ABSTRACT
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.
Subject(s)
Muramidase/chemistry , Water/chemistry , Cluster Analysis , Protein Conformation , SolutionsABSTRACT
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.
Subject(s)
Muramidase/chemistry , Dielectric Spectroscopy , Ethanol/chemistry , Gels/chemistry , Light , Muramidase/metabolism , Polystyrenes/chemistry , Protein Denaturation , Scattering, Radiation , Temperature , Water/chemistryABSTRACT
The dielectric properties of lysozyme aqueous solutions have been investigated over a wide frequency range, from 1 MHz to 50 GHz, where different polarization mechanisms, at a molecular level, manifest. The dielectric relaxation spectra show a multimodal structure, reflecting the complexity of the protein-water interactions, made even more intricate with the increase of the protein concentration. The deconvolution of the spectra into their different components is not unambiguous and is generally a delicate process which requires caution. We have analyzed the whole relaxation region, on the basis of the sum of simple Debye-type relaxation functions, considering three main contributions. Particular attention has been payed to the δ-dispersion, intermediate between the ß-dispersion (rotational dynamics of the protein) and the γ-dispersion (orientational polarization of the water molecules). This intermediate contribution to the dielectric spectrum is attributed to the orientational polarization of water molecules in the immediate vicinity of the protein surface (hydration water). Our measurements clearly demonstrate that, at least at high protein concentrations, the δ-dispersion has a bimodal structure associated with two kinds of hydration water, i.e., tightly bound and loosely bound hydration water. In the concentration range investigated, the existence of a three-modal δ-dispersion, as recently suggested, is not supported, on the basis of statistical tests, by the analysis of the dielectric relaxations we have performed and a bimodal dispersion is accurate enough to describe the experimental data. The amount of the hydration water has been evaluated both from the dielectric parameters associated with the δ-dispersion and from the decrement of the loss peak of the γ-dispersion. The relative weight of tightly bound and loosely bound hydration water is briefly discussed.
Subject(s)
Muramidase/chemistry , Water/chemistry , Animals , Chickens , Dielectric Spectroscopy , Egg White/chemistry , Muramidase/metabolism , SolutionsABSTRACT
The formation of aggregates in polyion-induced charged colloidal particles in aqueous suspension is characterized, under appropriate conditions, by two complementary effects, known as re-entrant condensation and charge inversion, which are considered as proof for the existence of a cluster phase. In this paper, we extend our previous investigation to a set of aqueous colloidal particle suspensions, such as polystyrene spheres, colloidal gold particles, and polylactic acid particles. These systems are characterized by the evolution of the average size of the aggregates and their surface electrical charge (charge inversion) by means of dynamic light-scattering measurements and laser Doppler electrophoretic techniques. The results, together with the previous ones concerning liposome particles, support the notion of a common behavior of this group of complex colloids characterized by short-ranged attractive interactions. The study provides some insights into these structures, which are potentially useful in biotechnological applications, such as multicompartmental carriers in nonviral drug delivery.
ABSTRACT
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.
Subject(s)
Colloids/chemistry , Ions , Lipids/chemistry , Polystyrenes/chemistry , Cluster Analysis , Drug Carriers , Drug Delivery Systems , Electrochemistry/methods , Fatty Acids, Monounsaturated/chemistry , Light , Liposomes/chemistry , Quaternary Ammonium Compounds/chemistry , Scattering, Radiation , Static Electricity , Surface Properties , TemperatureABSTRACT
Through elastic neutron scattering measurements, we investigated the thermal fluctuations of DNA enclosed by glycerol-water glassy matrices, at different levels of hydration, over the wide temperature range from 20 to 300 K. For all the samples, the extracted hydrogen mean square displacements (MSD) show a purely vibrational harmonic trend at very low temperatures, and a first onset of anharmonic dynamics above approximately 100 K. Such onset is consistent with the activation of DNA methyl group rotational motions. Then, at a certain temperature Td, the MSD show a second onset of anharmonicity, which corresponds to the DNA dynamical transition. The Td values vary as a function of the hydration degree of the environment. The crucial role of the solvent mobility to activate the DNA thermal fluctuations is proposed, together with a preferential hydration effect of the DNA phosphate groups. Finally, a comparison between the average mobility of homologous samples of DNA and the lysozyme protein is considered.
Subject(s)
DNA/chemistry , Glycerol/chemistry , Glycerol/pharmacology , Neutron Diffraction , Water/chemistry , Water/pharmacology , Animals , Elasticity , Movement/drug effects , Muramidase/chemistry , Transition TemperatureABSTRACT
We have investigated the effect of a cationic lipid [DOTAP] on both the thermotropic phase behavior and the structural organization of aqueous dispersions of dipalmitoyl-phosphatidylcholine [DPPC] by means of high-sensitivity differential scanning calorimetry and dynamic light scattering measurements. We find that the incorporation of increasing quantities of DOTAP progressively reduces the temperature and the enthalpy of the gel-to-liquid crystalline transition. We are further showing that, in mixed DOTAP-DPPC systems, the reduction of the phase transition temperature is accompanied by a reduction of the average size of the structures present in the aqueous mixtures, whatever the DOTAP concentration is. These results, which extend a previous investigation by Campbell et al. (Campbell, R. B.; Balasubramanian, S. V.; Straubinger, R. M.; Biochim. Biosphys. Acta 2001, 27, 1512.) limited to a DOTAP concentration below 20 mol %, confirm that the insertion of cationic head groups in zwitterionic phosphatidylcholine bilayers facilitates the formation of stable, relatively small, unilamellar vesicles. This self-assembling restructuring from an aqueous multilamellar structure toward a liposomal phase is favored by decreasing the phospholipid phase transition temperature and by increasing the temperature of the system. This reduction of the average size and the appearance of a stable liposomal phase is also promoted by a heating and cooling thermal treatment.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/chemistry , Fatty Acids, Monounsaturated/chemistry , Lipid Bilayers/chemistry , Quaternary Ammonium Compounds/chemistry , Calorimetry, Differential Scanning , Light , Liposomes/chemistry , Membrane Fluidity , Microscopy, Electron, Transmission , Scattering, RadiationABSTRACT
Interaction of DNA with oppositely charged objects, such as multivalent ions, cationic surfactants, cationic liposomes, basic proteins, and alcohols, up to nano- or mesoscopic particles, gives rise to a very interesting and fascinating phenomenology, where the shape, size, and stability of the resulting aggregates depend on a delicate balance between different driving forces, mainly of electrostatic origin. We have studied the cationic liposome-DNA complexes during the whole complexation process, below, close to, and above the isoelectric condition, where the number of cationic lipids equals the number of phosphate groups on the DNA chain. We took advantage of the combined use of dynamic light scattering, laser Doppler electrophoretic mobility, and radio-wave dielectric relaxation measurements in order to characterize both the structural parameters (hydrodynamic radius) and the electrical parameters (charge and counterion concentration) of the resulting structures. These structures are fundamentally of two types, clusters of liposomes stuck together by DNA chains (cluster phase in low-density colloidal suspension) and coexisting DNA coils and DNA globules, according to the procedure through which interactions occur (liposomes in excess DNA solution or DNA in excess liposome suspension).
Subject(s)
DNA/chemistry , DNA/ultrastructure , Drug Carriers/chemistry , Liposomes/chemistry , Models, Chemical , Models, Molecular , Refractometry/methods , Spectrum Analysis/methods , Computer Simulation , Nucleic Acid Conformation , Scattering, RadiationABSTRACT
In this paper, we report on the original global fit procedure of synchrotron small-angle X-ray scattering (SAXS) data applied to a model protein, met-myoglobin, in dilute solution during temperature- and pressure-induced denaturation processes at pH 4.5. Starting from the thermodynamic description of the protein unfolding pathway developed by Hawley (Hawley, S. A. Biochemistry 1971, 10, 2436), we have developed a new method for analyzing the set of SAXS curves using a global fitting procedure, which allows us to derive the form factor of all the met-myoglobin species present in the solution, their aggregation state, and the set of thermodynamic parameters, with their p and T dependence. This method also overcomes a reasonably poor quality of the experimental data, and it is found to be very powerful in analyzing SAXS data. SAXS experiments were performed at four different temperatures from hydrostatic pressures up to about 2000 bar. As a result, the presence of an intermediate, partially unfolded, dimeric state of met-myoglobin that forms during denaturation has been evidenced. The obtained parameters were then used to derive the met-myoglobin p, T phase diagram that fully agrees with the corresponding phase diagram obtained by spectroscopic measurements.
Subject(s)
Metmyoglobin/chemistry , Dimerization , Hydrogen-Ion Concentration , Models, Chemical , Pressure , Protein Denaturation , Scattering, Small Angle , Solutions/chemistry , Thermodynamics , X-Ray Diffraction/methodsABSTRACT
By means of combined calorimetric and dynamic light-scattering measurements, we have investigated the conformational behavior of DNA chains after thermal melting in the presence of a cationic surfactant at different concentrations, up to a surfactant-to-phosphate group molar ratio close to unity. Both the specific heat capacity, C(ex)(p) and the hydrodynamic radius R of the DNA chains provide support for the existence of two structural arrangements with different thermal stabilities, coexisting in the bulk solution. Although a component remains an elongated unfolded DNA chain originated in the thermal denaturation, the second component, consisting of DNA-surfactant complexes, assumes a compact structure with an average size of about 80 nm, whose thermal denaturation occurs at temperatures higher than 100 degrees C.
Subject(s)
DNA/chemistry , Surface-Active Agents/chemistry , Animals , Calorimetry, Differential Scanning , Cations/chemistry , Cattle , Cetrimonium , Cetrimonium Compounds/chemistry , Circular Dichroism , Light , Transition TemperatureABSTRACT
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.
ABSTRACT
The dynamics of lysozyme in the picosecond timescale has been studied when it is in dry and hydrated powder form and when it is embedded in glycerol, glycerol-water, glucose and glucose-water matrices. The investigation has been undertaken through elastic neutron scattering technique on the backscattering spectrometer IN13. The dynamics of dry powder and embedded-in-glucose lysozyme can be considered purely vibrational up to 100 K, where the onset of an anharmonic contribution takes place. This contribution can be attributed to the activation of methyl group reorientations and is described with an Arrhenius trend. An additional source of anharmonic dynamics appears at higher temperatures for lysozyme in hydrated powders and embedded in glycerol, glycerol-water and glucose-water matrices. This second process, also represented with an Arrhenius trend, corresponds to the so-called protein dynamical transition. Both the temperature where such a transition takes place and the magnitude of the protein mean square displacements depend on the environment. The dynamical response of the protein to temperature is put in relationship with its thermal stability.
Subject(s)
Glucose/chemistry , Glycerol/chemistry , Muramidase/chemistry , Neutrons , Scattering, Radiation , Water/chemistry , Animals , Chickens , Egg White , Elasticity , Environment , Models, Chemical , Models, Molecular , Neutron Diffraction , Protein Conformation , Temperature , ThermodynamicsABSTRACT
Through elastic neutron scattering we measured the mean-square displacements of the hydrogen atoms of lysozyme embedded in a glucose-water glassy matrix as a function of the temperature and at various water contents. The elastic intensity of all the samples has been interpreted in terms of the double-well model in the whole temperature range. The dry sample shows an onset of anharmonicity at approximately 100 K, which can be attributed to the activation of methyl group reorientations. Such a protein intrinsic dynamics is decoupled from the external environment on the whole investigated temperature range. In the hydrated samples an additional and larger anharmonic contribution is provided by the protein dynamical transition, which appears at a higher temperature Td. As hydration increases the coupling between the protein internal dynamics and the surrounding matrix relaxations becomes more effective. The behavior of Td that, as a function of the water content, diminishes by approximately 60 K, supports the picture of the protein dynamics as driven by solvent relaxations. A possible connection between the protein dynamical response versus T and the thermal stability in glucose-water bioprotectant matrices is proposed.
Subject(s)
Excipients/chemistry , Models, Chemical , Models, Molecular , Muramidase/chemistry , Neutron Diffraction , Solvents/chemistry , Computer Simulation , Glucose , Kinetics , Phase Transition , Protein Conformation , TemperatureABSTRACT
Two binary aqueous mixtures which contain the small amphiphilic molecules TMAO (trimethylamine-N-oxide) and TBA (tert-butyl alcohol) have been investigated by molecular dynamics simulations and NMR chemical shift and self-diffusion measurements. TMAO is an osmolyte, while TBA is a monohydrate alcohol. Both possess bulky hydrophobic groups and polar heads, namely, NO in TMAO and OH in TBA. The hydrophilic/hydrophobic content of these isosteric molecules strongly modulates the structure and dynamics of the hydration shell, which is thought to be responsible for the effects observed on proteins and phospholipids. Simulation results, especially on hydrogen-bond networking, spatial correlations, and self-diffusivity, are consistent with NMR data and agree well with previous numerical studies on similar solutions. The methods employed allow the elucidation of the microscopic features of the solutions. For TBA solutions, the hydration shell is found to have a low density and a large spatial spread, and thus, above the molar fraction of 0.03, reduction of hydrophobic hydration drives self-aggregation of the solute. This effect does not take place in TMAO solutions, where the hydration shell is more compact and stable, maintaining its structure over a wider range of solute concentrations.
Subject(s)
Computer Simulation , Magnetic Resonance Spectroscopy/methods , Methylamines/chemistry , Models, Chemical , Surface-Active Agents/chemistry , tert-Butyl Alcohol/chemistry , Magnetic Resonance Spectroscopy/standards , Reference Standards , Sensitivity and Specificity , Solutions/chemistry , Water/chemistryABSTRACT
The radiowave dielectric dispersions of DNA in different water-organic co-solvent mixtures have been measured in the frequency range from 100 kHz to 100 MHz, where the polarization mechanism is generally attributed to the confinement of counterions within some specific lengths, either along tangential or perpendicular to the polyion chain. The dielectric dispersions have been analyzed on the basis of two partially different dielectric models, a continuum counterion fluctuation model proposed by Mandel and a discrete charged site model, proposed by Minakata. The influence of the quality of the solvent on the dielectric parameters has been investigated in water-methanol and water-glycerol mixtures at different composition, by varying the permittivity (m) and the viscosity eta of the solvent phase. The analysis of the dielectric spectra in solvents where electrostatic and hydrodynamic interactions vary with the solvent composition suggests that both the two models are able, in principle, to account for the observed high-frequency dielectric behavior. However, while some certain assumptions are necessary about the polyion structure within the Mandel model, no structural prerequisite is needed within the Minakata model, where the polarization mechanism invoked considers a radial counterion exchange with the outer medium, which is largely independent of the local polyion conformation.
Subject(s)
DNA/chemistry , Electrochemistry , Glycerol/chemistry , Ions/chemistry , Methanol/chemistry , Water/chemistry , Electric Conductivity , Models, Chemical , Models, Theoretical , Thermodynamics , ViscosityABSTRACT
The compaction of DNA induced by two simple amphiphiles, cetyltrimethylammonium bromide [CTAB] and dodecyldimethylamine oxide [DDAO], has been investigated by means of combined viscosity and dynamic light scattering measurements, to demonstrate the formation of soluble DNA/surfactant complexes, undergoing a coil-globule transition, upon the increase of the amphiphile concentration. In both of the two systems investigated, the complexation process reaches a maximum for a value of the surfactant to DNA phosphate groups molar ratio of about X = 1. Below this critical concentration, the coil and the globule state coexist in the solution, as clearly shown by the bimodal size distribution obtained from the light scattering intensity correlation functions. Some suggestions are given to support a molecular mechanism responsible for the complex formation, both in the case of a cationic surfactant (CTAB) and of a pH-dependent neutral or cationic amphiphile (DDAO), where the hydrophobic interactions play an important role.
Subject(s)
Cations/chemistry , Chemistry, Physical/methods , DNA/chemistry , Nucleic Acid Conformation , Surface-Active Agents/chemistry , Animals , Cattle , Hydrogen-Ion Concentration , Light , Models, Statistical , Scattering, Radiation , Solubility , Thymus Gland/metabolism , Time FactorsABSTRACT
This work deals with a dielectric study at radio frequencies of the influence at room temperature of two organic molecules, known as cryo-protectants, ethylene-glycol and glycerol, on conformational and dynamic properties of two model proteins, lysozyme (lys) from chicken egg-white and ferricytochrome-c (cyt-c) from horse heart. Cyt-c is a compact globular protein whereas lys is composed of two structural domains, separated by the active site cleft. Measurements were carried out at the fixed temperature of 20 degrees C varying the concentration of the cosolvent up to 90% w/w. From the analysis of the dielectric relaxation of the protein solution, the effective hydrodynamic radius and the electric dipole moment of the protein were calculated as a function of the cosolvent concentration. The data show that glycerol does not modify significantly the conformation of both proteins and cyt-c is also stable in the presence of ethylene-glycol. On the contrary ethylene-glycol strongly affects the dielectric response of lysozyme denoting a specific effect on its conformation and dynamics. The data are coherently interpreted hypothesizing that glycol molecule wedges between and separates the two domains of lys making them rotationally independent.
Subject(s)
Cytochromes c/chemistry , Ethylene Glycol/chemistry , Muramidase/chemistry , Spectrum Analysis/methods , Water/chemistry , Animals , Chickens , Complex Mixtures/chemistry , Electric Conductivity , Horses , Myocardium/enzymology , Protein Conformation , Protein Structure, Tertiary , SolutionsABSTRACT
A neutron-scattering investigation of the internal picosecond dynamics of lysozyme solvated in glycerol as a function of temperature in the range 200-410 K has been undertaken. The inelastic contribution to the measured intensity is characterized by the presence of a bump generally known as "boson peak", clearly distinguishable at low temperature. When the temperature is increased the quasielastic component of the spectrum becomes more and more intrusive and progressively overwhelms the vibrational bump. This happens especially for T > 345 K when the protein goes through an unfolding process, which leads to the complete denaturation. The quasielastic term is the superposition of two components whose intensities and linewidths have been studied as a function of temperature. The slower component describes motions with characteristic times of approximately 4 ps corresponding to reorientations of polypeptide side chains. Both the intensity and linewidth of this kind of relaxations show two distinct regimes with a crossover in the temperature range where the melting process occurs, thus suggesting the presence of a dynamical transition correlated to the protein unfolding. Conversely the faster component might be ascribed to the local dynamics of hydrogen atoms caged by the nearest neighbors with characteristic time of approximately 0.3 ps.
Subject(s)
Glycerol/chemistry , Muramidase/chemistry , Elasticity , Enzyme Activation , Glycerol/analysis , Motion , Muramidase/analysis , Neutron Diffraction , Protein Conformation , Protein Denaturation , Protein Folding , Solutions , Temperature , Water/chemistryABSTRACT
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.
Subject(s)
Cytochrome c Group/chemistry , Scattering, Radiation , Animals , Circular Dichroism , Dimerization , Horses , Hydrogen-Ion Concentration , Models, Molecular , Monte Carlo Method , Myocardium/metabolism , Protein Conformation , Protein Denaturation , Protein Folding , Protein Structure, Secondary , Spectrophotometry , X-RaysABSTRACT
In this paper, we report a study of the effect of solvent viscosity on both translational and rotational dynamics of a simple model protein: the egg white lysozyme. For this, we investigated the dynamical properties of lysozyme in mixtures water-glycerol by means of parallel measurements of photon correlation spectroscopy (PCS) and dielectric spectroscopy at radiofrequencies (DS). In the framework of the Debye-Stokes-Einstein theory, the translational and rotational coefficients allow an estimation of hydrodynamic radius of the protein. A decoupling between translational and rotational dynamics, observed as a different estimation of hydrodynamic radius, is reported in the literature for some systems. In order to ascertain if this effect is present also in our sample, we performed PCS and DS measurements on lysozyme-water-glycerol solutions. The content of glycerol was in the range of 0-70% w/w, with a solvent viscosity from 0.9 to about 10 cpoise, and the protein concentration was up to 20 mg ml(-1). The average sizes of lysozyme, obtained by the two methods, are remarkably different at high protein concentrations. However, the values of hydrodynamic radius extrapolated to infinite dilution are coincident and independent of glycerol. These results indicate that the diffusive behavior of lysozyme in the water-glycerol mixture is coherent with the Debye-Stokes-Einstein hydrodynamic model.