuttle missions (the time duration of which was 210 hours). The apparatus for protein crystallization by vapor diffusion techniques was available and we have tried to use it for measurement of crystal dissolution rate under microgravity. The barrels of two syringes (20 ml x 2) were filled with unsaturated protein solutions (hen egg-white lysozyme (HEWL): 0.0wt%, 0.1wt%, 0.2wt%, 0.3wt%; NaCl 3wt% aqueous solution) and a crystal (HEWL: tetragonal form; 1 mm +/- 0.2 mm in diameter) was put on the bottom of the one syringe. Dissolution of the crystal was started by extruding the unsaturated solutions onto the syringe tip with the crystal. The crystal dissoluted in 40 ml droplets that are extruded from syringes. The temperature was kept at 20 degrees C. Just before the Space Shuttle begins returning to the earth, the protein solution was withdrawn back into the syringes by the astronaut, and the melting experiment was finished. In the one syringe the incompletely melted crystal was withdrawn as well. The solution concentration in the other syringe was measured. In the flight experiment, only the initial and final solution concentrations can be measured. In the control experiment on earth the solution concentrations in the course of dissolution were also measured. 20 undersaturated solutions (40 ml in volume) were prepared and a crystal (1 mm +/- 0.2 mm in diameter) was put in each solution. The solution concentration was measured at constant time intervals. When the crystal starts to dissolute [correction of dissolue], molecules in the crystal surface leave the crystal, the concentration around the crystal becomes high and there occurs a radial concentration gradient centered on the nucleus. Molecules diffuse along the gradient and go farther from the crystal. On earth, the concentration gradient might be disturbed by convection and the high concentration around the crystal might be disturbed more quickly than under microgravity. We might therefore expect that dissolution rate on the ground should be larger than under micro-gravity. However, our experimental results show that the expectation was not correct. What might the reason be? One possible reason is that there might be Marangoni convection effects in the apparatus for protein crystallization by vapor diffusion techniques used for the melting experiment. This effect has also been observed by Chayen et al.

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The accuracy of the structures of biological macromolecules determined by X-ray crystallography is of fundamental importance, both for the understanding of life processes and for medical applications. The resolution of the structure is thus critical, and is largely determined by the quality of single crystals. Here we report the results of applying a magnetic field and a magnetization force during growth of the snake muscle fructose-1,6-bisphosphatase and human estrogenic 17beta-hydroxysteroid dehydrogenase crystals. For both enzyme proteins, the quality of the crystals improved with repeated assay, and their data sets were collected at significantly higher resolutions. These results coincide with a mechanism involving the reduction of convection, due to both the hydrodynamics within a magnet and the partially reduced gravity induced by a magnetization force. The density difference between the crystal and solution becomes less significant, and the sedimentation speed of the crystals is also reduced in the presence of the magnetization force.

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We observed low-frequency Raman spectra of tetragonal lysozyme crystals and DNA films, with varying water content of the samples. The spectra are fitted well by sums of relaxation modes and damped harmonic oscillators in the region from approximately 1 cm(-1) to 250 cm(-1). The relaxation modes are due to crystal water, and the distribution of relaxation times is determined. In wet samples, the relaxation time of a small part of the water molecules is a little longer than that of bulk water. The relaxation time of a considerable part of the crystal water, which belongs mainly to the secondary hydration shell, is an order of magnitude longer than that of bulk water. Furthermore, the relaxation time of some water molecules in the primary hydration shell of semidry samples is shorter than we expected. Thus we have shown that low-frequency Raman measurements combined with properly oriented samples can give specific information on the dynamics of hydration water in the ps range. On the other hand, we concluded, based on polarized Raman spectra of lysozyme crystals, that the damped oscillators correspond to essentially intramolecular vibrational modes.

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Since the production of peroxynitrite may contribute to the pathophysiology of endotoxemia or sepsis, the quantities of the produced peroxynitrite were evaluated in rats after lipopolysaccharide (LPS) treatment by measuring plasma nitrotyrosine concentrations with a new method. The intraperitoneal administration of LPS caused a persistent increase in plasma nitrotyrosine concentrations, which reached a maximum with 6-fold level of the base line (105 pmol ml-1) at 24 h and gradually declined to 3-fold level of the base line at 7 days. However, plasma concentrations of nitrite and nitrate peaked at 18 h, returning to base line within 48 h. The effect of LPS on the increase in plasma concentration of nitrotyrosine was dose-dependent and consistent with that of nitrite and nitrate concentrations. On the other hand, intravenous injection of nitrotyrosine revealed a rapid clearance with a plasma half-life of 1.67 h. These results indicate that the elevation of plasma nitrotyrosine concentrations may persist for more than a week after LPS treatment, and that the determination of plasma nitrotyrosine concentrations may be useful to detect the previous peroxynitrite-dependent oxidative damages.

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Small angle neutron scattering (SANS) method was used to study lysozyme solutions, with particular interest in an understanding of the crystallization process at the initial stage. It is found that (1) in the unsaturated solution, the protein molecules aggregate with a continuous increase in size when NaCl concentration is increased, and (2) in the supersaturated solution, an irreversible change, superimposed on the former process, occurs when the supersaturation is realized. These facts indicate the usefulness of SANS in detecting changes of protein molecules in solution on the nanometer scale. The reliability of the SANS results are indicated by (1) comparing them with those of small angle X-ray scattering (SAXS), and (2) comparing the effect of D(2)O and H(2)O as solvent. Since the interparticle interaction is essential in the crystallization process and a simple Guinier plot analysis is not allowed, a more rigorous framework of analyzing data with interference function is developed, through which both average interparticle distance and particle size are estimated.

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The elementary processes of protein crystal growth were investigated by means of laser Michelson interferometry on the example of the (101) face of tetragonal hen egg-white (HEW) lysozyme. The method allows real-time in situ observations of the morphology of the growing protein crystal surface, as well as simultaneous precise measurement of growth rate and step velocity on identified growth-layer sources. At the critical supersaturation of 1.6 the growth mechanism was shown to transform from dislocation-layer generation to surface nucleation. Measurements on different growth hillocks, with material of a different source and at a different temperature, all indicated that for supersaturations lower than approximately 1 growth is hindered by the competitive adsorption of (most probably) other protein species contained in HEW, although the material is pure by most analytical methods. At supersaturations sigma < 0.4 other impurities sometimes led to cessation of growth. However, at sigma in the range 0.9 < sigma < 1.6 growth processes are determined by the kinetics of pure lysozyme. This enabled us to measure the step kinetic coefficient beta for crystallization of a protein substance for the first time: beta = 2.8 micro m s(-1). This also means that by working in this supersaturation range we can eliminate the impurity effects. Other means to reduce influence of impurities is to use, if possible, a higher crystallization temperature. It is shown that slow crystallization of proteins is due primarily to impedance of the elementary act of entering the growth site and not to the low concentration of the solution. The value of beta does not depend on temperature, indicating the decisive role of entropy, not energy barriers, in the crystallization of biological macromolecules.

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Solubility curves (a phase diagram) of an amorphous precipitate and the orthorhombic crystals of a membrane protein, the photoreaction center from Rhodobacter sphaeroides, were determined. With the phase diagram, the process of crystal growth of the membrane protein was elucidated within a general physicochemical framework, and justification was provided of hitherto empirically selected crystallization conditions.

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A differential equation model with a polynuclear growth mechanism was formulated for a theoretical understanding of protein crystallization. The model equation contains two parameters characterizing nucleation and growth: the number of protein molecules constituting a critical nucleus and the order of growth kinetics. This model was applied successfully to explain the experimental data on the protein concentration changes due to nucleation and crystal growth of tetragonal and orthorhombic hen egg-white lysozyme. It was shown that the critical nucleus most probably consists of three or four molecules. The range and extent of the validity of the present model and analysis are discussed.

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Concentration changes in supersaturated solutions during the nucleation and growth of the orthorhombic form of hen egg-white lysozyme crystals have been observed for 121 d at 35 degrees C and pH 4.6, and with 3% NaCl. The effect of a variation in the initial protein concentration on the rate of approach to solubility in equilibrium is analyzed, by applying a model, originally developed for the understanding of protein self-assembly. It is shown that the observed kinetics can be explained fairly well by this model, whose basic assumptions are that (a) the nucleation is induced by aggregation of i0 molecules into particular geometry, and (b) the growth proceeds via attachment of a monomer. The i0 value for this process is four, which agrees with the number of molecules in a unit cell. Similarity and dissimilarity of the observed crystal growth to that of low molecular weight substances are discussed.

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To express the solute-removing capacity of a dialyzer, two diagrams are used in combination: one to represent the relation between the mass transfer coefficient and the solute molecular weight (M), and the other between the extraction ratio and the number of mass transfer units. It is shown that membrane permeation properties can be deduced from the first diagram, a limiting factor of removal can be understood from the second diagram, both can be used to compare performances with different blood or dialysate flow rate (QB or QD) or membrane area, and this method of representing dialyzer performance is more informative than the conventional ones in which dialysance (DB) is plotted against QB or M. Discussion is also given on the conditions under which the relation between DB and log M, or log DB and log M, becomes linear. Numerical evaluation of DB/QB for cross-flow geometry is presented in the Appendix.

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The performance of hemodialyzers was expressed schematically using three dimensionless quantities. Compared with the usual representation in which the dialysance is plotted against the blood flow rate for each of the solutes of interest, this expression is considered to have the following advantages: (1) the function of a dialyzer to remove different solutes can be expressed by a single curve, (2) the effect of changing the membrane area, its permeability, and the flow rates on the performance can easily be understood, and (3) the effect of changing the blood flow rate on the ratio of the transport efficiency of two or more kinds of solutes can be evaluated. An estimate of the overall mass transfer coefficient is the only procedure necessary to move from the usual to the present expression. These analyses were applied to the performance of a commercial hemodialyzer, and it was verified that this representation could denote its operation fairly well.

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