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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).

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.

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.

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.

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.

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.

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

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 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.

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.

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.

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.

The surface tensions for n -butoxyethanol/water mixtures have been determined as a function of concentration for various temperatures in the range 2–55°C. Measurements of adiabatic compressibility were also performed on the same water/alcohol solutions at 25, 40 and 55°C. The two sets of experimental data are compared and discussed in terms of the presence of n -butoxyethanol microaggregates in solution.

The viscosities of water—AOT—carbon tetrachloride mixtures have been measured at 20°C for various AOT and H2O concentrations and molar ratios, [H 2 o] [AOT] ranging from 0 to 8. It is shown that an appropriate analysis of the data allows one to distinguish between solvation and micelle shape in their contribution to the viscosity of the solutions. Our results indicate that solvation rather than asymmetry is the principal cause of the observed deviations from the behavior of unsolvated spheres.

The critical micelle concentration of sodium dodecylsulfate in water—ethanol mixtures has been measured conductometrically as a function of both temperature (in the 10–40°C range) and of ethanol mole fraction in the water-rich region. The adiabatic compressibility of the plain water—ethanol mixtures in the same concentration and temperature range has also been determined. The free energy, enthalpy and entropy of micellization have been computed and are related with the possible structure of the solvent system. The present experimental data support the assumption that the dominant mechanism by which ethanol affects the micellization process is through its effect on the structure of water.

The effect of ethanol on the critical micelle concentration (CMC) of several surfactant aqueous solutions has been studied by surface tension methods. The results show that with increasing alcohol concentration the CMC values first reach a minimum at an intermediate ethanol mole fraction x2* ≈ 0.055 and then increase with increasing x2. The value of x2* is close to that at which structural changes in the mixture occur as inferred from compressibility and optical absorption measurements. The present experiments support the assumption that the dominant mechanism by which ethanol affects the micellization process is through its effect on the structure of water.

The interaction of copper(II) with adenosine triphosphate (ATP) has been studied as a function of pH in the range pH 3–12. Our approach is the study of the effect of binding both on the ATP ultraviolet absorption spectrum and on the optical d-d transition of copper ions. The results show that Cu(II)-ATP complexes exist in a variety of forms in equilibrium, the percentage of each species varying according to the state of ionization of the intrinsic adenine, phosphate and ribose groups. These results also show a close correlation between the rate of dephosphorylation of ATP in the presence of CU(II) ions and Cu(II) bonding to the adenine of ATP, thus supporting the hypothesis that the metal-ion/nucleic-base interactions are crucial for the observation of a metal-ion promoted dephosphorylation of ATP (D.H. Buisson and H. Sigel, Biochim. Biophys. Acta 343 (1974) 45).