Pressure-Temperature Stability, Ca(2+) Binding, and Pressure-Temperature Phase Diagram of Cod Parvalbumin: Gad m 1

Fish allergy is associated with IgE-mediated hypersensitivity reactions to parvalbumins, which are small calcium-binding muscle proteins and represent the major and sole allergens for 95% of fish-allergic patients. We performed Fourier transform infrared and tryptophan fluorescence spectroscopy to explore the pressure-temperature (p-T) phase diagram of cod parvalbumin (Gad m 1) and to elucidate possible new ways of pressure-temperature inactivation of this food allergen. Besides the secondary structure of the protein, the Ca(2+) binding to aspartic and glutamic acid residues was detected. The phase diagram was found to be quite complex, containing partially unfolded and molten globule states. The Ca(2+) ions were essential for the formation of the native structure. A molten globule conformation appears at 50 °C and atmospheric pressure, which converts into an unordered aggregated state at 75 °C. At >200 MPa, only heat unfolding, but no aggregation, was observed. A pressure of 500 MPa leads to a partially unfolded state at 27 °C. The complete pressure unfolding could only be reached at an elevated temperature (40 °C) and pressure (1.14 GPa). A strong correlation was found between Ca(2+) binding and the protein conformation. The partially unfolded state was reversibly refolded. The completely unfolded molecule, however, from which Ca(2+) was released, could not refold. The heat-unfolded protein was trapped either in the aggregated state or in the molten globule state without aggregation at elevated pressures. The heat-treated and the combined heat- and pressure-treated protein samples were tested with sera of allergic patients, but no change in allergenicity was found.     

Catalytic activity of beta-amylase from barley in different pressure/temperature domains

The depolymerization of starch by beta-amylase during exposure to hydrostatic pressure up to 700 MPa and within a temperature range from 20 to 70 degrees C has been investigated. Inactivation of the enzyme as well as alterations in conversion speed in response to combined pressure-temperature treatments were assessed by analyzing the kinetic rate constants. At 200 MPa a significant stabilization of the enzyme against heat inactivation was observed. However, high pressure also impedes the catalytic reaction and a progressive reduction of the conversion rate constants with increasing pressure was found at all temperatures investigated. For the overall reaction of maltose liberation from soluble starch in ACES buffer at pH 5.6 an optimum was identified at 106 MPa and at 63 degrees C, which is approximately 7 degrees C above the local maximum at ambient pressure (0.1 MPa). Gelatinization of nonsoluble starch granules in response to pressure-temperature (p-T) treatment has been inspected by phase-contrast microscopy and yielded circular curves of identical effect in the p-T plane.     

Isochoric preservation: A novel characterization method

Isochoric (constant volume) preservation is an alternative to traditional cryopreservation methods because it requires less cryoprotectant and is simple to operate. In order to validate that this method automatically minimizes the pressure for a given temperature, pressure and temperature data were collected from a specially designed pressure vessel. This vessel was then used to examine the effect of an isochoric environment on freezing point nucleation in an aqueous antifreeze protein solution, and to generate pressure-temperature phase diagrams for various cryoprotectant solutions. Our results show that the isochoric pressure vessel follows the pressure-temperature phase diagram of water, thereby minimizing the pressure for the given temperature. We also show that the nucleation temperature of the antifreeze protein in an isochoric vessel is lower than that of the isobaric method. Furthermore, the nucleation temperature decreased with increasing concentration in the isochoric vessel while the isobaric nucleation temperature showed no change. These results indicate that the isochoric environment imposes additional constraints on ice formation and warrants further study as these results may change when a different type of cryoprotectant is used. Finally, all of the cryoprotectant phase diagrams exhibited a similar pressure-temperature slope indicating that, regardless of the cryoprotectant used or the mechanism by which it suppresses freezing, isochoric freezing affects the molecules in the same manner. Together, all of these results indicate that the isochoric method of preservation is a valuable tool for characterizing the thermodynamic properties of cryoprotectants and has great potential as a cryopreservation method in the field of cryobiology.     

Theory of lipid monolayer and bilayer phase transitions: Effect of headgroup interactions

Summary Headgroup and soft core interactions are added to a lipid monolayer-bilayer model and the surface pressure-area phase diagrams are calculated. The results show that quite small headgroup interactions can have biologically significant effects on the transition temperature and the phase diagram. In particular, the difference in transition temperatures of lecithins and phosphatidyl ethanolamines is easy to reproduce in the model. The phosphatidic acid systems seem to require weak transient hydrogen bonding which is also conjectured to play a role in most of the lipid systems. By a simple surface free energy argument it is shown that monolayers under a surface pressure of 50 dynes/cm should behave as bilayers, in agreement with experiment. Although the headgroup interactions are biologically very significant, in fundamental studies of the main phase transition in lipids they are secondary in importance to the hydrocarbon chain interactions (including the excluded volume interaction, the rotational isomerism, and the attractive van der Waals interaction).     

Stability and catalytic activity of alpha-amylase from barley malt at different pressure-temperature conditions

The impact of high hydrostatic pressure and temperature on the stability and catalytic activity of alpha-amylase from barley malt has been investigated. Inactivation experiments with alpha-amylase in the presence and absence of calcium ions have been carried out under combined pressure-temperature treatments in the range of 0.1-800 MPa and 30-75 degrees C. A stabilizing effect of Ca(2+) ions on the enzyme was found at all pressure-temperature combinations investigated. Kinetic analysis showed deviations of simple first-order reactions which were attributed to the presence of isoenzyme fractions. Polynomial models were used to describe the pressure-temperature dependence of the inactivation rate constants. Derived from that, pressure-temperature isokinetic diagrams were constructed, indicating synergistic and antagonistic effects of pressure and temperature on the inactivation of alpha-amylase. Pressure up to 200 MPa significantly stabilized the enzyme against temperature-induced inactivation. On the other hand, pressure also hampers the catalytic activity of alpha-amylase and a progressive deceleration of the conversion rate was detected at all temperatures investigated. However, for the overall reaction of blocked p-nitrophenyl maltoheptaoside cleavage and simultaneous occurring enzyme inactivation in ACES buffer (0.1 M, pH 5.6, 3.8 mM CaCl(2)), a maximum of substrate cleavage was identified at 152 MPa and 64 degrees C, yielding approximately 25% higher substrate conversion after 30 min, as compared to the maximum at ambient pressure and 59 degrees C.     

Kinetics of pressure inactivation at subzero and elevated temperature of lipoxygenase in crude green bean (Phaseolus vulgaris L.) extract

Lipoxygenase (LOX) in crude green bean extract was irreversibly inactivated by pressure treatments combined with subzero or elevated temperature. LOX inactivation was described accurately assuming a first-order reaction. In the entire pressure-temperature domain studied (200 to 700 MPa and -10 to 60 degrees C), an increase in pressure at constant temperature enhanced the LOX inactivation rate, whereas at constant pressure, an increase in reaction rate was obtained by either increasing or decreasing temperature at 20 degrees C. At elevated pressure, LOX exhibited the greatest stability around 20 degrees C. Also the pressure dependence of the inactivation rate constants for LOX was the highest around 20 degrees C. On the basis of the estimated LOX inactivation rate constants, an iso-rate contour diagram as a function of pressure and temperature was constructed, and an empirical mathematical model describing the combined pressure-temperature dependence of the LOX inactivation rate constants was formulated.     

Protein stability and dynamics in the pressure-temperature plane

The pressure-temperature stability diagram of proteins and the underlying assumptions of the elliptical shape of the diagram are discussed. Possible extensions, such as aggregation and fibril formation, are considered. An important experimental observation is the extreme pressure stability of the mature fibrils. Molecular origins of the diagram in terms of models of the partial molar volume of a protein focus on cavities and hydration. Changes in thermal expansivity, compressibility and heat capacity in terms of fluctuations of the enthalpy and volume change of the unfolding should also focus on these parameters. It is argued that the study of water-soluble polymers might further our understanding of the stability diagram. Whereas the role of water in protein behaviour is unquestioned, the role of cavities is less clear.     

Hydrostatic Pressure and Proteins: Basic Concepts and New Data

In this paper we review some basic facts about the reversible and irreversible effects of high pressure on proteins. The effects include changes in intra- or intermolecular interactions (noncovalent bonds), in conformation and in solvation. Particular attention is directed to the interpretation of data where pressure-temperature dependency is an important phenomenon. Using model reactions, we have formulated a putative interpretation of physiological problems; we use these to explain how biological systems maintain activity when the temperature decreases and pressure increases, as in the case of barophilic micro-organisms in the deep sea world.     

Coarse-grained strategy for modeling protein stability in concentrated solutions. II: phase behavior

We use highly efficient transition-matrix Monte Carlo simulations to determine equilibrium unfolding curves and fluid phase boundaries for solutions of coarse-grained globular proteins. The model we analyze derives the intrinsic stability of the native state and protein-protein interactions from basic information about protein sequence using heteropolymer collapse theory. It predicts that solutions of low hydrophobicity proteins generally exhibit a single liquid phase near their midpoint temperatures for unfolding, while solutions of proteins with high sequence hydrophobicity display the type of temperature-inverted, liquid-liquid transition associated with aggregation processes of proteins and other amphiphilic molecules. The phase transition occurring in solutions of the most hydrophobic protein we study extends below the unfolding curve, creating an immiscibility gap between a dilute, mostly native phase and a concentrated, mostly denatured phase. The results are qualitatively consistent with the solution behavior of hemoglobin (HbA) and its sickle variant (HbS), and they suggest that a liquid-liquid transition resulting in significant protein denaturation should generally be expected on the phase diagram of high-hydrophobicity protein solutions. The concentration fluctuations associated with this transition could be a driving force for the nonnative aggregation that can occur below the midpoint temperature.     

Pressure-induced inactivation of E. coli β-galactosidase: influence of pH and temperatur

In order to assess the feasibility of a high-pressure immunodesorption process using a β-galactosidase-anti-/3-galactosidase complex as a model, the influence of high hydrostatic pressure on the inactivation of E. coli /3-galactosidase has been investigated. The irreversible activity loss of β-galactosidase was studied as a function of pH and temperature for pressures comprised between atmospheric pressure and 500 megapascal (MPa; 1 MPa = 10 bar). This enabled us to establish a practical pressure-temperature diagram of stability for this enzyme. The stability domains determined thus appeared to be strongly dependent on the pH under atmospheric pressure of the phosphate buffer employed for pressurisation. Therefore, to interpret meaningfully this result, the influence of pressure on the pH-activity curve of β-galactosidase was investigated by using a high-pressure stopped-flow device. It appeared that the pH-activity curve of this enzyme was also reversibly affected by pressures lower than 150 MPa. An interpretation of these results in relation to the high-pressure induced changes of ionisation constants is proposed. For our practical purpose, the implications for the elaboration of a high-pressure immunodesorption process using /3-galactosidase as a tag, are discussed.     

Effects of pressure-induced membrane phase transitions on inactivation of HorA, an ATP-dependent multidrug resistance transporter, in Lactobacillus plantarum

The effects of pressure on cultures of Lactobacillus plantarum were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of L. plantarum induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature diagram for cell membranes was established. Cells grown at 37 degrees C and pressure treated at 15 degrees C lost >99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membranes of these cells were in the gel phase region at ambient pressure. In contrast, cells grown at 15 degrees C and pressure treated at 37 degrees C lost >99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membranes of these cells were in the liquid crystalline phase region at ambient pressure. The kinetic analysis of inactivation of L. plantarum provided further evidence that inactivation of HorA is a crucial step during pressure-induced cell death. Comparison of the biological findings and the membrane state during pressure treatment led to the conclusion that the inactivation of cells and membrane enzymes strongly depends on the thermodynamic properties of the membrane. Pressure treatment of cells with a liquid crystalline membrane at 0.1 MPa resulted in HorA inactivation and cell death more rapid than those of cells with a gel phase membrane at 0.1 MPa.     

Prediction of ice content in biological model solutions when frozen under high pressure

High pressure is, at least, as effective as cryoprotective agents (CPAs) and are used for decreasing both homogenous nucleation and freezing temperatures. This fact gives rise to a great variety of possible cryopreservation processes under high pressure. They have not been optimized yet, since they are relatively recent and are mainly based on the pressure–temperature phase diagram of pure water. Very few phase diagrams of biological material are available under pressure. This is owing to the lack of suitable equipment and to the difficulties encountered in carrying out the measurements. Different aqueous solutions of salt and CPAs as biological models are studied in the range of 0°C down to ‐35°C, 0.1 up to 250 MPa, and 0–20% w/w total solute concentration. The phase transition curves of glycerol and of sodium chloride with either glycerol or sucrose in aqueous solutions are determined in a high hydrostatic pressure vessel. The experimental phase diagrams of binary solutions were well described by a third‐degree polynomial equation. It was also shown that Robinson and Stokes' equation at high pressure succeeds in predicting the phase diagrams of both binary and ternary solutions. The solute cryoconcentration and the ice content were calculated as a function of temperature and pressure conditions during the freezing of a binary solution. This information should provide a basis upon which high‐pressure cryopreservation processes may be performed and the damages derived from ice formation evaluated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Phase behavior and hydration of silk fibroin

The osmotic stress method was applied to study the thermodynamics of supramolecular self-assembly phenomena in crystallizable segments of Bombyx mori silkworm silk fibroin. By controlling compositions and phases of silk fibroin solution, the method provided a means for the direct investigation of microscopic and thermodynamic details of these intermolecular interactions in aqueous media. It is apparent that as osmotic pressure increases, silk fibroin molecules are crowded together to form silk I structure and then with further increase in osmotic pressure become an antiparallel beta-sheet structure, silk II. A partial ternary phase diagram of water-silk fibroin-LiBr was constructed based on the results. The results provide quantitative evidence that the silk I structure must contain water of hydration. The enhanced control over structure and phase behavior using osmotic stress, as embodied in the phase diagram, could potentially be utilized to design a new route for water-based wet spinning of regenerated silk fibroin.     

Theory of lipid bilayer phase transitions as detected by fluorescent probes

Summary We present a quantitative theory that relates the fluorescence intensityvs. temperature (I vs. T) profile of a fluorescent-labeled two-component lipid bilayer to the phase diagram of the bilayer and the partition coefficientK of the fluorophore between fluid and solid phases of the bilayer. We show how the theory can be used to evaluateK from experimentalI vs. T profiles and the appropriate phase diagrams as well as to understand the different shapes ofI vs. T profiles obtained with particular fluorophores and phase diagrams. Using calculatedI vs. T graphs, we discuss the meaning of parameters, such as midpoint of the phase transition and onset and termination of a transition, which are often used to characterize phase transitions on the basis of fluorescence intensityvs. temperature profiles.     

Theory of cooperative transitions in protein molecules. I. Why denaturation of globular protein is a first-order phase transition

A theory of equilibrium denaturation of proteins is suggested. According to this theory, a cornerstone of protein denaturation is disruption of tight packing of side chains in protein core. Investigation of this disruption is the object of this paper. It is shown that this disruption is an "all-or-none" transition (independent of how compact is the denatured state of a protein and independent of the protein-solvent interactions) because expansion of a globule must exceed some threshold to release rotational isomerization of side chains. Smaller expansion cannot produce entropy compensation of nonbonded energy loss; this is the origin of a free-energy barrier (transition state) between the native and denatured states. The density of the transition state is so high that the solvent cannot penetrate into protein in this state. The results obtained in this paper make it possible to present in the following paper a general phase diagram of protein molecule in solution.     

Effects of Pressure-Induced Membrane Phase Transitions on Inactivation of HorA, an ATP-Dependent Multidrug Resistance Transporter, in Lactobacillus plantarum

The effects of pressure on cultures of Lactobacillus plantarum were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of L. plantarum induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature diagram for cell membranes was established. Cells grown at 37°C and pressure treated at 15°C lost >99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membranes of these cells were in the gel phase region at ambient pressure. In contrast, cells grown at 15°C and pressure treated at 37°C lost >99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membranes of these cells were in the liquid crystalline phase region at ambient pressure. The kinetic analysis of inactivation of L. plantarum provided further evidence that inactivation of HorA is a crucial step during pressure-induced cell death. Comparison of the biological findings and the membrane state during pressure treatment led to the conclusion that the inactivation of cells and membrane enzymes strongly depends on the thermodynamic properties of the membrane. Pressure treatment of cells with a liquid crystalline membrane at 0.1 MPa resulted in HorA inactivation and cell death more rapid than those of cells with a gel phase membrane at 0.1 MPa.     

Methods for Screening Cloud Point Temperatures

A novel and simple method for the measurement of cloud point temperatures of solutions is presented. Cloud point determination, which is currently used to establish the phase diagrams of protein solutions, is indicative of proteins interactions and constitutes a useful tool for food products engineering. We describe a novel experimental setup that allows screening of a large number of physical-chemical conditions in one measurement and the determination of cloud point temperatures both above and below ambient temperature. We use a simple method to avoid solvent evaporation and condensation, so that the set-up can be used for solutions prepared with a volatile solvent. We present the operating parameter range and the precision of the measurement. The optical properties of the system are calibrated with solutions of known transmittance, and the determination of cloud point temperatures is validated on a standard non-ionic surfactant solution. Finally, we demonstrate the efficiency of the method by determining the phase diagram of a wheat protein extract, soluble in a water/ethanol mixture. Complemented with differential scanning calorimetry measurements, the liquid-liquid phase transition can be determined up to a protein concentration of 250 g/L, a range inaccessible with conventional methods for this protein extract.     

Kinetics of combined pressure-temperature inactivation of avocado polyphenoloxidase

Irreversible combined pressure-temperature inactivation of the food quality related enzyme polyphenoloxidase was investigated. Inactivation rate constants (k) were obtained for about one hundred combinations of constant pressure (0.1-900 MPa) and temperature (25-77.5 degrees C). According to the Eyring and Arrhenius equation, activation volumes and activation energies, respectively, representing pressure and temperature dependence of the inactivation rate constant, were calculated for all temperatures and pressures studied. In this way, temperature and pressure dependence of activation volume and activation energy, respectively, could be considered. Moreover, for the first time, a mathematical model describing the inactivation rate constant of a food quality-related enzyme as a function of pressure and temperature is formulated. Such pressure-temperature inactivation models for food quality-related aspects (e.g., the spoilage enzyme polyphenoloxidase) form the engineering basis for design, evaluation, and optimization of new preservation processes based on the combined effect of temperature and pressure. Furthermore, the generated methodology can be used to develop analogous kinetic models for microbiological aspects, which are needed from a safety and legislative point of view, and other quality aspects, e.g., nutritional factors, with a view of optimal quality and consumer acceptance.     

Component and state separation in DMPC/DSPC lipid bilayers: a Monte Carlo simulation study

In this paper a two-state, two-component, Ising-type model is used to simulate the lateral distribution of the components and gel/fluid state acyl chains in dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) lipid bilayers. The same model has been successful in calculating the excess heat capacity curves, the fluorescence recovery after photobleaching (FRAP) threshold temperatures, the most frequent center-to-center distances between DSPC clusters, and the fractal dimensions of gel clusters (Sugar, I. P., T. E. Thompson, and R. L. Biltonen, 1999. Biophys. J. 76:2099-2110). Depending on the temperature and mole fraction the population of the cluster size is either homogeneous or inhomogeneous. In the inhomogeneous population the size of the largest cluster scales with the size of the system, while the rest of the clusters remain small with increasing system size. In a homogeneous population, however, every cluster remains small with increasing system size. For both compositional and fluid/gel state clusters, threshold temperatures-the so-called percolation threshold temperatures-are determined where change in the type of the population takes place. At a given mole fraction, the number of percolation threshold temperatures can be 0, 1, 2, or 3. By plotting these percolation threshold temperatures on the temperature/mole fraction plane, the diagrams of component and state separation of DMPC/DSPC bilayers are constructed. In agreement with the small-angle neutron scattering measurements, the component separation diagram shows nonrandom lateral distribution of the components not only in the gel-fluid mixed phase region, but also in the pure gel and pure fluid regions. A combined diagram of component and state separation is constructed to characterize the lateral distribution of lipid components and gel/fluid state acyl chains in DMPC/DSPC mixtures. While theoretical phase diagrams of two component mixtures can be constructed only in the case of first-order transitions, state and component separation diagrams can be constructed whether or not the system is involved in first-order transition. The effects of interchain interactions on the component and state separation diagrams are demonstrated on three different models. The influences of state and component separation on the in-plane and off-plane membrane reactions are discussed.     

Phase behavior of an intact monoclonal antibody

Understanding protein phase behavior is important for purification, storage, and stable formulation of protein drugs in the biopharmaceutical industry. Glycoproteins, such as monoclonal antibodies (MAbs) are the most abundant biopharmaceuticals and probably the most difficult to crystallize among water-soluble proteins. This study explores the possibility of correlating osmotic second virial coefficient (B(22)) with the phase behavior of an intact MAb, which has so far proved impossible to crystallize. The phase diagram of the MAb is presented as a function of the concentration of different classes of precipitants, i.e., NaCl, (NH4)2SO4, and polyethylene glycol. All these precipitants show a similar behavior of decreasing solubility with increasing precipitant concentration. B(22) values were also measured as a function of the concentration of the different precipitants by self-interaction chromatography and correlated with the phase diagrams. Correlating phase diagrams with B(22) data provides useful information not only for a fundamental understanding of the phase behavior of MAbs, but also for understanding the reason why certain proteins are extremely difficult to crystallize. The scaling of the phase diagram in B(22) units also supports the existence of a universal phase diagram of a complex glycoprotein when it is recast in a protein interaction parameter.