A. Bonincontro

Dynamics of DNA Adsorption on and Release from SDS−DDAB Cat−Anionic Vesicles: a Multitechnique Study

DNA adsorption and release from cat-anionic vesicles made of sodium dodecylsulfate-dodecyldimethylammonium bromide (SDS-DDAB) in nonstoichiometric amounts was investigated by different electrochemical, spectroscopic, and biomolecular strategies. The characterization of the vesicular system was performed by dynamic light scattering, which allowed estimating both its size and distribution function(s). The interaction dynamics was followed by dielectric spectroscopy and zeta-potential, as well as by agarose gel electrophoresis, AGE. Also, circular dichroism, CD, measurements were carried out, to ascertain possible structural rearrangements of DNA, consequent to the interactions with the cat-anionic vesicles. CD demonstrates that vesicle-bound DNA retains its native conformation. The results obtained by the aforementioned techniques are consistent and indicate that binding saturation is obtained at a [DNA/vesicles] charge ratio close to 0.8, considering only the excess surface charges on the vesicles. This result is apparently in contradiction with a purely electrostatic approach and is tentatively ascribed to the distance between charges on the biopolymer and the vesicle surface, respectively. A possible interpretation is discussed. The nucleic acid can be completely retrieved from the vesicles upon addition of adequate amounts of SDS, which is the defective surfactant in the vesicular system. Precipitation of the poorly soluble SD-DDA salt results in an almost complete release of DNA.

Dielectric dispersions of colloidal particles in aqueous suspensions with low ionic conductivity

Abstract The dielectric behavior of polystyrene aqueous suspensions with low ionic conductivity has been investigated in the frequency range 5 kHz to 30 MHz. The observed dispersions have been studied as due to the overlapping of two distinct processes, on the basis of the Schwarz-Schurr model. The characteristic parameters of the model, as a function of temperature, have been valued.

INFLUENCE OF PH ON LYSOZYME CONFORMATION REVEALED BY DIELECTRIC SPECTROSCOPY

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.

Interactions between single-walled carbon nanotubes and lysozyme.

Dispersions of single-walled and non-associated carbon nanotubes in aqueous lysozyme solution were investigated by analyzing the stabilizing effect of both protein concentration and pH. It was inferred that the medium pH, which significantly modifies the protein net charge and (presumably) conformation, modulates the mutual interactions with carbon nanotubes. At fixed pH, in addition, the formation of protein/nanotube complexes scales with increasing lysozyme concentration. Electrophoretic mobility, dielectric relaxation and circular dichroism were used to determine the above features. According to circular dichroism, lysozyme adsorbed onto nanotubes could essentially retain its native conformation, but the significant amount of free protein does not allow drawing definitive conclusions on this regard. The state of charge and charge distribution around nanotubes was inferred by combining electrophoretic mobility and dielectric relaxation methods. The former gives information on changes in the surface charge density of the complexes, the latter on modifications in the electrical double layer thickness around them. Such results are complementary each other and univocally indicate that some LYS molecules take part to binding. Above a critical protein/nanotube mass ratio, depletion phenomena were observed. They counteract the stabilization mechanism, with subsequent nanotube/nanotube aggregation and phase separation. Protein-based depletion phenomena are similar to formerly reported effects, observed in aqueous surfactant systems containing carbon nanotubes.

Temperature dependence of DNA dielectric dispersion at radiofrequency.

We have studied the dielectric behavior of DNA aqueous solutions at various ionic strengths and in the presence of the specific DNA ligand ethidium bromide, in the frequency range 1 MHz-1 GHz, at different temperatures ranging from 5 to 40 degrees C. The activation enthalpies of the dielectric relaxations studied were obtained by Arrhenius plots of In(tau T)-1 vs. T-1. The results are consistent with a counterion fluctuation model as previously developed by Mandel and colleagues.

Electrical conductivity and dielectric dispersion of E. coli 70S ribosomes and of 30S and 50S subunits: effects of magnesium ions

Abstract Electrical conductivity and dielectric dispersions have been measured on suspensions of 30S, 50S subunits and 70S ribosomal particles from E. coli, in the presence and absence of magnesium ions. Conductivity measurements show a net contribution due to the disperzed phase. Ribosomal particles act in general as obstructants to the ionic motions in the solution, but besides this effect, there is an intrinsic contribution to the overall conductivity which is more pronounced in the absence than in the presence of magnesium ions. This effect suggests the existence of an interfacial conductivity, modulated by bound magnesium ions. The intrinsic conductivity is higher in the 30S subunit than in the 50S. Dielectric dispersions show a consistent difference between 30S and 50S, indicating that the exposure of ribosomal RNA should be greater in the 30S than in the 50S. Structural information is derived from the conductivity measurement, via the Looyenga equation.

Radiowave dielectric dispersions of colloidal particles in KCl and LiCl electrolyte solutions

Abstract The permittivity ϵ′ and dielectric loss ϵ″ of a colloidal suspension in KCl and LiCl electrolyte solution in the frequency range 5 kHz to 10 MHz and in the temperature interval 0 to 50°C have been measured. The absence of the Maxwell—Wagner effect, due to the particular values of the ionic conductivity, permits us to define with great accuracy the parameters describing the dielectric dispersion of counterions on the basis of the Schwarz—Schurr model. The surface charge density σo and the two surface conductivities λ and λo as a function of temperature have been obtained. No anomaly in the thermal dependence of these parameters has been found whereas our previous measurements evidenced a marked anomaly in the temperature dependence of the dielectric loss ϵ″ at microwave frequency. This circumstance may be of interest in understanding the molecular configuration of interfacial water.

Non-linear Dependence of the Dielectric Properties of Chick Embryo Myoblast Membranes Exposed to A Sinusoidal 50 Hz Magnetic Field

Primary chick embryo myoblasts can be a useful tool for studying the developmental events which accompany myoblast differentiation, particularly myoblast membrane fusion. To determine whether the electrical properties and/or fusion in these systems are affected by 50 Hz magnetic fields, chick embryo myoblast cultures were exposed to B-field intensities ranging from 1 to 10 mT. The electrical parameters of the myoblasts, i.e. membrane conductivity, membrane permittivity and the conductivity of the cell interior (cytosol) were determined by the analysis of conductivity dispersion data in the radio frequency range (10 kHz-100 MHz). Preliminary results indicate that the time of fusion (60 h) is not affected by these fields, but that the absolute values of the two membrane electrical parameters are affected. In particular, a B-field intensity-dependent decrease was observed. The maximum effect resulted after a 1 h exposure to a magnetic flux density of about 5 mT. The conductivity of the cytosol remained unchanged. These data seem to indicate that exposure to 50 Hz magnetic fields affects both static and dynamic membrane properties in primary chick embryo myoblasts.

Interaction of bovine serum albumin with gemini surfactants

The interactions between bovine serum albumin and cationic gemini surfactants were investigated as a function of concentration, under different pH conditions. The investigation deals with dielectric relaxation, dynamic light scattering, zeta-potential, circular dichroism, and UV spectroscopy. The interactive behavior of the anionic form is quite different from the cationic species. It indicates that protein-surfactant interactions are mostly electrostatic in nature and depend on the state of charge of bovine serum albumin. The results indicate the presence of both hydrophobic and electrostatic contributions in the interactions of gemini with bovine serum albumin. Comparison of dynamic light scattering, dielectric relaxation, electrophoretic mobility, and optical circular dichroism allows drawing some preliminary hypotheses on the different contributions to surfactant binding and supports former studies on the formation of complexes between the bovine serum albumin and the above species.

Dielectric spectroscopy as a probe for the investigation of conformational properties of proteins

In this brief paper, we review recent and significant results obtained in our laboratory by dielectric spectroscopy (DS). This is a multi purpose and very sensitive approach to investigate structural features of biological systems. DS at radiofrequencies is particularly powerful in the study of structural and conformational properties of proteins. We report on results obtained on three well-known proteins: lysozyme, cytochrome-c and metmyoglobin, which represent very useful models for folding/unfolding studies. The influence of pH and temperature as well as presence of trehalose as a co-solvent, was determined by estimation of the effective hydrodynamic radius and electric dipole moment of the protein in solution. In particular, trehalose was shown to affect the alkaline transition of cytochrome. Conformational effects on the three above-mentioned proteins were observed in a temperature range near the physiological ones. Dynamical properties of lysozyme in mixtures water-glycerol are also discussed. Parallel measurements of photon correlation spectroscopy (PCS) and DS indicated that both translational and rotational diffusive behavior are coherent with the Debye-Stokes-Einstein hydrodynamic model.