Electrical properties of hydrated collagen. I. Dielectric properties

The effect of water on the low-frequency (10²-10⁵ H_z) complex permittivitv of native, sold-state collagen has been investigated experimentally. Measurements at ambient temperature show that dry collagen exhibits essentially no frequency or temperature dependence. As water is absorbed, both dielectric constant and loss factor increase simultaneously and rise sharply upward at a hydration level which may be associated with the completion of the primary absorption layer as determined from independent water absorption studies. The behaviour is qualitatively identical to that observed for other proteins and related materials. Temperature-dependent measurements made under vacuum conditions in the range ?196°C to +100°C are characteristic of the dielectric properties of the water in the sample. Dehydration produced by successive temperature recycling to the maximum temperature effectively eliminates any temperature or frequency dependence. A maximum in the temperature-dependent curves is found at about +40°C and is explained as the superposition of two processes: (1) the transition of water molecules from bound to free states, and (2) the difffusion of water molecules out of the system. The dielectric constant of dry collagen, after desorption at ambient temperature, is about 4.5. Desorption at elevated temperatures reduced the room temperature value to about 2.3 and the liquid nitrogen temperature value to a number indistinguishable from the optical value of n² = 2.16.     

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.     

Electrical conduction in hydrated collagen. I. Conductivity mechanisms

The electrical conductivity of bovine Achilles tendon with various amounts of adsorbed water was measuredas a function of temperature. The conduction appeared to be fully determined by the water of hydration. The current is probably primarily carried by protons at water contents up to 45% and by small ions at water contents beyond 65%. In both ranges of water content, a linear relation between activation energy and water and content was found. As to the lower range, this is explained by the action of Coulombic forces during the separation of proton–hydroxyl ion pairs. In two regions of water content a linear relation between the logarithm of the pre-exponential factor and the activation energy was found. There are, however, indications that at certain water contents the dissociation constant of the adsorbed water is several orders of magnitude higher than in liquid water.     

Piezoelectric and related properties of hydrated collagen.

Two piezoelectric constants (polarization per unit stress, d=d'-id', and polarization per unit strain, e=e'-ie'), the elastic constant, and dielectric constant are determined for oriented collagen at different hydration levels at 10 Hz from -150 to 50 degrees C. With no hydration (approximately 0% H2O), d' increases slightly with higher temperatures, while e' decreases slightly. Near 11 wt% H2O, both d' and e' increase then decrease around 0 degrees C, and is probably caused by an increase of the dielectric constant and the ionic conductivity in the nonpiezoelectric phase. Hydration greater than 25 wt%, d' and e' decrease above -50 degrees C which is considered to be due to a greater ionic conductivity surrounding the piezoelectric phase.     

Electrical conduction in collagen. II. Some aspects of hydration

Determinations of the amount of bound water in hydrated proteins yield strongly diverging values. The cause of this is the continuity of the transition from bound to free water, and the diffeernt sensitivities to water structure of the measuring techniques. Only the methods that aim at the determination of the amount of water, whose phase remians unchanged duing freezing, yield similar values. The value for collagen as deduced from conductivity data is about 50% water of the dry weight. It is believed that this water interacts with adsorptive groups on the macromolecules, whereas the freezable water occurs in capillaries.     

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.     

High-angle electron diffraction of frozen hydrated collagen.

By using the techniques developed by Taylor et al. [(1975) J. Mol. Biol. 92, 165-167] (freezing of the hydrated specimen before its insertion into the electron microscope and keeping it frozen throughout the diffraction experiment), it was possible to obtain a high-angle electron-diffraction pattern from collagen fibrils. This pattern is in good agreement with that obtained by high-angle X-ray diffraction. Electron diffraction will be very useful to study collagen, because the diffraction pattern from a carefully selected area of one fibril is now feasible.     

Polypeptide models of collagen. II. Solution properties of (Pro-Gly-Phe)n

The conformation of (Pro-Gly-Phe)_n in trifluoroethanol was investigated using CD, nmr and ir techniques. After making appropriate correction for the contribution of the phenylalanine chromophore to the observed CD spectra of the polytripeptide at several temperatures, it is found that (Pro-Gly-Phe)_n can exist in a partially triple-helical conformation in this solvent a t low temperatures. The nmr and ir data support this conclusion. In conjunction with recent theoretical sutdies, our data offer an explanation for the preferential occurrence of the Phe residue in position 2 of the tripeptide sequence Gly-R₂-R₃, in collagen.     

X-ray diffraction studies on the structure of hydrated collagen

The temperature dependence of the humidity-sensitive spacing, d, related to the lateral packing of collagen molecules was measured for fully hydrated collagen. In the vicinity of 0°C, a sudden change in d was observed, which was reversible with temperature. In the diffraction profile, below 0°C, a set of diffraction peaks identified with the hexagonal crystalline form of ice was observed. With the reduction in water content, the intensity of the set of diffraction peaks decreased and was found to be zero at a water content of 0.38 g/g collagen. These results were considered to be caused by the frozen water in collagen fibril below 0°C. According to the water content dependence of d, it was considered that up to a certain water content water absorbed would be stowed in the intermolecular space of collagen and above that water content water molecules would aggregate to make pools, i. e., extrafibrillar spaces. The unfreezable bound water was considered to be located in the intermolecular space of collagen. Size of the extrafibrillar space, determined from the intensity analysis of a smallangle x-ray scattering pattern, corroborates the speculation that the water showed in the extrafibrillar space is freezable and free. The formation of the hexagonal crystalline form of ice in the extrafibrillar space was considered to cause the sudden change in d at 0°C.     

NMR-relaxation in hydrated collagen from the spotted dogfish]

Collagen samples from dog-fish egg case at different water content were studied by the 1H NMR relaxation method. The dependences of the proton spin-lattice and spin-spin relaxation rates on the concentration of water in hydrated native collagen were measured. The fractions of water protons of different mobility and their corresponding spin-spin and spin-lattice relaxation rates were determined in a multi-phase model of water protons in natural biopolymer-water systems. The correlation times were calculated as the characteristics of molecular motion in hydrated collagens with different content of absorbed water. The results obtained were compared with literature data of pulse NMR studies of molecular mobility in other collagen fibers.     

Association of chondroadherin with collagen type II.

Chondroadherin is a cell binding, leucine-rich repeat protein found in the territorial matrix of articular cartilage. Several members of the leucine-rich repeat protein family present in the extracellular matrix of e.g. cartilage have been shown to interact with collagen and influence collagen fibrillogenesis. We show that complexes of monomeric collagen type II and chondroadherin can be released under non-denaturing conditions from articular cartilage treated with p-aminophenylmercuric acetate to activate resident matrix metalloproteinases. Purified complexes as well as complexes formed in vitro between recombinant chondroadherin and collagen type II were studied by electron microscopy. Chondroadherin was shown to bind to two sites on collagen type II. The interaction was characterized by surface plasmon resonance analysis showing K(D) values in the nanomolar range. Both chondroadherin and collagen interact with chondrocytes, partly via the same receptor, but give rise to different cellular responses. By also interacting with each other, a complex system is created which may be of functional importance for the communication between the cells and its surrounding matrix and/or in the regulation of collagen fibril assembly.     

The state of water on hydrated collagen as studied by pulsed NMR

The spin-lattice relaxation time (T₁) of water adsorbed on collagen fibers was determined at six frequencies and temperatures varying from 25° to ?80°C. Care was taken to eliminate the contributions to the signal of protons other than those in the adsorbed water. Quantitative calculations were made on T₁ and the results were compared with the experimental data. It is suggested that a maximum of about 0.50–0.55 g water per g collagen forms a hydration layer, which cannot be frozen down to ?90°C and exhibits a distribution of motional correlation times. For collagen samples containing a larger quantity of adsorbed water, the additional water molecules behave like ordinary isotropic water, having a single correlation time and a freezing temperature of about ?10°C.     

Electrical birefringence study of monodisperse collagen solutions

Acid-soluble collagen solution in 0.1 M acetic acid are studied by electrical birefringence. Specific birefringence is independant of concentration (for c < 100 mg/l.) and follows Kerr's law at low fields. Birefringence decays present a single relaxation time, and reversing pulse experiments show a very low contribution of induced moment compared to permanent dipole orientation. This result is also confirmed by birefringence rise measurements.     

Temperature-dependent dielectric properties of slightly hydrated horn keratin

With an aim to reveal the mechanism of protein-water interaction in a predominantly two phase model protein system this study investigates the frequency and temperature dependence of dielectric constant epsilon' and loss factor epsilon' in cow horn keratin in the frequency range 30 Hz to 3 MHz and temperature range 30-200 degrees C at two levels of hydration. These two levels of hydration were achieved by exposing the sample to air at 50% relative humidity (RH) at ambient temperature and by evacuating the sample for 72 h at 105 degrees C. A low frequency dispersion (LFD) and an intermediate frequency alpha-dispersion were the two main dielectric responses observed in the air-dried sample. The LFD and the high frequency arm of the alpha-dispersion followed the same fractional power law of frequency. Within the framework of percolation cluster model these dispersions, respectively have been attributed to percolation of protons between and within the clusters of hydrogen-bonded water molecules bound to polar or ionizable protein components. The alpha-dispersion peak, which results from intra-cluster charge percolation conformed to Cole-Cole modified Debye equation. Temperature dependence of the dielectric constant in the air-dried sample exhibited peaks at 120 and 155 degrees C which have been identified as temperatures of onset of release of water bound to polar protein components in the amorphous and crystalline regions, respectively. An overall rise in the permittivity was observed above 175 degrees C, which has been identified as the onset of chain melting in the crystalline region of the protein.