The dipolar origin of protein relaxation

1. A set of parameters is proposed to check the interpretation of the dielectric behaviour of protein solutions as a rigid-dipole relaxation of prolate ellipsoids of revolution in the frequency range between 20 kHz and 10 MHz. Besides the delta(b)-function of Scheraga, another analogous function (delta(a)) is presented to establish size and shape of globular proteins. A study of the influence of solvent viscosity on the dielectric dispersion also gives strong evidence in favour of rigid-dipole relaxation. 2. Measurements of the dielectric dispersion of monomer solutions of bovine serum albumin and transferrin are reported. Monomers of bovine serum albumin were obtained by fractionation on Sephadex G-150. Low-conductivity solutions of both proteins are obtained by passage through an ion-exchange resin. 3. Computer analysis of the experimental dispersion curves by use of a two-term Debye dispersion gives valuable information about transferrin and leads to an axial ratio 4.5 for a prolate ellipsoid of revolution. The dielectric increment of bovine serum albumin is very low and no conclusive results have yet been obtained.     

Electrodynamics of interactions in electrolyte solutions and their biological significance

Attention is drawn to the fact that the interaction of charges in aqueous solutions of electrolytes, such as media having physiological characteristics, depends not only on the distance between interacting charges but also on the frequency that determines their dynamics. This fact has significant consequences for some biological processes and their kinetics. The analysis of reasons for charge shielding, including the dynamic effects, shows that, even at distances exceeding the Debye length, electric interactions in systems similar to physiological are effective provided that charges move with frequencies higher than 250 MHz. For each electrolyte solution, the threshold frequency (Maxwell frequency) can be found, which determines the transition from the conducting to the dielectric mode of interactions of charges in physiological solutions.     

Promising insights into the kosmotropic effect of magnetic nanoparticles on proteins: The pivotal role of protein corona formation

Increasing concerns about biosafety of nanoparticles (NPs) has raised the need for detailed knowledge of NP interactions with biological molecules especially proteins. Herein, the concentration-dependent effect of magnetic NPs (MNPs) on bovine serum albumin and hen egg white lysozyme was explored. The X-ray diffraction patterns, zeta potential, and dynamic light scattering measurements together with scanning electron microscopy images were employed to characterize MNPs synthesized through coprecipitation method. Then, we studied the behavior of two model proteins with different surface charges and structural properties on interaction with Fe₃O₄. A thorough investigation of protein–MNP interaction by the help of intrinsic fluorescence at different experimental conditions revealed that affinity of proteins for MNPs is strongly affected by the similarity of protein and MNP surface charges. MNPs exerted structure-making kosmotropic effect on both proteins under a concentration threshold; however, binding strength was found to determine the extent of stabilizing effect as well as magnitude of the concentration threshold. Circular dichroism spectra showed that proteins with less resistance to conformational deformations are more prone to secondary structure changes upon adsorption on MNPs. By screening thermal aggregation of proteins in the presence of Fe₃O₄, it was also found that like chemical stability, thermal stability is influenced to a higher extent in more strongly bound proteins. Overall, this report not only provides an integrated picture of protein–MNP interaction but also sheds light on the molecular mechanism underling this process.     

The optical birefringence of DNA solutions induced by oscillatory electric and hydrodynamic fields

The optical birefringence induced in DNA solutions by both oscillating hydrodynamic fields (flow birefringence) and oscillating electric fields (Kerr effect) is measured over a wide frequency range. The observed frequency response of the birefrigence is compared with theories for rigid ellipsoidal particles and for Gaussian chains. DNA at 6 × 10⁵ molecular weight is found to exhibit rigid particle hydrodynamic behavior, while DNA at 5 × 10⁶ molecular weight behaves like a flexible chain. Characterization of the hydrodynamic relaxation spectra for the DNA's by oscillatory flow birefringence allows precise comparison between theory and the experimental Kerr effect response. The dielectric model for DNA contains both permanent and dispersionless induced dipole moments. The dielectric behavior of DNA has the character of a permanent dipole but with anomalous low-frequency dispersion in the Kerr effect. The existing theories do not adequately describe this dispersion. A fluctuation dipole mechanism with relaxation times comparable to those associated with the hydrodynamic motion could possibly demonstrate the observed polar behavior.     

Electrodynamics of interactions in electrolyte media. Possible consequences in biological functions

Attention is drawn to the fact that electric charge interactions in aqueous electrolyte solutions, such as physiological media, depend not only on the distance between the interacting charges but also on the frequency at which they move. This fact has important consequences for some biological processes, particularly the kinetic ones. Consideration of charge screening, including the dynamic effects, shows that electrical interactions in quasi-physiological systems are effective even above Debye’s length, provided that charges move at frequencies higher than ～250 MHz. For each electrolyte solution, it is possible to define a threshold frequency, the Maxwell frequency, as the point of crossover between a conducting and a dielectric regime in the solution.     

Complex permittivity of representative biological solutions in the 2–67 GHz range

The main purpose of this study is to provide experimental data on the complex permittivity of some biological solutions in the 2–67 GHz range at room and human body temperatures. The permittivity measurements are performed using an open‐ended coaxial probe. Permittivity spectra of several representative monomolecular solutions of proteins, amino acids, nucleic acids, and carbohydrates are analyzed and compared. Furthermore, measurements have also been performed for complex biomolecular solutions, including bovine serum albumin (BSA)–DNA–glucose mixture, culture medium, and yeast extract solution. The results demonstrate that for concentrations below 1%, the permittivity spectra of the solutions do not substantially differ from that of distilled water. Measurements carried out for 4% and 20% BSA solutions show that the presence of proteins results in a decrease in permittivity. For highly concentrated RNA solutions (3%), a slight increase in the imaginary part of the permittivity is observed below 10 GHz. Experimental data show that free water permittivity can be used for modeling of the culture medium above 10 GHz. However, at lower frequencies a substantial increase in the imaginary part of the permittivity due to ionic conductivity should be carefully taken into account. A similar increase has also been observed for the yeast extract solution in the lower frequency region of the considered spectrum. Above 10 GHz, the high concentration of proteins and other low‐permittivity components of the yeast extract solution results in a decrease in the complex permittivity compared to that of water. Obtained data are of utmost importance for millimeter‐wave dosimetry studies. Bioelectromagnetics 33:346–355, 2012. © 2011 Wiley Periodicals, Inc.     

Absorption and fluorescence spectra of hypericin sodium salt in complexes with albumins

The spectra of absorption and fluorescence of hypericin sodium salt (Na-Hy) in organic solvents and in complexes with human serum albumin, bovine serum albumin, and lipoproteins of low and high density have been studied. It was shown that, as the proton donor properties of the solvent enhance, the absorption and fluorescence maxima shift toward the blue region, and as the proton-accepting properties increase, the maxima shift toward the red region. The absorption spectra of complexes of Na-Hy with bovine serum albumin significantly differ from those of complexes of this ligand with human serum albumin, which is evidenced by a lesser width of absorption bands and a lower value of the Stokes shift. The positions of the absorption and fluorescence maxima and the value of the Stokes shift for the complex of Na-Hy with human serum albumin increases when D₂O instead of common water is used as a solvent. It was concluded that H-bonds of hypericin play a significant role in the interaction with human serum albumin.     

On the mechanism of the cold ethanol precipitation method of plasma protein fractionation

Given the negligible difference in the value of the dielectric constant of water at 20°C and that of ethanol solutions at low temperatures, the often advanced expanation for the precipitation of plasma proteins by the cold ethanol process, as being due to a reduction of the dielectric constant and the resulting increase in interprotein charge interactions, is not tenable. It is shown by a surface-thermodynamic approach that, upon dehydration by ethanol, isoelectric serum albumin molecules as well as isoelectric serum gamma globulin molecules will attract each other to a sufficient degree by van der Waals forces to become insoluble in the ethanol-water mixtures used.     

Dynamics of water in supercooled aqueous solutions of glucose and poly(ethylene glycol)s as studied by dielectric spectroscopy

The dielectric behaviour of aqueous solutions of glucose, poly(ethylene glycol)s (PEGs) 200 and 600, and poly(vinyl pyrrolidone) (PVP) has been examined at different concentrations in the frequency range of 10(6)-10(-3) Hz by dielectric spectroscopy and by using differential scanning calorimetry down to 77 K from room temperature. The shape of the relaxation spectra and the temperature dependence of the relaxation rates have been critically examined along with temperature dependence of dielectric strength. In addition to the so-called primary (alpha-) relaxation process, which is responsible for the glass-transition event at T(g), another relaxation process of comparable magnitude has been found to bifurcate from the main relaxation process on the water-rich side, which continues to the sub-T(g) region, exhibiting relaxation at low frequencies. The sub-T(g) process dominates the dielectric measurements in aqueous solutions of higher PEGs, and the main relaxation process is seen as a weak process. The sub-T(g) process was not observed when water was replaced by methanol in the binary mixtures. These observations suggest that the sub-T(g) process in the aqueous mixtures is due to the reorientational motion of the 'confined' water molecules. The corresponding dielectric strength shows a noticeable change at T(g), indicating a hindered rotation of water molecules in the glassy phase. The nature of this confined water appears to be anomalous compared to most other supercooled confined liquids.     

Effect of the Medium Dielectric Strength on the Activity of Alpha Chymotrypsin

The rates of hydrolysis of TrEE, TEE, and ATEE¹ by α-chymotrypsin were determined in media of variable dielectric strength. Many substances which modify the dielectric constant of the medium, exert additional specific effects on the reaction rate, noticeable at more or less elevated concentrations. Notwithstanding, it is possible to differentiate the dielectric and specific effects by comparing the rates in solvents of distinct nature at relatively low concentrations. Thus, the effect of varying the dielectric strength could be studied within wider ranges (ΔD = 20 with TrEE and ca. 28 with ATEE) than in the previous study of trypsin (ΔD = 12). The dielectric effect on α-chymotrypsin is the opposite of that observed with trypsin. In both cases there is a linear relationship between the logarithm of the rate of hydrolysis and the reciprocal of the dielectric constant. The slope is negative with α-chymotrypsin and positive with trypsin. According to expressions relating the dielectric constant to the rate in non-enzymatic reactions, the behavior of α-chymotrypsin is like that of a negative ion, while trypsin behaves as a positive ion. The enzyme activity appears to depend upon the arrangement of charges in the enzyme and substrate molecules, rather than on the presence of certain atomic groupings in the substrate.     

Quantitation of pH-induced Aggregation in Binary Protein Mixtures by Dielectric Spectroscopy

This paper presents a quantitative approach for measuring pH-controlled protein aggregation using dielectric spectroscopy. The technique is demonstrated through two aggregation experiments, the first between ??-lactoglobulin (??-Lg) and hen lysozyme (HENL) and the second between bovine serum albumin (BSA) and HENL. In both experiments, the formation of aggregates is strongly dependent on the solution pH and is clearly indicated by a decrease in the measured permittivity when the second protein is added. A quantifiable lower-bound on the ratio of proteins involved in the aggregation process is obtained from the permittivity spectra. Lower-bound aggregation ratios of 83?% for ??-Lg/HENL at pH?6.0 and 48?% for BSA/HENL at pH?9.2 were consistent with turbidity measurements made on the same solutions.     

Thermal denaturation of globular proteins. Fourier transform-infrared studies of the amide III spectral region.

Fourier transform-infrared (FT-IR) spectra are reported for the amide III spectral region of the native and thermally denatured forms of chymotrypsinogen, ribonuclease, bovine serum albumin, and lysozyme. Chymotrypsinogen denatures into structures containing substantial contributions from beta-sheets, while lysozyme and bovine serum albumin show increased amounts of random-coil forms. The changes observed for ribonuclease are quite small. Bovine serum albumin shows at least six bands in the 1,260-1,320 cm-1 region which undergo large intensity changes upon thermal denaturation, and hence are assignable to alpha-helical amide III modes. The large number of observed bands suggests that slight variations in helical geometry, symmetry, or interactions result in changed amide III frequencies, so that simple correlations between narrow frequency ranges and secondary structures may not be applicable for this mode. A widened frequency range is suggested as diagnostic for helical structures.     

Effect of salt concentration of buffer on the binding of sodium dodecyl sulfate and on the viscosity behavior of the protein polypeptide derived from bovine serum albumin in the presence of the surfactant

The complex between SDS and a protein polypeptide derived from bovine serum albumin was characterized with respect to binding of SDS and viscosity behavior. The amount of bound SDS increased from 1.0 to 2.2 g/g with increase of the buffer concentration from 10 to 220 mM. A logarithmic plot of the amount of bound SDS against the buffer concentration gave a linear relation like in the plot where the number of SDS molecules constituting a spherical micelle of SDS is plotted similarly. The increase in the buffer concentration up to 25 mM, from 25 to 100 mM and beyond 100 mM, was accompanied by a sharp rise, monotonic decrease and levelling-off of the intrinsic viscosity in the respective region. In the region 45-175 mM, a linear relation was found between the intrinsic viscosity and reciprocal square root of the buffer concentration. The observed changes can be interpreted as follows: (1), the electrostatic repulsion between charges introduced by the bound SDS caused the initial increase; (2), shielding of the charges as the result of ion condensation with further increase in ionic strength caused the viscosity drop and subsequent levelling-off. The characteristics of the plots are consistent with the necklace model proposed previously for such complexes in which SDS is bound to a protein polypeptide forming micelle-like clusters and which behave like a flexible polyelectrolyte (Shirahama, K., Tsujii, K. and Takagi, T. (1974) J. Biochem. 75, 309-319).     

Terahertz-infrared spectroscopy of <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1 extracellular matrix

Employing optical spectroscopy we have performed a comparative study of the dielectric response of extracellular matrix and filaments of electrogenic bacteria Shewanella oneidensis MR-1, cytochrome c, and bovine serum albumin. Combining infrared transmission measurements on thin layers with data of the terahertz spectra, we obtain the dielectric permittivity and AC conductivity spectra of the materials in a broad frequency band from a few cm^?1 up to 7000 cm^?1 in the temperature range from 5 to 300 K. Strong absorption bands are observed in the three materials that cover the range from 10 to 300 cm^?1 and mainly determine the terahertz absorption. When cooled down to liquid helium temperatures, the bands in Shewanella oneidensis MR-1 and cytochrome c reveal a distinct fine structure. In all three materials, we identify the presence of liquid bound water in the form of librational and translational absorption bands at ≈ 200 and ≈ 600 cm^?1, respectively. The sharp excitations seen above 1000 cm^?1 are assigned to intramolecular vibrations.     

Phase behavior of aqueous solutions of bovine serum albumin in the presence of dextran, at rest, and under shear

The demixing conditions for aqueous solutions of bovine serum albumin (BSA, fraction V) and for joint solutions of BSA plus dextran (DEX, M(w) = 2000 kg/mol) were determined by turbidimetric measurements as a function of composition, temperature, and shear rate. Aqueous solutions of BSA phase separate upon heating. Within the region of BSA concentrations between 0.05 and 32 wt %, the demixing temperature, T1, falls from ca. 65 degrees C to an almost constant value of 45 degrees C. Adding DEX to the BSA solutions reduces the homogeneous region of the mixture drastically where the amount of DEX required to lower T1 to 25 degrees C decreases rapidly as the concentration of BSA is raised. Experiments concerning the influences of shear have been performed for the ternary system up to 500 s(-1). They demonstrate that the content of dextran determines the sign of the effect. At low DEX concentrations, the mechanical field favors the homogeneous state (shear-induced mixing), whereas the opposite effect (shear-induced demixing) is observed at high DEX concentrations. Possible reasons for this observation are discussed.     

Dynamics of human serum albumin studied by acoustic relaxation spectroscopy

Sonic absorption spectra of solutions of human serum albumin (SA) in water and in aqueous phosphate buffer systems have been measured between 0.2 and 2000 MHz at different temperatures (15-35 degrees C), pH values (1.8-12.3), and protein concentrations (1-40 g/L). Several spectra, indicating relaxation processes in the whole frequency range, have been found. The spectra at neutral pH could be fitted well with an analytical function consisting of the asymptotic high frequency absorption and two relaxation contributions, a Debye-type relaxation term with discrete relaxation time and a term with asymmetric continuous distribution of relaxation times. Both relaxation contributions were observed in water and in buffer solutions and increased with protein concentration. The contribution represented by a Debye-type term is practically independent of temperature and was attributed to cooperative conformational changes of the polypeptide chain featuring a relaxation time of about 400 ns. The distribution of the relaxation times corresponding to the second relaxation contribution was characterized by a short time cutoff, between about 0.02 and 0.4 ns depending on temperature, and a long time tail extending to microseconds. Such relaxation behavior was interpreted in terms of solute-solvent interactions reflecting various hydration layers of HSA molecules. At acid and alkaline pH, an additional Debye-type contribution with relaxation time in the range of 30-100 ns exists. It seems to be due to proton transfer reactions of protein side-chain groups. The kinetic and thermodynamic parameters of these processes have been estimated from these first measurements to indicate the potential of acoustic spectra for the investigation of the elementary kinetics of albumin processes.     

Dielectric behavior and atomic structure of serum albumin

After 1946, serum albumin was available for studies. Its residue sequence and internal disulfide bonding was developed by 1976. We began to make dielectric dispersion studies and apply Perrin's equations for rotational relaxation times around the two axes of revolution in 1938. These data indicated that albumin should have an elongated shape. In 1992 atomic structure data indicated the molecule was heart-shaped. A similar 1998 study of albumin complexed with fatty acid showed that the molecule was substantially rearranged. We found that the dielectric constant of albumin solutions was sensitive to fatty acid content, making this property an attractive probe in stop–flow kinetic studies. Such studies show that the fatty acid reaction is a two-step process. The fatty acid first binds to exterior sites in a diffusion-limited second order reaction complete in 1 ms. Then a first order rearrangement reaction with 400 ms half-life follows. Thus the highly specialized serum albumin sequence of amino acid residues determines not only the structure of the unligated molecule, but also the distinctive structures of the numerous multiligated molecules.     

High-frequency dielectric study of side-chain dynamics in poly(lysine) aqueous solutions

The high-frequency dielectric properties of poly(lysine) of different chirality in aqueous solutions have been measured in the frequency range from 1 MHz to 1.8 GHz. The dielectric spectra show the existence of relatively small dielectric dispersions at around 100 MHz that have been attributed to internal motion in the polymer chain, due to side-chain polar groups. Our results indicate that the local structure of the chain and its possibility to undergo a conformational transition induced by pH does not modify the main feature of the side-chain dynamics, the dielectric strength being largely proportional to the concentration of charged groups. A similar behavior has been found in poly(alpha-glutamate) and in poly(gamma-glutamate) aqueous solutions, where the dielectric parameters appear to be related to the change in the charge density on the main chain, rather than to the accompanying conformational helix-coil transition.     

A 59Co NMR relaxation probe of macromolecule anionic binding sites

⁵⁹Co NMR linewidth measurements are reported which show that hexacyanocobaltate-(III) ion may be used as a sensitive probe of protein interactions with anions in aqueous solutions. Applications are demonstrated to bovine serum albumin where the probe ion binding is monitored as a function of pH and is displaced from the protein sites by hexacyanoferrate(III) ion. The general utility of complex metal ions is suggested as a generally useful approach to the analysis of ion-macromolecule interactions.