Dielectric behavior of water in biological solutions: studies on myoglobin, human low-density lipoprotein, and polyvinylpyrrolidone

The dielectric behavior of the aqueous solutions of three widely differing macromolecules has been investigated: myoglobin, polyvinylpyrrolidone (PVP), and human serum low-density lipoprotein (LDL). It was not possible to interpret unambiguously the dielectric properties of the PVP solution in terms of water structure. The best interpretation of the dielectric data on the myoglobin and LDL solutions was that, in both cases, the macromolecule attracts a layer of water of hydration one or two water molecules in width. For LDL, this corresponds to a hydration factor of only 0.05 g/g, whereas for myoglobin the figure is nearer 0.6 g/g. With myoglobin, part of the water of hydration exhibits its dispersion at frequencies of a few GHz, and the rest disperses at lower frequencies, perhaps as low as 10-12 MHz. The approximate constancy of the width of the hydration shell for two molecules as dissimilar in size as LDL and myoglobin confirms that the proportion of water existing as water of hydration in a biological solution depends critically on the size of the macromolecules as well as on their concentration.     

Time-domain reflectrometry studies of water binding and structural flexibility in chymotrypsin

Time-domain dielectric spectroscopy has been employed to probe the hydration properties and structural flexibility of chymotrypsin (EC 3.4.21.1). The dielectric properties of the hydrated protein above 100 MHz have been used to identify two categories of protein-bound water, the first being irrotationally bound to the protein with a second, relatively weakly bound, having a rotational freedom comparable with that of normal bulk water. A dielectric dispersion observed, centred at 12 MHz, has been attributed to the relaxation of the polar components of the protein structure. This dielectric loss became increasingly significant above a transition in the hydration dependence, where water is relatively weakly bound to the chymotrypsin. This is discussed in terms of the formation of water clusters on the protein surface which screen electrostatic interactions between protein-charged groups.     

Dynamics of Biological Macromolecules: Not a Simple Slaving by Hydration Water

We studied the dynamics of hydrated tRNA using neutron and dielectric spectroscopy techniques. A comparison of our results with earlier data reveals that the dynamics of hydrated tRNA is slower and varies more strongly with temperature than the dynamics of hydrated proteins. At the same time, tRNA appears to have faster dynamics than DNA. We demonstrate that a similar difference appears in the dynamics of hydration water for these biomolecules. The results and analysis contradict the traditional view of slaved dynamics, which assumes that the dynamics of biological macromolecules just follows the dynamics of hydration water. Our results demonstrate that the dynamics of biological macromolecules and their hydration water depends strongly on the chemical and three-dimensional structures of the biomolecules. We conclude that the whole concept of slaving dynamics should be reconsidered, and that the mutual influence of biomolecules and their hydration water must be taken into account.     

Hydration Water, Charge Transport and Protein Dynamics

The hydration water of proteins is essential to biological activity but its properties are not yet fully understood. A recent study of dielectric relaxation of hydrated proteins [A. Levstik et al., Phys. Rev E.60 7604 (1999)] has found a behavior typical of a proton glass, with a glass transition of about 268 K. In order to analyze these results, we investigate the statistical mechanics and dynamics of a model of `two-dimensional water' which describes the hydrogen bonding scheme of bounded water molecules. We discuss the connection between the dynamics of bound water and charge transport on the protein surface as observed in the dielectric measurements.     

Long-range DNA-water interactions

DNA functions only in aqueous environments and adopts different conformations depending on the hydration level. The dynamics of hydration water and hydrated DNA leads to rotating and oscillating dipoles that, in turn, give rise to a strong megahertz to terahertz absorption. Investigating the impact of hydration on DNA dynamics and the spectral features of water molecules influenced by DNA, however, is extremely challenging because of the strong absorption of water in the megahertz to terahertz frequency range. In response, we have employed a high-precision megahertz to terahertz dielectric spectrometer, assisted by molecular dynamics simulations, to investigate the dynamics of water molecules within the hydration shells of DNA as well as the collective vibrational motions of hydrated DNA, which are vital to DNA conformation and functionality. Our results reveal that the dynamics of water molecules in a DNA solution is heterogeneous, exhibiting a hierarchy of four distinct relaxation times ranging from ∼8 ps to 1 ns, and the hydration structure of a DNA chain can extend to as far as ∼18 Å from its surface. The low-frequency collective vibrational modes of hydrated DNA have been identified and found to be sensitive to environmental conditions including temperature and hydration level. The results reveal critical information on hydrated DNA dynamics and DNA-water interfaces, which impact the biochemical functions and reactivity of DNA.     

Dynamics of uncrystallized water and protein in hydrated elastin studied by thermal and dielectric techniques

Dynamics of uncrystallized water and protein was studied in hydrated pellets of the fibrous protein elastin in a wide hydration range (0 to 23 wt.%), by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption–desorption measurements (ESI) were performed at room temperature. The glass transition of the system was studied by DSC and its complex dependence on hydration water was verified. A critical water fraction of about 18 wt.% was found, associated with a reorganization of water in the material. Three dielectric relaxations, associated to dynamics related to distinct uncrystallized water populations, were recorded by TSDC and DRS. The low temperature secondary relaxation of hydrophilic polar groups on the protein surface triggered by hydration water for almost dry samples contains contributions from water molecules themselves at higher water fractions (ν relaxation). This particular relaxation is attributed to water molecules in the primary and secondary hydration shells of the protein fibers. At higher temperatures and for water fraction values equal to or higher than 10 wt.%, a local relaxation of water molecules condensed within small openings in the interior of the protein fibers was recorded. The evolution of this relaxation (w relaxation) with hydration level results in enhanced cooperativity at high water fraction values, implying the existence of “internal” water confined within the protein structure. At higher temperatures a relaxation associated with water dynamics within clusters between fibers (p relaxation) was also recorded, in the same hydration range.     

Hydration of gluten: a dielectric, calorimetric, and fourier transform infrared study

The nature of the hydration of proteins and the subsequent implications for functionality is a matter of importance in both pharmaceutical and food applications. Most published studies rely on the use of one technique and attempt to characterize the system. Few studies have used combinations of techniques. In this paper we report on the use of infrared, dielectric, and calorimetric methods to examine the hydration process of wheat gluten. This has been the subject of considerable study by other techniques and has been well characterized by our group. Results show that in both the infrared and dielectric measurements there is a change in behavior at about 35% water content. This is also the water content below which lowering the temperature of the sample does not result in ice formation. We suggest that at this water content the protein amide groups are fully hydrated, and beyond this point addition of water results in protein dilution rather than further hydration.     

Dielectric properties of slightly hydrated collagen: time-water content superposition analysis

The frequency dependences of the dielectric constant, ε′, and the loss factor, ε″, in collagen were measured at several water contents from 0.1 to 0.3 g/g collagen over a frequency range of 30 Hz to 100 kHz and at a temperature of 20°C. Remarkable dispersion was observed at the lower frequencies for higher water contents. According to accumulated results on the thermodynamic and structural investigations, the dispersion has some analogy to the surface conduction proposed by B. V. Hamon [(1953) Aust. J. Phys. 6 , 304–315]. An empirical relation bewteen ε″ and frequency, f, ε″ ∝? fⁿ, where 0 < n < 1, suggests that the dielectric and conductive properties of hydrated collagen are related to carrier jumps between neighboring sites. For the polarization mechanism of this dispersion, we supposed a model of the transfer of protons between absorbed water molecules, and found that the time–water content superposition procedure is applicable to slightly hydrated collagen. The results derived from the superposition procedure show that the water content, ?, is related to the conductivity, σ, or the dielectric loss factor by the following equations: σ (?, f) = const. × ?^nm?1f^1?n and ε″ (?, f) = const. ?^nmf^?n, respectively, where m is a constant independent of ? and f. These results agree with that derived by another treatment of the same data. The role of water molecules in the conduction and polarization in slightly hydrated collagen is considered to be not far from that assumed in the model.     

DFT and MP2 investigations of <Emphasis Type="SmallCaps">L</Emphasis>-proline and its hydrated complexes

A theoretical study of L-proline-nH₂O (n = 1–3) has been performed using the hybrid DFT-B3LYP and MP2 methods together with the 6-311++G(d,p) basis set. The results show that the P2 conformer is energetically favorable when forming a hydrated structure, and the hydration of the carboxyl group leads to the greatest stability. For hydrated complexes, the adiabatic and vertical singlet–triplet excitation energies tend to decrease with the addition of water molecules. The hydration energy indicates that in the hydrated complexes the order of stability is: binding site 2 > binding site 1 > binding site 3, and binding site 12 > binding site 23 > binding site 13. As water molecules are added, the stabilities of these hydrated structures gradually increase. In addition, an infrared frequency analysis indicated that there are some differences in the low-frequency range, which are mainly dominated by the O–H stretching or bending vibrations of different water molecules. All of these results should aid our understanding of molecular behavior and provide reference data for further studies of biological systems.     

Concentration-dependent effects on fully hydrated DNA at terahertz frequencies

Using terahertz time-domain spectroscopy (THz-TDS), the frequency-dependent dielectric constant of deoxyribonucleic acid (DNA) in solution was measured. The response of the buffer solution is dominated by two Debye modes in this frequency range, and, from an analysis of the concentration dependence, the presence of the DNA increases the main relaxation time and dielectric constant. This reflects the fact that the water in the hydration layer is more tightly bound under the influence of the DNA molecule in comparison to bulk water. This dynamical slowing down with increasing DNA concentration is similar to what is observed with purine nucleotides, but opposite to the behavior of pyrimidine nucleotides. In addition, a suspension model was used with the concentration-dependent data to isolate the dielectric response of the hydrated DNA molecule. The data for the hydrated DNA molecule is still dominated by a Debye response. It is also possible to determine the thickness of the hydration layer, and the DNA molecule influences the surrounding water out to 16 or 17 Å, which corresponds to about six effective hydration layers.     

A novel approach for using dielectric spectroscopy to predict viable cell volume (VCV) in early process development

Online monitoring of viable cell volume (VCV) is essential to the development, monitoring, and control of bioprocesses. The commercial availability of steam‐sterilizable dielectric‐spectroscopy probes has enabled successful adoption of this technology as a key noninvasive method to measure VCV for cell‐culture processes. Technological challenges still exist, however. For some cell lines, the technique's accuracy in predicting the VCV from probe‐permittivity measurements declines as the viability of the cell culture decreases. To investigate the cause of this decrease in accuracy, divergences in predicted vs. actual VCV measurements were directly related to the shape of dielectric frequency scans collected during a cell culture. The changes in the shape of the beta dispersion, which are associated with changes in cell state, are quantified by applying a novel “area ratio” (AR) metric to frequency‐scanning data from the dielectric‐spectroscopy probes. The AR metric is then used to relate the shape of the beta dispersion to single‐frequency permittivity measurements to accurately predict the offline VCV throughout an entire fed‐batch run, regardless of cell state. This work demonstrates the possible feasibility of quantifying the shape of the beta dispersion, determined from frequency‐scanning data, for enhanced measurement of VCV in mammalian cell cultures by applying a novel shape‐characterization technique. In addition, this work demonstrates the utility of using changes in the shape of the beta dispersion to quantify cell health. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:479–487, 2014     

A Novel Method for Single Sample Multi-Axial Nanoindentation of Hydrated Heterogeneous Tissues Based on Testing Great White Shark Jaws

Nanomechanical testing methods that are suitable for a range of hydrated tissues are crucial for understanding biological systems. Nanoindentation of tissues can provide valuable insights into biology, tissue engineering and biomimetic design. However, testing hydrated biological samples still remains a significant challenge. Shark jaw cartilage is an ideal substrate for developing a method to test hydrated tissues because it is a unique heterogeneous composite of both mineralized (hard) and non-mineralized (soft) layers and possesses a jaw geometry that is challenging to test mechanically. The aim of this study is to develop a novel method for obtaining multidirectional nanomechanical properties for both layers of jaw cartilage from a single sample, taken from the great white shark (Carcharodon carcharias). A method for obtaining multidirectional data from a single sample is necessary for examining tissue mechanics in this shark because it is a protected species and hence samples may be difficult to obtain. Results show that this method maintains hydration of samples that would otherwise rapidly dehydrate. Our study is the first analysis of nanomechanical properties of great white shark jaw cartilage. Variation in nanomechanical properties were detected in different orthogonal directions for both layers of jaw cartilage in this species. The data further suggest that the mineralized layer of shark jaw cartilage is less stiff than previously posited. Our method allows multidirectional nanomechanical properties to be obtained from a single, small, hydrated heterogeneous sample. Our technique is therefore suitable for use when specimens are rare, valuable or limited in quantity, such as samples obtained from endangered species or pathological tissues. We also outline a method for tip-to-optic calibration that facilitates nanoindentation of soft biological tissues. Our technique may help address the critical need for a nanomechanical testing method that is applicable to a variety of hydrated biological materials whether soft or hard.     

Dynamic hydration numbers for biologically important ions

The role of ionized groups in biological systems is determined by their affinity for water [Biophys. J. 72 (1997) 65-76]. The tightly bound water associated with biologically important ions increases their apparent size. We define the apparent dynamic hydration number of an ion here as the number of tightly bound water molecules that must be assigned to the ion to explain its apparent molecular weight on a Sephadex G-10 size exclusion column, and report the first accurate determination of tightly bound water for 23 ions of biological significance, including H(+) and HO(-). We also calculate the radius of the equivalent hydrated sphere (r(h)) for each ion. We find that the ratio of the hydrated volumes of two ions approximates the ratio of the square of the charges of the same two ions. Since the 'ionic strength' of the solution also depends upon the square of the charges on the ions, our results suggest that ionic strength effects may largely arise from local effects related to the hydrated volume of the ion--that is, from space filling, osmotic, water activity, surface tension and hydration shell overlap effects rather than from long-range electric field effects.     

Dielectric properties of developing rabbit brain at 37 degrees C

The dielectric properties of developing rabbit brain were measured at 37 degrees C between 10 MHz and 18 GHz using time domain and frequency domain systems. The results show a variation with age of the dielectric properties of brain. An analysis of the data suggests that the water dispersion in the brain of newly born animals can be represented by a Debye equation. This dispersion increases in complexity with age, and there is evidence of a smaller additional relaxation process centered around 1 GHz. It is concluded that the principal contribution to this subsidiary dispersion region arises from water of hydration.     

A model for water motion in crystals of lysozyme based on an incoherent quasielastic neutron-scattering study

This paper reports an incoherent quasielastic neutron scattering study of the single particle, diffusive motions of water molecules surrounding a globular protein, the hen egg-white lysozyme. For the first time such an analysis has been done on protein crystals. It can thus be directly related and compared with a recent structural study of the same sample. The measurement temperature ranged from 100 to 300 K, but focus was on the room temperature analysis. The very good agreement between the structural and dynamical studies suggested a model for the dynamics of water in triclinic crystals of lysozyme in the time range approximately 330 ps and at 300 K. Herein, the dynamics of all water molecules is affected by the presence of the protein, and the water molecules can be divided into two populations. The first mainly corresponds to the first hydration shell, in which water molecules reorient themselves fivefold to 10-fold slower than in bulk solvent, and diffuse by jumps from hydration site to hydration site. The long-range diffusion coefficient is five to sixfold less than for bulk solvent. The second group corresponds to water molecules further away from the surface of the protein, in a second incomplete hydration layer, confined between hydrated macromolecules. Within the time scale probed they undergo a translational diffusion with a self-diffusion coefficient reduced approximately 50-fold compared with bulk solvent. As protein crystals have a highly crowded arrangement close to the packing of macromolecules in cells, our conclusion can be discussed with respect to solvent behavior in intracellular media: as the mobility is highest next to the surface, it suggests that under some crowding conditions, a two-dimensional motion for the transport of metabolites can be dominant.     

Dielectric studies on water in solutions of purified lecithin vesicles

The complex permittivity of sonicated aqueous solutions of purified dimyristoylphosphatidylcholine has been measured as a function of frequency between 3 kHz and 40 GHz. The dielectric spectrum of the samples shows two dispersion/absorption regions, one centered at about 80 MHz the other at about 20.GHz (30°C). Otherwise than in previous studies no additional dispersion/absorption process has been found at frequencies below 10 MHz.The complex dielectric spectrum of the samples is discussed with respect to the dynamical state of solvent water in solutions of single-bilayer vesicles. The main relaxation time of the solvent water, τ₁ ((2πτ₁)^?1 ≈ 20 GHz), is smaller than that of pure water, τ_W, at the same temperature. This effect results from the action of internal depolarizing fields which obviously overcompensate and enhancement of τ₁ due to specific solute/solvent interactions (hydration) as had been previously found with micellar solutions of lysolecithins.It cannot be excluded, that some solvent water shows unusual dynamical behaviour. If there exists a substantial amount of such motionally perturbed water, however, it must be characterized by a relaxation time close to that of the phosphorylcholine zwitterions, τ₂ ((2πτ₂)^?1 ≈ 80 MHz).     

Hydrodynamic characterization of the size and shape of atropinesterase from Pseudomonas putida

Atropinesterase from Pseudomonas putida has been investigated by means of different ultracentrifugation methods under native and denaturing conditions. The following quantities were determined: sedimentation coefficient, translational diffusion and friction coefficient, partial specific volume and molecular weight. From these data the size, shape and hydration of the enzyme molecule in solution were estimated. The results suggest that atropinesterase is a globular protein which consists of a single polypeptide chain with a molecular weight of about 30,000. In solution under non-denaturing conditions, it occurs mainly as a dimer which hydrodynamically behaves as a rigid impenetrable particle. Calculations based on the spheroid model indicate that this particle resembles a hydrated sphere with a diameter of 6.1 +/- 0.2 nm and a hydration of 0.4 +/- 0.1 g of H2O/g of protein rather than a significantly less hydrated ellipsoid of revolution. Under denaturing conditions dissociation into monomers takes place. The effects of sodium dodecyl sulphate (SDS) on size and shape suggest that dimerization results from side-by-side association of two elongated monomers rather than from end-to-end association. Approximately 57 molecules of SDS are bound per dimer before dissociation occurs concomitant with the additional binding of about 19 molecules of detergent.     

Probing the membrane potential of living cells by dielectric spectroscopy

In this paper we demonstrate a quantitative way to measure the membrane potential of live cells by dielectric spectroscopy. We also show that the values of the membrane potential obtained using our technique are in good agreement with those obtained using traditional methods—voltage-sensitive dyes. The membrane potential is determined by fitting the experimental dielectric dispersion curves with the dispersion curves obtained from a theoretical model. Variations in the membrane potential were induced by modifying the concentration of potassium chloride in the solution of the cell suspension in the presence of valinomycin. For exemplification of the method, E. coli was chosen for our experiments.     

Dynamic properties of water bound to matrices of natural DNA and model complexes

From experimental data on the hydration energetics of nucleic acids obtained by differential scanning calorimetry under isothermal conditions, dielectric relaxation time tau d and "free volume" Vf occupied by water molecules in hydration shells of natural DNA and model polyribonucleotides were calculated. In addition, systems consisting of dinucleotides ApA, TpT, UpU, TpU, UpT and water clusters of various sizes (from 20 to 400 water molecules) were studied by Monte Carlo computer simulation. It was shown that, as water content in systems increases, the dynamic characteristics of bound water obtained with both methods approached the values for bulk water.