Dielectric properties of hydrated collagen

Dielectric measurements have been carried out on partially hydrated collagen in the frequency ranges 100 kHz–5 MHz, 100 MHz–1 GHz, and 8–23 GHz. In the low-frequency range, a dispersion was observed around 100 kHz which results from inhomogeneous conductivity of the samples. A dielectric relaxation was observed aroud 0.3 GHz using time-domain-spectroscopy techniques. This relaxation can be considered to originate from mobile side chains. Microwave measurements indicate that the water relaxation may extend into the 10-GHz region. An apparent discrepancy between the main water relaxation time and the average rotational correlation time of water as measured by nmr line widths was resolved by the assumption that a fraction of the water molecules is bound to the collagen with residence times on the order of 10^?6 sec, whereas the remainder of the water is only weakly bound and exhibits rotational rates on the order of 10^?10 sec.     

13C magnetic resonance evidence for anisotropic molecular motion in collagen fibrils

Relaxation times and integrated intensities have been obtained from dipolar decoupled ¹³C magnetic resonance spectra of reconstituted fibrils of chick calvaria collagen enriched at the glycine C^a and C′ positions. The data obtained are consistent with a model in which collagen molecules reorient about the long axis of the helix with a rotational diffusion constant (R₁) of ~10⁷ s^?1, a value similar to that expected for the helix in solution. Data obtained from natural abundance ¹³C spectra of native (crosslinked) calf achilles tendon and rat tail tendon provide evidence of rapid anisotropic reorientation for at least 75% of the carbons in these tissues. Hence, our preliminary data indicate that, in these materials, the intermolecular interactions in the fibrilar collagen lattice can accommodate rapid reorientation at a majority of carbon sites.     

Mobility of water bound to biological membranes: A proton NMR relaxation study

Water proton nuclear magnetic resonance relaxation measurements have been obtained for aqueous suspensions of red cell membranes. These data support a model in which water molecules are exchanging rapidly between a bound phase with restricted motions and a free phase with dynamic properties similar to liquid water. From this model and these data, estimates are obtained for the relaxation time for bound phase water. Possible relaxation mechanisms for bound phase water are discussed and some support is found for an intermolecular interaction modulated by translational motions characterized by a diffusion constant of 10^?9 cm²/s.     

Nuclear magnetic resonance relaxation time and self-diffusion coefficient measurements of water in frog ovarian eggs (Rana pipiens)

Self-diffusion coefficient measurements of water in untreated ovarian eggs of Rana pipiens using nuclear magnetic resonance indicate that cytoplasmic water has reduced translational mobility compared with pure water. Using a simple two-state model, we find that ～67% is “relatively immobile.” Consideration of the nuclear magnetic resonance spin-lattice and spin-spin relaxation times indicates that the decreased mobility can largely be ascribed to hydration. Our value for the self-diffusion coefficient (6.8 × 10^?6 cm²/sec) is lower than those reported by other investigators using isotopic water exchange techniques on frog eggs chemically treated to remove the membrane. However, the results reported here are in agreement with unpublished data on untreated frog eggs implying that chemical treatment has modified the cytoplasm in some manner.     

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.     

Nuclear magnetic resonance investigation of the helix in random coil transformation in poly-alpha-amino acids. I

High-resolution nuclear magnetic resonance spectra at 100 MHz and 220 MHz have been obtained on two samples of poly-L -alanine of differing molecular weights (2500 and 42 500) in the chloroform–trifluoroacetic acid system under various conditions of solvent composition, temperature, and polypeptide concentration. Separate helix and random coil peaks are observed for the α-CH and peptide NH backbone proton resonances, thereby permitting the determination of helix content. This observation of separate peaks demonstrates that the lifetimes of the helix and random coil portions of poly-L -alanine have lower limits of about 10^?1 sec. It is suggested that solvent–peptide versus peptide–peptide hydrogen bond competition, coupled with a destabilizing effect of the trifluoroacetic acid on the helix, is responsible for the helix–random coil transformation.     

Electrical properties of hydrated collagen. II. Semiconductor properties

The dc conductivity of hydrated bovine Achilles' tendon collagen has been determined as a function of hydration over a limited temperature range. At ambient temperature the conductivity changes from 10^?15 (Ω cm)^?1 in the dry state to about 10^?8 (Ω cm)^?1 at ～24% water content by weight. For all temperatures the conductivity increases exponentially with hydration obeying σ(h) = A exp (βh), where h is a measure of the hydration, A is independent of temperature, and the parameter β ～ T^?1. It is shown that the data may be described by an impurity-type mechanism in which the effective activation energy for the process is dependent on temperature and hydration. Conduction is assumed to be electronic with the impurity (water) acting as a donor. In the solid state the effect of water on the conductivity is reversible indicating the absence of chemical alteration of the hydrated collagen.     

Multinuclear magnetic resonance studies of collagen molecular structure and dynamics

We have measured the percentages of cis and trans Gly-Pro and X-Hyp peptide bonds in thermally unfolded type I collagen. ¹³C-nmr solution spectra show that 16% of the Gly-Pro and 8% of the X-Hyp bonds are cis in unfolded chick calvaria collagen. These results support the hypothesis that cis–trans isomerization is that rate-limiting step in the propagation of the collagen triple helix. We have used multinuclear solid-state nmr to study the molecular dynamics of the collagen backbone in tendon, demineralized bone, and intact bone as a function of temperature, hydration, and pH. These studies show that collagen backbone motions are characterized by a broad distribution of correlation times, τ, covering the range from 10^?4 to 10^?9 s. In the case of nonmineralized collagen, the root-mean-square fluctuations in azimuthal angle, γ_rms, range from ca. 10° when τ ～ 10^?9 s to ca. 30° when τ < 10^?4 s; in the case of bone collagen, γ_rms values are about half as large as those found in nonmineralized collagen. Backbone motions are negligible at temperatures below ?25°C. This is also the case at 22°C when demineralized bone collagen is lyophilized. In contrast, flexibility of hydrated demineralized bone collagen greatly increases as pH is lowered from 7 to 2. The more limited flexibility observed at neutral pH is a consequence of the intermolecular interactions that contribute to fibril organization and strength. However, the fibrils retain significant flexibility at physiological pH, enabling them to distribute stress and dissipate mechanical energy.     

Structure of hydrated Na+ ions around a region of A- or B-DNA helix

A molecular-dynamics simulation was used to carry out an introductory study of the hydration of a section of a rigid single A- or B-DNA helix with one Na⁺ counterion per nucleotide. Four Na⁺ ions and four nucleotides and periodic boundary conditions were used to mimic an infinite helix. The atoms of the helix and the Na⁺ ions were assumed to be Lennard-Jones spheres that also carried charges. Stillinger four-point charge model water molecules were used. We carried out five calculations, for 26 and 46 water molecules in B-DNA and 20, 32, and 46 in A-DNA fragments. The arrangements of the Na⁺ ions are found to have some similarities to those obtained by Clementi and Corongiu. In the calculations with 46 water molecules, we found that two Na⁺ ions can be bridged by about two water molecules and form a hydrated bound pair, which in turn forms a bridge between the guanine N7 and a near phosphate group. These bound pairs may be important in stabilizing the helix structure of DNA molecules.     

Determination of the relaxation time of the helix–coil transition of poly(γ-benzyl-L-glutamate) by the nmr technique

NMR measurements of poly(γ-benzyl-L -glutamate) are reported in several different strengths of magnetic field to determine the relaxation time of the helix–coil transition. Nmr spectra of various samples had line shapes varying from the double to single, depending on the extent of the polydispersity of the sample. This result indicated that the correct line shape of a polypeptide is obscured in the overlapping of multipeaks, which are due to the heterogeneity of the molecular weight in the sample. Thus, the conventional line-shape analysis could not be applied to the kinetic study of the helix–coil transition of polypeptides without consideration of this polydispersity effect on the line shape. To overcome this difficulty, we measured linewidths of nmr spectra for fairly monodisperse samples, using various nmr spectrometers, having field strengths from 60 to 220 MHz. The results were analyzed by a quadratic equation, which involves an additional term proportional to the frequency difference of two sites. The equation differs from the conventional quadratic equation, usually utilized in the case of the fast-exchange limit, only in this additional term. This modification is required to evaluate correctly the unusual broadening of the linewidth resulting from the polydispersity effect and to determine the relaxation time reflected in nmr. Nmr spectra of three samples (DP-35, 85, and 250) were measured by 220-, 100-, and 60-MHz spectrometers in trifluoroacetic acid/chloroform at 28°C and linewidths were analyzed. Relaxation times of the helix–coil transition obtained at the transition midpoint are 2.5 × 10^?4, 7 × 10^?4, and 1.1 × 10^?3 sec, for DP-35, 85, and 250, respectively.     

Physical properties of type i collagen in solution: Structure of α-Chains and β- and γ-components and two-component mixtures

The structure of thermally denatured Type I collagen has been studied using laser light scattering. The results indicate that the diffusion coefficients of α-chains and β- and γ-components are 1.550 ± 0.08 × 10^?7, 1.000 ± 0.05 × 10^?7, and 0.835 ± 0.04 × 10^?7 cm²/sec, respectively, at temperatures between 20 and 40°C. It is concluded from diffusion data that these species have hydrodynamic radii of about 13.8 nm (α-chain), 21.5 nm (β-component), and 25.7 nm (γ-component), consistent with previous studies of thermal denaturation by light scattering. It is also concluded, based on volume calculations, that a large volume increase occurs when the triple helix unfolds. Homodyne correlation functions for two component mixtures of α-chains and β-and γ-components appeared to decay exponentially. In all but one case discussed the correlation function could be fitted with a single component having a translational diffusion coefficient which was an intensity weighted average of the diffusion coefficient of each component present.     

Li+ counterion self-diffusion in ordered DNA

The anisotropic self-diffusion coefficient of ⁷Li⁺ (I = 3/2) counterions has been studied in hydrated, macroscopically oriented Li-(B)DNA fibers at relatively high water contents, corresponding to approximate DNA-DNA helix axis distances of 22–35 Å, using the pulsed field gradient hmr spin-echo method. Self-diffusion coefficients parallel (D_∥) and perpendicular (D_?) to the DNA helix axis increase with increasing salt content and with increasing DNA-DNA helix axis distance. The observed anisotropy D_∥/D_? decreases from 1.6 to 1.2 with the DNA-DNA separation increasing from 22 to 35 Å in the salt-free sample. This result can be understood by the obstruction effect caused by the DNA molecules themselves. The values of the Li⁺ self-diffusion coefficients in the most water-rich system with no added salt (corresponding to an approximate distance of 35 Å between the DNA helix axes) were D_∥ ～ 1.15 × 10^?10 m² s^?1 and D_? ～ 0.98 × 10^?10 m² s^?1, compared to 9.14 × 10^?10 m² s^?1 for the diffusion of Li⁺ in an aqueous solution of LiCl (～ 2.1M). The possible occurrence of restriction effects in the DNA fibers have also been studied by determining the self-diffusion coefficient at different effective diffusion times. The self-diffusion coefficient of Li⁺ in the sample with the largest DNA-DNA helix axis distance seems to be independent of the effective diffusion time, which indicates that the lithium ions are not trapped within impermeable barriers. The possibility of diffusion through permeable barriers has also been investigated, and is discussed. © 1994 John Wiley & Sons, Inc.     

A study of dextran hydration in dilute,aqueous solution by the proton magnetic relaxation method

The times of proton magnetic relaxation in dilute (<1%), aqueous solutions of dextrans, having a molecular weight range of 17 x 10³?500 x 10³, are highly sensitive to the temperature—time prehistory of the samples investigated. Reliable results have been obtained only after preliminary heating of the solutions at 100° for 30 min. On the basis of the model of “two states of water in a solution”, the dependence of the degree of hydration of a dextran on its molecular weight has been obtained. In the molecular weight range 17 x 10³?110 x 10³, only a fraction of the D-glucose residues are hydrated, the degree of hydration increasing with the molecular weight. The data obtained are considered to be a consequence of intersegmentary interaction in a dextran macromolecule.     

25OHD, a circulating vitamin D metabolite in fish.

Serum and hepatic 25-hydroxyvitamin D (25OHD), and serum calcium, phosphate, 25OHD₃ binding capacity and binding affinity were measured in male and female trout. Both serum and hepatic 25OHD levels are decreased in female trout with elevations in protein bound calcium and phosphate. Whereas the apparent dissociation constant (Kd) for serum binding of 25OHD₃ of 1.0–2.0 × 10^?9M is similar in males and females, the 25OHD₃ binding capacity of hypercalcemic spawning trout (1.39 × 10^?7M) is significantly less than that of male fish (1.88 × 10^?7M). At circulating serum concentrations of 25OHD which average 9.5 × 10^?9M only 5–7% of trout serum 25OHD binding sites are occupied.     

A Nuclear Magnetic Resonance Study of Hydrated Systems Using the Frequency Dependence of the Relaxation Processes

A practical method is described for determining some characteristics of the spectrum of proton mobilities in a hydrated system from the frequency dependence of the nuclear magnetic resonance (NMR) relaxation processes. The technique is applied to water in association with agarose and gelatin. The results for agarose are consistent with the hypothesis that a fraction of the protons is distributed over states of reduced mobility and exchanges rapidly with the remaining fraction which is attributed to water in the normal state. No variation in the characteristics of the modified fraction could be detected for water concentrations in the range 1.2-50 g H₂O/g agarose. Within the modified fraction, higher mobilities are more common than low mobilities; at 1.2 g H₂O/g agarose, not more than 10% of the proton population has mobilities more than 100 times smaller than normal. The modified proton fraction is tentatively identified with agarose hydroxyl protons and possibly water molecules bound to the polymer. Proton states with mobilities intermediate between water and ice have also been detected in hydrated gelatin. As in agarose, higher mobilities are the most common. In contrast to agarose, the characteristics of the modified proton states are markedly dependent on water concentration. They are tentatively attributed to gelatin protons coupled for spinlattice relaxation with those of the bulk phase by exchange and spin diffusion.     

The dynamics of the DNA hydration shell at gigahertz frequencies

We have used Brillouin scattering to measure the linewidths and frequencies of GHz acoustic phonons in Na- and Li-DNA films as a function of temperature between 300 and 140 K for samples that were dry, lightly, and heavily hydrated. The linewidths decrease with falling temperature and water contents, indicating that coupling to a water relaxation is the main source of phonon damping. The strength of the relaxation was determined using measurements of the phonon linewidth as a function of frequency, and confirmed by comparison of measured and calculated spectral profiles. The relaxation strength is anisotropic, being greater for phonons propagating perpendicular to the helix axis. The hydrated DNA exhibits both a rapid relaxation (≤ 10^?11 s per radian) giving rise to a classical f² damping, and a slower motion with a relaxation time that varies from ～ 4 × 10^?11 s per radian (primary hydration shell) to ～ 2 × 10^?12 s per radian (secondary hydration shell) at room temperature. In the frequency interval that bounds these relaxation times (～ 4 to 80 GHz) we expect degrees of freedom associated with the primary hydration shell to be important. The sample with primary hydration follows a simple Arrhenius behavior with ΔH ～ 5 kcal mole^?1. The effective activation energy for the sample with secondary hydration is somewhat higher (indicating a more cooperative water relaxation) and varies strongly with temperature. The elastic moduli change much more than can be accounted for by relaxation, indicating the importance of water motion in softening interatomic potentials. The extent of the softening caused by the “unfreezing” of water motion is similar to the degree of softening caused by hydrating the sample.     

81 Br-NMR studies of carbonic anhydrase

⁸¹Br nmr measurements have been made on high (bovine; BCA) and low (human-B; HCAB) specific activity forms of carbonic anhydrase and on a chemically modified form of the human enzyme (carboxyamidomethyl; CAM-HCAB). The high specific activity form of the enzyme, BCA, exhibits a ⁸¹Br line broadening which is determined by the lifetime of Br^? bound to the zinc ion of the enzyme. The low specific activity form of the enzyme, HCAB, under similar conditions of concentration, pH, etc., does not exhibit a ⁸¹Br nmr line broadening. $\text{Cl}{}^{\mathrm{?}}\text{Br}{}^{\mathrm{?}}$ competitive binding studies, using ³⁵Cl nmr, suggests that the failure to observe ⁸¹Br broadening is due to an increase in the lifetime of a zinc bound Br^?. An increase in this lifetime by a factor of 10–100 over that exhibited by BCA is sufficient to abolish the line broadening. A modified form of HCAB, CAM-HCAB, does, however, exhibit a ⁸¹Br nmr line broadening. Estimates of the lifetime of zinc bound Br^?, τ_M, are 4 × 10^?7 sec. for CAM-HCAB at pH 8 and 1 × 10^?7 sec. for BCA at pH 7. The lifetime for Br^? bound to HCAB is estimated to be ≥10^?6 sec.     

The effect of the bilayer phase transition on the carbonyl carbon-13 chemical shift anisotropy

Proton enhanced (PE), natural abundance carbon-13 magnetic resonance spectra have been obtained of the carbonyl groups in hydrated dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. A four-fold change in the overall linewidth results on passing from the fluid to crystalline phase. The carbonyl resonance provides a sensitive measure of the changes in mobility experienced by the lipid molecule above and below the phase transition temperature. The spectral shapes derived from both the fluid (T = 45°C) and crystalline (T = 15°C) phases indicate that even in the crystalline phase sufficient molecular motion is present to average the chemical shielding tensor. It is suggested that this motion in the L_β′ phase is a result of dislocations and packing faults diffusing in the plane of the bilayer. Because of the small size of the chemical shielding interaction (approx. 3 kHz for ω₀ = 22.63 MHz) lipid diffusion coefficients of order 10^?10 cm²/sec observed in the L_β′ phase [1] are effective in averaging the shielding tensor.A comparison is made with the perturbation suffered by the carbonyl groups in the L_α phase in the presence of substantial amounts of cholesterol or the polypeptide gramicidin A.     

Effect of hydration upon the thermal stability of tropocollagen and its dependence on the presence of neutral salts

The endotherm enthalpy changes ΔH_D and temperatures T_D of thermal denaturation of tropocollagen fibers were measured by DSC calorimetry as functions of water content. The denaturation temperatures decrease with increasing water content. The enthalpy change values increase sharply in the range 0–28% of water content, where a maximum of 14.3 cal g^?1 is reached. The effect of water uptake on the enthalpy term is explained by water bridge formation within the collagen triple helix. Evidence is given for the existence of approximately three intercatenary water bridges per triplet at the enthalpy maximum, their H-bond energy amounting to approximately 4000 kcal/mol of protein. In the 30–60% range of water content, ΔH_D decreases by 2 cal^?1 probably due to interactions between secondary water structures and the stabilizing intrahelical water bonds. The influence of two neutral potassium salts, with a structure-stabilizing and a structure-breaking anion (F^? and I^?), on the hydration dependence of ΔH_D and T_D was also studied. It was shown that the primary hydration is not influenced by these ions, but that T_D and ΔH_D are altered in an ion specific way in the presence of interface and bulk water. Hydrophobic interactions do not explain the experimental results. A reaction mechanism of the effects of ions upon the structural stability of collagen is proposed and discussed in terms of interactions of the medium water molecules with the intrahelical water bonds, and in terms of proton-donor/proton-acceptor equilibria between peptide groups, hydrated ions, and intrahelical water molecules.     

Interactions des ions métalliques avec le DNA. IV. Fixation de i'ion cuivrique sur le DNA

The binding of cupric ion (Cu⁺⁺) to DNA was followed by spectrophotometry, melting profiles, and hydrodynamic techniques, in 0. 1M NaClO₄ and at pH 5. 6. A small amount of Cu⁺⁺ is bound specifically to bases (about 1 Cu⁺⁺ per 20 nucleotides), in agreement with polarographic and EPR data. A preferential stabilization of G–C pairs and only a slight increase of the flexibility of the molecule were observed. In 5 × 10^?3M NaClO₄, a higher number of nonhomogeneous binding sites is found by spectrophotometry. It is concluded that at least two types of sites are available for Cu⁺⁺. The first one, where Cu⁺⁺ is chelating N₇ of purines to phosphate, is observed only at low ionic strength and destabilizes the double helix. The second exists mainly at 0, 1M or higher ionic strength. All the sites are identical and could be attributed to two successive guanine residues in the same strand. Similar behavior was found for other divalent cations, e. g., Fe⁺⁺, Mn⁺⁺, and Co⁺⁺.