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Water close to proteins plays a key role in determining their structural and functional properties. Despite being a subject of considerable interest, the characterization of hydration water, as far as its total amount is concerned, is still controversial and its influence on protein structure and folding is not yet fully understood. In this work, we have investigated the dielectric properties of lysozyme aqueous solutions over the frequency range where the orientational polarization relaxation of the aqueous phase occurs (from 500 MHz to 50 GHz). Measurements extend over a wide concentration range, up to 300 mg/mL, corresponding to a volume fraction of the order of 0.4. The analysis of the dielectric spectra, based on the decrease of the dielectric increment of the γ-dispersion as a function of protein concentration, allows us to estimate the total amount of hydration water (both bound water and loosely bound water) present in the system investigated. We observe a decrease of the hydration number as a function of the protein concentration. This behavior is well accounted for by considering the formation of small equilibrium clusters with aggregation number of some units, as recently reported by Stradner et al. (1) on the basis of small-angle X-ray and neutron scattering measurements.

The critical micelle concentration (cmc) and ionisation degree (α), of micelles of cetyltrimethylammonium bromide (CTABr), cetyltrimethylammonium chloride (CTACl), cetyltripropylammonium bromide (CTPABr) and cetyltripropylammonium chloride (CTPACl) have been measured over a narrow temperature range at 2 degree intervals using electrical conductivity. CTPACl and CTPABr are very soluble in water and were measured in the temperature range 275.15-323.15K. The Krafft temperatures for CTABr and for CTACl are 293.15K and 284.15K, respectively and established a lower temperature limit for our studies on these two surfactants. The cmc vs temperature curves have a smooth minimum near room temperature and α linearly increases with temperature. The changes of cmc and α with temperature are smaller than those associated with the modification of head group size or counterion nature. Using these results, basic thermodynamic quantities associated with the phenomena of micellization have been evaluated. Thermodynamic properties of the surfactant solutions were discussed in terms of temperature dependence of the free energy, enthalpy and entropy of micellization. A close similarity between the effects of change in temperature on protein folding and micellization process appears from the data.

Brillouin light scattering from acoustic phonons has been used to investigate the elastic properties of Nafion 115 membranes in the GHz range of frequencies. Measurements were carried out as a function of temperature from 0 to 60 °C on samples with different hydration. Detection of longitudinal bulk waves travelling either parallel to the free surface or at an angle with respect to the normal to the surface enabled us to evaluate the refractive index and the longitudinal modulus of the membranes. A marked decrease of the latter with increasing either the temperature or the hydration degree was found. Moreover, a comparison of the above results with those obtained from measurement of the Young's modulus ( E ) in the quasi-static regime (stress–strain curves) shows that the high-frequencies values of E are an order of magnitude larger than in quasi-static conditions, reflecting the viscoelastic character of Nafion.

The conformation response of calf thymus DNA and β-lactoglobulin (BLG) to solvent conditions altered by varying amounts of ethanol (EtOH) and tert-butanol (TBA) has been monitored by viscosimetry and circular dichroism measurements. A correlation between the effects of EtOH and TBA on DNA condensation and BLG denaturation clearly appears from the data, suggesting that a similar molecular mechanism is responsible for the alcohol effects on these two very different systems and processes. These alcohol-induced transitions appear in the concentration range (alcohol mole fraction X = 0.22 and X = 0.07 for EtOH and TBA, respectively) where changes in the association state of the alcohol molecule occur (see compressibility and IR data).

Photon correlation spectroscopy and circular dichroism have been used to study the role of hydration in the structure and thermostability of the model protein lysozyme in water-glycerol mixtures. Two cases have been considered: water-rich and glycerol-rich regimes of concentrations. We follow the thermal denaturation both by optical spectral changes and hydrodynamic radius variations. This methodology allows us to emphasize the relevant role played by hydrophobic interactions during the process in aqueous solutions and, in glycerol, to distinguish the non-cooperative melting of secondary structure, supporting the view of a protein transition to a molten globule-like state.

The influence of methanol on the activity, stability and internal dynamics of myoglobin (Mb) has been investigated to obtain information about the effect of alcohol on protein behaviour. Spectroscopic investigations show that there are no significant changes in conformational structures of the protein within the range of alcohol concentrations where the catalytic activity ( k cat ) and thermal stability ( T m ) of Mb decrease. Neutron scattering measurements indicate that the characteristic times related to protein internal fluctuations increase when the methanol molar fraction in the mixtures increases. This suggests a direct relationship between the protein internal motion rate and the catalytic activity.

The hydrolysis of methylnaphthalene-2-sulfonate (MeONs) is inhibited at high concentrations of cetyltrimethylammonium methanesulfonate (CTAOMs) above the critical micellar concentration, cmc, and accelerated at low concentration of it, below cmc. UV–vis spectra of MeONs were recorded in CTAOMs solutions, at different concentration, below and above cmc, and the substrate interacts differently with CTAOMs below and above the cmc. The hydrolysis of MeONs and its spectrum were investigated in two other systems for comparison purposes, using the same concentration range of CTAOMs: the zwitterionic tetradecyldimethylammonium propane sulfonate (SB3-14) surfactant, and the non-micellizing salt tetramethylammonium methanesulfonate (TMAOMs). The reaction is slightly monotonically inhibited in dilute SB3-14 and it is little affected by TMAOMs, whereas the spectrum of MeONs is not affected by the dilute solutions of both these two systems. The acceleration in dilute CTAOMs and the change of the MeONs spectra below and above cmc gives indication of premicelle formation in dilute CTAOMs.

We have investigated the formation of a cluster phase in low-density colloidal systems formed by charged solid charged particles stuck together by an oppositely charged polyion. In analogy with what we have previously observed in the case of soft charged particles, also in this case the same basic phenomenology occurs, consisting of the presence of the two well-known characteristic phenomena of this class of colloids, that is, reentrant condensation and charge inversion. With the aim of comparing the cluster formation in both soft and solid charged particles, we have, in previous works, employed cationic liposomes (soft particles, lipidic vesicles built up by dioleoyltrimethylammonium propane [DOTAP] lipid) and, in the present work, polystyrene particles (solid particles) covered by the same lipidic bilayer as the one of the soft particles, so that the two classes of particles share electrostatic interactions of the same nature. These charged particle clusters, where the single aggregating particles maintain their integrity without undergoing a structural rearrangement, join to a class of different aggregated structures (lamellar or inverse hexagonal phases) observed as well in the polyion-induced aggregation of oppositely charged mesoscopic particles, in particular, lipidic vesicles. Our results show that the formation of relatively large, equilibrium clusters of particles which maintain their integrity, stuck together by a polyion which acts as an electrostatic glue, is one of the many facets of the complex phenomenology underlying the interactions of charged particles with oppositely charged objects.

Through elastic neutron scattering measurements, we investigated the picosecond dynamics of DNA in the hydrated powder state or embedded in glycerol glassy matrix from 20 K to 300 K. We calculated the relaxational contribution of the mean square displacements (MSD) of DNA hydrogen atoms. We found the existence of a linear relationship between the inverse of the biomolecule relaxational MSD and the logarithm of the bulk viscosity of the surrounding environment. From the comparison with the case of lysozyme in the same environments, for which the validity of the relationship was already verified, possible differences and analogies concerning the biomolecule-to-solvent dynamical coupling can be stressed. (C) 2007 Elsevier B.V. All rights reserved.

We performed a neutron scattering Study to investigate file dynamical behaviour of water absorbed in Nafion at low hydration levels (lambda=6, lambda=moles of water/moles of sulphonic acid sites) as a function of temperature ill file range 200-300 K. To Single Out file signal of the confined water, file measurements were performed oil samples hydrated with both H2O and D2O in the same temperature ran,,e. Due to the strong incoherent scattering cross section of hydrogen atoms with respect to deuterium. in the difference spectra the contribution from the Nafion membrane is subtracted out and most of the spectra originates from absorbed water. The estimated dynamical Susceptibility exhibits features that resemble those of bulk water. In particular, the spectra display a bump at around 1 meV, possibly related to the alpha relaxation, the intensity of which is markedly affected by the temperature change. Two features due to the phonon-like collective hydrogen bond network dynamics are visible at approximately 7 meV and 25 meV.

We performed a neutron scattering study to investigate the dynamical behavior of water absorbed in Nafion at low hydration level as a function of temperature in the range 200-300 K. To single out the spectral contribution of the confined water, the measurements were done on samples hydrated with both H(2)O and D(2)O. Due to the strong incoherent scattering cross section of hydrogen atoms with respect to deuterium, in the difference spectra, the contribution from the Nafion membrane is subtracted out and the signal originates essentially from protons in the liquid phase. The main quantities we extracted as a function of the momentum transfer are the elastic incoherent structure factor (EISF) and the line width of the quasielastic component. Their trend suggests that the motion of hydrogen atoms can be schematized as a random jumping inside a confining region, which can be related to the boundaries of the space where water molecules move in the cluster they form around the sulfonic acid site. Through the calculated EISF, we obtained information on the size of such a region, which increases up to 260 K and then attains a constant value. Above this temperature, the number of water protons that are dynamically activated in the accessible time window increases with a faster rate. The jump diffusion dynamics is characterized by a typical jumping time which is stable at 5.3 ps up to approximately 260 K and then gradually decreases. The ensemble of the findings indicates that, within the limits of the energy resolution of the present experiment, water absorbed in the Nafion membrane undergoes a dynamical transition at around 260 K. We discuss the possible relationship of this dynamical onset with the behavior of the electrical conductivity of the membrane as a function of the temperature.

Through a neutron scattering experiment, we studied the dynamics of lysozyme embedded in glucose–water matrices. Two distinct quasielastic contributions, corresponding to motions in the sub- and picosecond timescales, are revealed after the subtraction of the inelastic intensity, as estimated at low temperature. Their characteristic times are hydration and temperature independent, and quite similar to those revealed when the environment around the biomolecule surface is pure water or glycerol. The momentum transfer dependence of each quasielastic signal has been interpreted in terms of the rotational diffusion model. The faster component corresponds to a more localized rotational motion on a radius of 1 A, while the slower contribution is associated to a larger radius of 1.9 A. The behavior of the fraction of moving protein hydrogen atoms as a function of temperature and hydration suggests that the protein undergoes a dynamical transition just at conditions corresponding to the dynamical onset of the enclosing matrix.

A recent neutron scattering investigation on lysozyme embedded in glycerol–water mixtures has shown that solvent dynamics is crucial in determining the dynamical properties of the biomolecule itself (Biophys. J. 83 (2002) 1157). To better understand the role played by solvent, we have performed an elastic incoherent neutron scattering (INS) experiment as a function of the temperature on pure glycerol. To directly compare the dynamics of the solvated protein and that of the pure solvent, we settled the same experimental conditions and applied the same data analysis procedure as in Paciaroni et al. (Biophys. J. 83 (2002) 1157). By using a double-well model and taking into account for the global molecular diffusion, we exploited the measured intensity to estimate the mean square displacements (MSD) of glycerol hydrogen atoms. We found that the total MSD deviate from the low-temperature vibrational harmonic trend at approximately T =235 K, consistent with the value of the critical temperature reported in literature (Phys. Rev. E 55 (1997) 3183). The present investigation suggests that the internal dynamics of glycerol molecules, i.e. the vibrations and reorientations of hydrogen atoms relative to the molecular centre of mass (c.o.m.), can be put in relationship with the protein dynamics.

Quasielastic neutron scattering has been used to investigate the dynamical behavior of H(2)O in water/tert-butyl alcohol solutions. The measurements were made at fixed temperature (293 K) as a function of tert-butyl alcohol molar fraction, x, in the range 0-0.042. The data have been compared to those of pure water in the temperature range 269-293 K. The effect of tert-butyl alcohol addition on water dynamics is equivalent to that obtained by lowering the temperature of pure water by an amount proportional to the alcohol concentration. The temperature dependence of the diffusivity parameters in pure water and their concentration dependence in tert-butyl alcohol/water solutions can be rescaled to a common curve attributing to each solution a concentration-dependent "structural temperature" lower than the actual thermodynamic one. These results can be understood in terms of Stillinger's picture of water structuring and of other more recent theoretical pictures that emphasize the influence of the geometrical properties of hydrogen bond networks on water mobility.

We performed an inelastic and quasielastic neutron scattering investigation of the molecular dynamics of lysozyme solvated in glycerol, at different water contents h (g water/g lysozyme). At low temperature, dry lysozyme solvated with glycerol shows a low-frequency vibrational bump located at around 4 meV. Contrary to the behaviour of strongly hydrated protein powders, this peak is well visible also at room temperature and persists in the experimental spectra at all the hydration degrees. The quasielastic contribution increases remarkably with h, thus suggesting that water molecules perform their plasticizing action by allowing the onset of confined diffusive motions of protein groups. The quasielastic signal could be decomposed in two Lorentzian components, representing motions with characteristic times of, respectively, 15 and 0.8 ps. The Lorentzian curves exhibit constant widths but variable intensities as a function of the hydration degree. The trend of the estimated quasielastic structure factors suggests that a preferential hydration effect may take place in lysozyme solvated with glycerol and water.

Divalent ions, as Mg 2+ , strongly interact with polyelectrolytes, in particular with DNA and this binding involves two phosphate groups. In our laboratory we studied this interaction by dielectric spectroscopy. At radiofrequencies DNA shows a well-defined dielectric relaxation, due to monovalent counterion oscillations along the surface of the biopolymer. The dielectric response of the biopolymer is thus modified by the interaction with magnesium. In this communication we report on measurements at radiofrequencies, performed on Na-DNA aqueous solutions in the presence of increasing amounts of Mg 2+ ions. The dielectric relaxation is reduced as the magnesium content increases. This effect tends to saturate when the molar ratio Mg 2+ /nucleotide approaches the value of 0.25. A significant parameter i.e. length of the biopolymer subunit, estimated by dielectric relaxation time, appears to be modified, denoting also the existence of a structural effect. This new approach proved to be a valid tool in studying interaction processes among nucleic acids and divalent ions or, in general, charged macromolecules.

We performed an elastic neutron scattering investigation of the molecular dynamics of lysozyme solvated in glycerol, at different water contents h (grams of water/grams of lysozyme). The marked non-Gaussian behavior of the elastic intensity was studied in a wide experimental momentum transfer range, as a function of the temperature. The internal dynamics is well described in terms of the double-well jump model. At low temperature, the protein total mean square displacements exhibit an almost linear harmonic trend irrespective of the hydration level, whereas at the temperature T(d) a clear changeover toward an anharmonic regime marks a protein dynamical transition. The decrease of T(d) from approximately 238 K to approximately 195 K as a function of h is reminiscent of that found in the glass transition temperature of aqueous solutions of glycerol, thus suggesting that the protein internal dynamics as a whole is slave to the environment properties. Both T(d) and the total mean square displacements indicate that the protein flexibility strongly rises between 0.1 and 0.2h. This hydration-dependent dynamical activation, which is similar to that of hydrated lysozyme powders, is related to the specific interplay of the protein with the surrounding water and glycerol molecules.

A highly sensitive scanning microcalorimeter and a low scanning rate (0.1 K/min) has been used to determine the thermodynamic properties of the main phase transition of distearoylphosphatidylcholine (DSPC) in presence of ethanol in the 0–0.20 ethanol mole fraction (x2) range. These measurements revealed previously unreported features of DSPC/water/ethanol phase behavior and visualized distinct changes in the main phase transition mechanism induced by addition of ethanol. It is found that the well known interdigitated phase in DSPC induced by ethanol above x2 =0.01 abruptly disappears at x2 =0.12. The abrupt change in the DSPC phase behavior at x2 = 0.12 shows some analogies with the observed one relative to the conformation response of DNA molecule to solvent condition altered by varying amounts of ethanol. As inferred from previous adiabatic compressibility and IR data in water/ethanol mixtures, a correlation between changes in properties of the solvent, and the effect of ethanol on both the conformational properties of DNA and the main phase transition of DSPC clearly appears from the data. © 2002 Elsevier Science B.V. All rights reserved.

We report the study of the effect of tert-butyl alcohol (TBA) and trimethylamine-N-oxide (TMAO) on a typical process governed by the hydrophobic interaction, such as the micellization of a surfactant in aqueous solution. The behaviour of the critical micelle concentration (c.m.c.) of the sodium dodecyl sulfate (SDS) was investigated as a function of the added amounts of TBA or TMAO by conductivity measurements. The hydration properties of the two solutes was studied by IR spectroscopy, in the region of the frequency of the O–H stretching band for H2O and of the methylic band for TBA and TMAO. The overall behaviour of the TBA/H2O and TMAO/H2O solutions is also studied by compressibility measurements. The data were discussed in terms of the hydration properties of TBA and TMAO and of the thermodynamical properties of the mixtures. Significant correlations between these properties and the effect of TBA and TMAO on the micellization process are shown.

The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U1 unfolded state, whereas the successive addition of Cl− ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A1 and A2 states, respectively). A second unfolded state (U2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle x-ray scattering. The results show that in the acidic-unfolded U1 form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U1 appear structurally different: in the A1 state cyt-c is dimeric and less compact, whereas in the A2 form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.

This work concerns a comparison of the hydration properties and self-association behavior in aqueous solution of two biologically relevant simple molecules: the trimethyl-amine-N-oxide (TMAO) and the tert-butyl alcohol (TBA). These molecules are geometrically very similar, having the same hydrophobic moiety and different polar groups. Both molecules were used as a model to study hydrophobic behavior in water solution. In particular, water perturbation induced by TBA and TMAO molecules was studied as a function of the solute molar fraction X2 (0 < X2 < 0.05) by using the IR absorption bands due to the vibrational modes of water in the 4000−2600 cm-1 frequency region. Furthermore, possible clustering effects in aqueous solution of the TBA and TMAO hydrophobic groups were investigated by studying the behavior of the alkyl CH stretching band in the 3200−2800 cm-1 frequency region as a function of X2. The OH stretching absorption data show, in agreement with molecular dynamics simulation results and other sug...

The overall atomic mobility of AOT reverse micelles in CCl 4 as a function of the molar ratio W =[D 2 O]/[AOT] has been studied with an elastic neutron scattering experiment. The measured intensity suggests that, for small contents of water, the average reverse micelles mobility increases as a function of the hydration. A marked departure of the estimated mean square displacements from low hydration conditions has been revealed above W =1. Such a hydration-dependent dynamical activation is in agreement with previous NMR and dielectric measurements. The dependence of the atomic mean square displacements as a function of W has been explained by taking into account for the existence of two different dynamical contributions from more mobile completely-hydrated and less mobile partially-hydrated headgroups.

The technique of intensity photon correlation spectroscopy has been utilized to investigate the native conformation of lysozyme in water/ethanol and in water/ tert -butanol mixtures as a function of alcohol concentration in the water-rich region of composition (cosolvent mole fraction x 2 x 2 =0.02 and a maximum at x 2 =0.06 in water/ethanol mixtures and by a minimum at x 2 =0.005 and a maximum at x 2 =0.02 in water/ tert -butanol mixtures. These values of x 2 are close to those at which structural changes in the mixtures occur as inferred from compressibility and infrared absorption measurements. The results are discussed in connection to the effect of alcohol in modulating solvent-mediated interactions.

This work concerns a comparison of the hydration properties of two simple molecules of biological relevance: trimethyl-amine- N -oxide (TMAO) and tert -butyl alcohol (TBA). These molecules are geometrically very similar, having the same hydrophobic moiety and different polar groups. Both molecules have been used as a model to study hydrophobic behaviour in water solutions. In the present work, dielectric relaxation of water/TBA and water/TMAO mixtures have been studied at a cosolvent mole fraction, x 2 =0.02. Water perturbation induced by TBA and TMAO molecules has been studied also at x 2 =0.02 and 0.05 by using the absorption bands due to the vibrational modes of water in the infrared region. The data show, in agreement with molecular dynamics simulation results, that TMAO and TBA interaction with water are remarkably different and that water molecules are more tightly coordinated by TMAO than by TBA.

The technique of intensity photon correlation spectroscopy has been utilised to investigate the native conformation of lysozyme in water/ethanol mixture as a function of alcohol concentration in the water-rich region of composition (cosolvent mole fraction x2

This work deals with a study of alkaline transition of ferricytochrome-c in water and in water−trehalose solutions using two different techniques: spectrophotometry in the region of the heme absor...

The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 °C, while the other two occur at 63 °C and 74 °C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63 °C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.

The technique of intensity photon correlation spectroscopy was utilised to investigate the native conformation of lysozyme in water–ethanol and water–tert-butanol mixtures as a function of alcohol concentration in the water-rich region of composition (cosolvent mole fraction x2

Previous studies from our laboratory demonstrated the existence of at least two levels of structural complexity in E. coli 70S ribosomes. Ribosomal RNA seems to be principally involved in the overall stability of these structures. In this paper we present an investigation of ribosomes subjected to treatment with RNase. The study is based on both differential scanning microcalorimetry and dielectric spectroscopy. In the thermograms obtained on treated ribosomes only the low temperature peak of the two typical denaturation events observed in native ribosomes, is promptly eliminated by the enzyme treatment. Dielectric spectroscopy measurements carried out on the same samples indicate an alteration of the dielectric behavior previously shown to consist of two subsequent relaxation processes. In fact, only the low frequency relaxation is affected by the treatment. The second one, observed at higher frequency, remains unaltered. The same effect on the dielectric parameters is observed if the ribosome particles are heated and then cooled prior to measurement. These results are consistent with the idea that two different structures are present within the ribosome. One is very stable and withstands both temperature and RNase treatment while the second is promptly abolished by both treatments. Data presented here strongly suggest that the RNA domains exposed to the solvent play a fundamental role in the stability of the 3-D structure of the ribosome particle.

The effect of monohydric alcohols on the micellization of sodium dodecyl sulphate (SDS) and on the thermal unfolding of lysozyme has been studied by DSC as a function of alcohol concentration in the water-rich region of composition. The results show that the effects due to alcohols on these very different systems and processes are strikingly similar and are closely linked with structural and dynamical properties of water–alcohol mixtures themselves. The data have been interpreted on the basis of the assumption that the addition of short chain alcohol affects both the thermal unfolding of proteins as the micellization process modifying the extent of enthalpy and entropy contribution associated with structural reorganization of water in these processes.

The effect of substituting Na+ counterion of Aerosol-OT [sodium bis(2 ethylhexyl) sulfosuccinate] with doubly charged counterion Cu2+ has been studied for the Cu(AOT)2/H2O/CCl4 system by infrared a...

The conformational response of calf thymus DNA to solvent conditions altered by varying amounts of ethanol, 2-propanol and tert-butanol has been monitored by circular dichroism. These measurements reveal above a critical cosolvent mole fraction x 2 ∗ typical for each alcohol, a condensed form of the macromolecule with unusually large ellipticity in the 250–300 nm region similar to the well-known ψ(−) forms induced by above-critical concentrations of neutral polymers and salt. This alcohol induced transition is completely reversible and it appears in the concentration range ( x 2 ∗ =0.135, 0.076, 0.055 for ethanol, 2-propanol and tert-butanol, respectively) where change in the association state of the alcohol molecules occurs as inferred from compressibility and IR data. Both DNA condensation and alcohol association processes start when almost all water is involved in hydration structures. The present data clearly indicate the important role that water and solvation play in the self-association of alcohol molecules and in the conformation of nucleic acids.

In this paper, we report on IR and dielectric spectroscopy of Ca(AOT)2 reverse micelles containing water pools of different sizes. We investigate the effect of changing the nature of the counterion...

We report dielectric measurements at radiofrequencies on lysozyme in aqueous solution at two values of pH (3.5 and 6) as a function of temperature in the interval 5–55°C. From the analysis of the dielectric relaxation of the protein solution, the effective hydrodynamic radius r and the electric dipole moment μ of the protein were calculated. The results show that temperature causes continuous gradual changes of r and μ with a maximum at 25–30°C where the Gibbs free energy for native lysozyme shows an analogous trend. We suggest that the gradual variations of r and μ are the manifestation of a redistribution of microscopic state populations of the protein within the same macroscopic native state.

Istituto Nazionale di Fisica della Materia and Dipartimento di Fisica, Universita` di Perugia I-06100 Perugia, Italy Received 30 October 1997; revised manuscript received 24 July 1998The high-frequency dielectric response of sodium bis 2-ethylhexyl sulfosuccinate AOT reverse micelles inn-heptane has been measured in the range 0.01–20 GHz as a function of volume fraction of a dispersedphase at a fixed value of the water to surfactant molar ratio W 11. A relaxation phenomenon has beenobserved that is connected to the AOT ion pair diffusion at the water-surfactant interface. This dielectricrelaxation has been found to be markedly affected by the interparticle interactions that have been modulated byadding different amounts of NaCl. In order to reveal the connections between attractive interactions among thedroplets and the stereochemical configurations of AOT molecules, an ir study has been performed on thecarbonyl stretching band of AOT molecules in the micellar aggregates. A comparison with dielectric datashows that a correlation exists between the intensity of attractive interactions and the composition of AOTrotamers in the microemulsion system. These results support the hypothesis that attractive intermicellar inter-actions are favored by a particular orientation and packing of nonpolar tails of AOT molecules in the micellarmicroaggregate. S1063-651X 98 10312-4PACS number s : 82.70. y, 77.22.Gm, 77.84.Nh, 63.50. xI. INTRODUCTION

We investigated the thermal degradation of E. coli ribosomes by differential scanning microcalorimetry. The 70S particles show two distinctive and irreversible peaks upon thermal degradation. Free rRNA in solution produces, on the contrary, an unstructured denaturation profile. The thermal analysis of 50S particles shows a profile substantially identical to that observed in 70S, while 30S particles produce an unstructured denaturation pattern. Therefore the thermal behavior of the 70S particle is essentially attributable to the denaturation of the 50S subunit. Our data validate previous observations that the 50S has a more rigid structure as compared to 30S, which behaves as a 'floppy' particle. In addition our data suggest that protein/RNA interactions play a significant role to stabilize three-dimensional structures of the ribosome.

In this paper we report permittivity measurements in the frequency range 105–108 Hz, at the fixed temperature of 20°C, on lysozyme dissolved in water and in a water–ethanol mixture (ethanol concentration 0.1 molar fraction) varying the pH of the solutions from acid (≅pH 2) to basic (≅pH 10). The experimental data were fitted with the Cole–Cole equation and from the dispersion parameters the effective hydrodynamic radius and the electric dipole moment of the protein in different conditions of pH were calculated. The results confirm a conformational effect induced on lysozyme by ethanol, reported in our previous work. For pH in the range 4–6, where the enzymatic activity of lysozyme reaches its maximum, our results indicate a stable conformation of the protein. Out of this pH interval aggregation and expansion processes are present.

In this paper, we report dielectric spectroscopy and optical measurements on lysozyme in water-ethanol mixtures as a function of alcohol concentration in the water-rich region of composition (cosolvent mole fraction X ⩽0.25). Dielectric measurements show a well defined relaxation curve in the MHz region, from which we obtain the electric dipole moment μ and the effective hydrodynamic radius r of the protein. UV melting measurements with estimation of the van't Hoff enthalpy are also reported. Molar ellipticity measurements were performed to test the α-helix content. The results indicate that alcohol promotes a more tightly folded conformation of the protein. Our measurements show a correlation between the effects of ethanol on the conformational properties of lysozyme and changes in the properties of solvent, as inferred from previous adiabatic compressibility and IR data on water-ethanol mixtures.

Thermal denaturation of lysozyme has been studied at pH = 3 in water/ethanol and water/tert-butyl alcohol mixtures in the water rich region of composition (mole fraction of cosolvent x2 < 0.12) by high-sensitivity differential scanning calorimetry. The results show that on increasing alcohol concentration, the enthalpy and entropy of denaturation of lysozyme first reach a maximum at an intermediate composition x2 = typical for each alcohol ( ≅ 0.06 for ethanol and ≅ 0.02 for tert-butyl alcohol) and then decrease with increasing x2. In addition, two enthalpy−entropy compensation patterns each having its own compensation temperature (Tc) clearly appear from the data: a compensation data line obtained with rising x2 in the 0− range (with Tc = 281 ± 6 K) followed by a compensation line with Tc = 403 ± 14 K after x2 passes the value. The value of is close to that at which a change in the nature of solvent component interaction occurs as inferred from compressibility and IR absorption measurements. The data ha...

This paper reports on critical micelle concentrations and enthalpies of micellization of n-butoxyethanol in water at three different temperatures (5, 25, and 45 °C) as determined by means of a sensitive microcalorimeter. The experimental enthalpies are compared with those calculated from the temperature dependence of the critical micelle concentration by using the phase separation model of micelle formation. Enthalpies and entropies of micelle formation are both positive and decrease as the temperature is raised indicating the hydrophobic effect in the formation of n-butoxyethanol microaggregates. These calorimetric results provide no indications of intermicellar interactions, while indicating a monomer−monomer repulsive interaction in the premicellar region.

The effect of replacing the Na + counterion of Aerosol OT by Cu 2+ on the hydration mechanism of AOT reverse micelles was studied by infrared spectroscopy. The IR properties of ternary systems formed by Cu(AOT) 2 /[H 2 O]/CCl 4 are reported and compared with the corresponding Na(AOT) system. The results show significant differences in interactions of Na + and Cu 2+ counterions with H 2 O molecules and the SO 3 − headgroup of AOT.

The dielectric properties of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles in CCl4 and n-heptane have been measured in the frequency range 0.02–20 GHz as a function of W = [ H 2 O ] [ AOT ] . A relaxation phenomenon has been observed reflecting some properties inherent the single particle dynamics. This dielectric relaxation has been found to be markedly affected by interparticle interactions. Our data, according to previous results, show that interactions in these systems depend on the oil chain length, on the molar ratio W and are significantly affected by the addition of small amounts of electrolyte. In order to achieve a deeper insight into the microscopic origin of these interactions, IR absorptions in the CO stretching region of AOT molecules have been recorded to give information on the relative population of different AOT rotational isomers in the various examined systems. The data show a clear correlation between the presence of attractive interactions and the composition of AOT rotamers, supporting the hypothesis that the intermicellar interactions are favored by a particular orientation and packing of non-polar tails of AOT molecules in the micellar microaggregate.

Our recent studies related to the properties of alcohol/water mixtures, show the occurrence of some kind of molecular aggregation in the water rich region of composition beyond a threshold alcohol concentration x 2 ∗ . The observed behaviour suggests that for x 2 2 ∗ the alcohol molecules are essentially dispersed and surrounded by “water cages” where the short range order and microdynamic of water molecules are changed with respect to those of pure water. Alcohol molecules are in mutual contact at higher concentration, only, when almost all water is involved in hydration shells of alcohol molecules. In addition, these studies show that the stabilisation of micellar structure and nucleic acids conformation in water/alcohol mixtures is closely linked to the properties and anomalous behaviour of the solvent systems. Accordingly, these results support the hypothesis that the dominant mechanism by which an alcohol affects these processes is through its effect on the structure of water. The main results of these investigations are reviewed and discussed in this paper.

Most of the previous dielectric studies on microemulsions and related systems have been conducted at relatively low frequencies where the dielectric response is sensitive to dynamic percolation phenomena. There is a lack of experimental studies at microwave frequencies where water plays a central role in dielectric relaxation. In this paper the dielectric complex permittivity of water/Aerosol OT/n-heptane microemulsions has been measured by a frequency domain coaxial technique in the range 0.02–20 GHz as a function of molar ratioW = [water]/[AOT] at low volume fraction of the dispersed phase (ϕ=0.1 and ϕ=0.2). The data show two dielectric dispersions: The first located in the 100 MHz frequency region and the second at frequencies higher than 20 GHz. The evolution of the dielectric parameters of these relaxations has been studied as a function of the molar ratioW in the range 0

The structure of water in bis(2-ethylhexyl)sodium sulfosuccinate (AOT) micelles has been studied as a function of the [H2O]/[AOT] ratio (W) by using the absorption IR due to O−H stretching modes in the 3800–3000 cm−1 range. Three systems have been studied: water/AOT/carbon tetrachloride, water/AOT/n-heptane and water/AOT iso-octane. Experimental spectra are presented and discussed for the O−H stretching region both of H2O and isotopically diluted HDO molecules in D2O. We have restricted ourselves to the region of small amounts of water (0

Depolarized Rayleigh-wing light scattering experiments on aqueous solutions of 2-butoxyethanol (${\mathrm{C}}_{4}$${\mathit{E}}_{1}$) as a function of temperature and concentration are reported. The measured spectra give information on the rotational dynamics of water molecules. Their analysis confirms the findings of surface-tension data performed in the same experimental conditions, namely, that the ethoxylated alcohol molecules have an amphiphilic character that can give rise to micellar structures. The water rotational motion is dominated by the presence of such aggregates and by the hydration of the oxyethylene groups of the alcohol molecules.

The dielectric behavior of native and heat-denatured lysozyme in ethanol-water solutions was examined in the frequency range from 1 MHz to 2 GHz, using frequency-domain dielectric relaxation spectroscopy. Because of the conformational changes on unfolding, dielectric methods provide information on the denaturation process of the protein and, at protein concentration high enough, on the subsequent aggregation and gelation. Moreover, the time evolution of the protein aggregation and gelation was monitored measuring, by means of dynamic light scattering methods, the diffusion coefficient of micro-sized polystyrene particles, deliberately added to the protein solution, which act as a probe of the viscosity of the microenvironment close to the particle surface. All together, our measurements indicate that heat-induced denaturation favors, at high concentrations, a protein aggregation process which evolves up to the full gelation of the system. These findings have a direct support from IR measurements of the absorbance of the amide I band that, because of the unfolding, indicate that proteins entangle each other, producing a network structure which evolves, in long time limit, in the gel.