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.     

Effect of water on piezoelectric,dielectric, and elastic properties of bone

The complex piezoelectric constant (d = d′ ? id″), elastic constant (c = c′ + ic″), and dielectric constant (ε = ε′ ? iε″) were measured at a frequency of 10 Hz over the temperature range from ?150 to 50°C and for a range of hydration up to 0.26 g/g for decalcified bone and up to 0.084 g/g for bone. For decalcified bone, ε′ and ε″ increased with increasing hydration with a deflection at the critical hydration h_c = 0.08 g/g;d′ at ?150°C increased below h_c but decreased above h_c with increasing hydration; c′ increased below ?60°C but decreased above ?60°C with increasing hydration; and the peak temperatures of ε″, d″, and c″ below ?50°C agree with each other and decreased with increasing hydration with a deflection at h_c. For bone, similar hydration and temperature dependences were observed for ε and c. However, the dependence of d on hydration and temperature was different from that of decalcified bone, reflecting a two-phase structure consisting of collagen fibers and mineral hydroxyapatite. The critical hydration for bone was 0.04 g/g.     

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.     

Temperature dependence of dielectric constant at 10 GHz of Na-DNA gels

Permittivity ε′ and dielectric loss ε″ of aqueous Na-DNA gels have been measured at 10 GHz in the temperature interval ?15 to + 45°C. The experimental results are analyzed in terms of a three-component equation (Na-DNA, interfacial water, bulk water) and yield a value of 35 water molecules/nucleotide interacting with DNA. According to theoretical and experimental data the presence of strongly bonded and weakly bonded water is considered. The modified water exhibits a mean dielectric relaxation time two-or threefold greater than that of bulk water.     

The dielectric method of investigating bound water in biological material: An appraisal of the technique

Three independent dielectric methods for the measurement of water of hydration (bound water) in a biological material are described and discussed comparatively. For well-defined aqueous solutions of biological molecules, hydration can be obtained from direct observations made on the δ dispersion or from measurement of the dielectric values of the β dispersion. For whole tissue, however, neither of these two methods is applicable, and to deduce the hydration, it is necessary to use the third technique in which the volume of the hydrated biological particle is obtained by measuring the effect of it on the known dielectric properties of pure water. The hydration can then be calculated by deducting the volume of the anhydrous particle from the experimentally determined volume of the hydrated particle. Owing to possible systemmatic errors the uncertainty in the absolute hydration value associated with this technique is rather larger than that obtained with the other two dielectric methods. For studying the differences between hydration in similar tissues, however, this objection disappears.     

The molecular details of collagen hydration

Nuclear magnetic resonance and dielectric data on hydrated collagen are interpreted in terms of Ramachandran's hydration model. It is found that all data are compatible with this model, indicating two specific binding sites per three amino acids in the threefold collagen helix. Sorption data have been interpreted according to the multilayer theory of Guggenheim and used to derive the fraction of bound water in the primary sites. From magnetic resonance anisotropies structural details of the position of the water molecules can be derived under the assumption that both sites are equally occupied. The residence time of a water molecule in one of these sites in moderately hydrated collagen (45 g H₂O/100 g collagen) is 1.2 × 10^?6 sec. The remainder of the water is weakly bound and consists of rapidly exchanging species with rotational correlation time shorter than 10^?10 sec. The sites are 50% occupied at a water content of 10 g/100 g collagen and may contribute significantly to the stability of the collagen threefold helix.     

Proton transfer and polarizability of hydrogen bonds in proteins coupled with conformational changes. I. Infrared investigation of poly(glutamic acid) with various N Bases

The nature of hydrogen bonds formed between carboxylic acid residues and histidine residues in proteins is studied by ir spectroscopy. Poly(glutamic acid) [(Glu)_n] is investigated with various monomer N bases. The position of the proton transfer equilibrium OH…?N ? O^?…?H⁺N is determined considering the bands of the carboxylic group. It is shown that largely symmetrical double minimum energy surfaces are present in the OH…?N ? O^?…?H⁺N bonds when the pK_a of the protonated N base is two values larger than that of the carboxylic groups of (Glu)_n. Hence OH…?N ? O^?…?H⁺N bonds between glutamic and aspartic acid residues and histidine residues in proteins may be easily polarizable proton transfer hydrogen bonds. The polarizability of these bonds is one to two orders of magnitude larger than usual electron polarizabilities; therefore, these bonds strongly interact with their environment. It is demonstrated that water molecules shift these proton transfer equilibria in favor of the polar proton boundary structure. The access of water molecules to such bonds in proteins and therefore the position of this proton transfer equilibrium is dependent on conformation. The amide bands show that (Glu)_n is α-helical with all systems. The only exception is the (Glu)_n-n-propylamine system. When this system is hydrated (Glu)_n is α-helical, too. When it is dried, however, (Glu)_n forms antiparallel β-structure. This conformational transition, dependent on degree of hydration, is reversible. An excess of n-propylamine has the same effect on conformation as hydration.     

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.     

A high-resolution 13C-nmr study of collagenlike polypeptides and collagen fibrils in solid state studied by the cross-polarization–magic angle-spinning method. Manifestation of conformation-dependent 13C chemical shifts and application to conformational characterization

We have recorded high-resolution ¹³C-nmr spectra of collagen fibrils in the solid state by the cross-polarization–magic-angle-spinning(CP–MAS)method and analyzed the spectra with reference to those of collagenlike polypeptides. We used two kinds of model polypeptides to obtain reference ¹³C chemical shifts of major amino acid residues of collagen (Gly, Pro, Ala, and Hyp): the 3₁-helical polypeptides [(Gly)_nII, (Pro)_nII, (Hyp)_n, and (Ala? Gly? Gly)_nII], and the triple-helical polypeptides [(Pro? Gly? Pro)_n and (Pro? Ala? Gly)_n]. Examination of the ¹³C chemical shifts of these polypeptides, together with our previous data, showed that the ¹³C chemical shifts of individual amino acid residues are the same, within experimental error (±0.5 ppm), among different polypeptides with different primary sequences, if the conformations are the same. We found that the ¹³C chemical shifts of Ala residues of the 3₁-helical (Ala? Gly? Gly)_n and triple-helical (Pro? Ala? Gly)_n are significantly displaced, compared with those of the α-helix, β-sheet, and silk I form, and can be utilized as excellent probes to examine conformational features of collagen-like polypeptides. Further, the ¹³C chemical shifts of Gly and Pro residues in the triple-helical polypeptides are substantially displaced from those found in (Gly)_nII and (Pro)_nII of the 3₁-helix, reflecting further conformational change from the 3₁-helix to the supercoiled triple helix. In particular, the ¹³C chemical shifts of Gly C ? O carbons of the triple-helical polypeptides are substantially displaced upfield (4.1–5.1 ppm), with respect to those of the 3₁-helical polypeptides. These displacements are interpreted by that Gly C ? O of the former is not involved in NH …? O ? C hydrogen bonds, while this carbon of the latter is linked by these kinds of hydrogen bonds. On the basis of these ¹³C chemical shifts, as reference data for the collagenlike structure, we were able to assign the ¹³C-nmr peaks of Gly, Ala, Pro, and Hyp residues of collagen fibrils, which are in good agreement with the values expected from the model polypeptides mentioned above. We also discuss a plausible conformational change of collagen fibrils during denaturation.     

The conformational properties of α,β‐dehydroamino acids with a C‐terminal ester group

α,β‐Dehydroamino acid esters occur in nature. To investigate their conformational properties, a systematic theoretical analysis was performed on the model molecules Ac‐ΔXaa‐OMe [ΔXaa = ΔAla, (E)‐ΔAbu, (Z)‐ΔAbu, ΔVal] at the B3LYP/6‐311+ + G(d,p) level in the gas phase as well as in chloroform and water solutions with the self‐consistent reaction field‐polarisable continuum model method. The Fourier transform IR spectra in CCl₄ and CHCl₃ have been analysed as well as the analogous solid state conformations drawn from The Cambridge Structural Database. The ΔAla residue has a considerable tendency to adopt planar conformations C5 (?, ψ ≈ ? 180°, 180°) and β2 (?, ψ ≈ ? 180°, 0°), regardless of the environment. The ΔVal residue prefers the conformation β2 (?, ψ ≈ ? 120°, 0°) in a low polar environment, but the conformations α (?, ψ ≈ ? 55°, 35°) and β (?, ψ ≈ ? 55°, 145°) when the polarity increases. The ΔAbu residues reveal intermediate properties, but their conformational dispositions depend on configuration of the side chain of residue: (E)‐ΔAbu is similar to ΔAla, whereas (Z)‐ΔAbu to ΔVal. Results indicate that the low‐energy conformation β2 is the characteristic feature of dehydroamino acid esters. The studied molecules constitute conformational patterns for dehydroamino acid esters with various side chain substituents in either or both Z and E positions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Thermal properties of collagen in helical and random coiled states in the temperature range from 4° to 300°K

The low-temperature heat capacity of collagen (in the hydrated and dehydrated states) and the large entropy of collagen in the coiled state relative to the same protein in the helical state were investigated. The heat capacity for collagen in the solid state in the temperature range 4°–50° K changes proportionally to the square of temperature (C_p ～ T²). Above 50°K there is a linear dependence (C_p ～ T). The differences in the character of temperature dependence of heat capacity for the hydrated and dehydrated collagen show the importance of the specific interaction of water molecules with polypeptide chains of this protein. The peculiarities of the temperature dependence of the heat capacity difference (ΔC_p) of hydrated denatured (random coiled) and hydrated native (helical) collagen are observed at 15°, 120°, and 240°K. These differences are caused by the varying degree of ordering of the hydrate water molecules in native and denatured collagen macromolecules. At all temperatures (4°–300°K) the entropy of the random coiled state is higher than that of collagen in the native state and at 298°K ΔS = ∫ (ΔC_p/T)dT = 0.8 cal/100 g °K.     

Ageing of the human corneal stroma: structural and biochemical changes

High and low angle X-ray diffraction patterns from the corneal stroma give information about the mean intermolecular spacing of the collagen molecules and the mean interfibrillar spacing of the collagen fibrils, respectively. X-ray data were collected, using a high intensity synchrotron source, from human corneas and sclera at approximately physiological hydration. The spacings were measured as a function of tissue age. Between birth and 90 years there is an increase in the cross-sectional area associated with each molecule in corneal collagen from approx. 3.04 nm² to 3.46 nm², and an increase in scleral collagen from approx. 2.65 nm² to 3.19 nm². These changes may be due to an increase in the extent of non-enzymatic cross-linking between collagen molecules over the age range. We have investigated this possibility by measuring collagen glycation using the thiobarbituric acid assay and the subsequent advanced glycation end-products (AGEs) using fluorescence emission. The results obtained have shown an age-related increase in glycation and AGEs in both tissues. We have also demonstrated a decrease in the interfibrillar spacing of corneal collagen with increasing age which may be related to changes in the proteoglycan composition of the interfibrillar matrix.     

The role of water in the crystal structure of N-acetyl-L-4-hydroxyproline

The crystal structure of N-acetyl-L -4-hydroxyproline (Hyp) was determined by direct methods. (The crystal is orthorhombic with the space group P2₁2₁2₁.) The acetyl group is in the trans conformation and the pyrrolidine ring puckers at C^γ (C^sC^γ envelope), as in most Hyp residues. According to the rotation angle ψ = ?30°, the N-acetyl-L -4Hyp has the same conformation as an α-helix of prolyl residues. The crystal packing is stabilized by hydrogen bonds between three different molecules and the same molecule of water. One of the water bridges involves the carbonyl of the N-acetyl group of one molecule and the hydrogen atom of the 4-OH group of another. Such an arrangement has been proposed to explain the high stability of (Gly-L -Pro-L -4Hyp)_n. A second bridge involves the two hydrogens of the water molecule and the carbonyl groups of two neighbouring molecules, as already proposed in a dihydrated model of collagen. These experimental features, which are discussed in relation to the different models of collagen, allow us to propose an hypothetical arrangement for the water molecule which is strongly retained in the triple helix of (Gly-L -Pro-L -4Hyp)_n.     

Molecular configuration of amylose and its complexes in aqueous solutions. Part IV. Determination of DP of amylose by measuring the concentration of free iodine in solution of amylose–iodine complex

In aqueous solutions of the amylase–iodine complex the concentration of free iodine [I_f]_v after reaching equilibrium (or closely approximating it) is determined by the following factors: temperature, pH, concentration of iodide ions and amylose, and DP of amylose. In the present paper the role of temperature, amylose concentration, and DP has been investigated. At half-saturation of amylose by iodine, the reciprocal value of free iodine defines the equilibrium constant: 1/[I_f]_v = K. The relation between [I_f]_v, in normality and temperature is the following: 5 + log [I_f]_v = ?(2.132/T) + 8.52, for DP _n = 1290, 0.4 mg. amylose in 100 ml. 0.1N HCl. The value of the energy of activation E_a between 2 and 52°C. is 9.72 kcal./mole. The influence of amylose concentration [Am] on photometrically determined [I_f]_v, at 20°C, in the range of 0.1–1.2 mg./100 ml. 0.1 N HCl for DP _n = 1290 is: 5 + log [I_f]_v = 0.209 ? 0.047 log [Am]. At [Am] = 0.6 mg. amylose/ 100 ml. 0.1 N HCl and 20°C, the value of [I_f]_v depends on DP _n as follows: 5 + log [I_f]_v = 0.085 = + 0.222 log (10⁴/DP _n). These above equations are summarized by the relation: [I_f]_v = exp {16.865 ? (E_a/RT)}[Am]^0.047(10⁴/DP _n)^0.222 ×10^?5 Considering that the determination of [I_f]_v by automatic photometric titration can be performed quickly and with appropriate reproducibility, this method is convenient for a rapid empirical and approximate determination of DP of amylose on a microscale. The iodine-binding capacity [IBC] as well as the value of λ_max, have been also investigated as functions of DP _n, by photometric and by amperometric titration.     

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.     

On the electric polarization of DNA

Dielectric dispersion of DNA was studied in the frequency range 100 Hz–100 kHz at four different temperatures (6–30°C). The dielectric increment ε₀–ε_∞ increased with the rise of temperature. The relaxation time, on the other hand, decreased. Both the increase in dielectric increment and the decrease in relaxation time could not be explained on the basis of the counterion polarization theory. Dipole moment was estimated from Kirkwood theory. It was found to decrease systematically with temperature. Even at 0°C there was a dipole moment of 10⁴D.     

Theoretical π-π* circular dichroic spectra of helical poly(glycine) and poly(L-alanine) as functions of backbone torsion angles

Circular dichroic spectra and oscillator strengths of the π-π transition near 190 nm are calculated for helical (Gly)₆ and (Ala)₆ at 30° intervals of the backbone torsion angles (?,ψ) over the range -180° ≤ ? ≤ -60°, ?60° ≤ ψ ≤ 180°, using the partially dispersive normal mode treatment of the dipole interaction model. Polarizabilities of atoms and the NC′O group are those determined semiempirically in previous studies. Calculations for (Ala)₆ at (?,ψ) angles corresponding to the α-helix, the poly(Pro) II helix, a collagen single helix, a poly-(MeAla) helix, and single β-helices are found to agree well with most of the available experimental data.     

Implementation of subcellular water mapping by electron energy loss spectroscopy in a medium-voltage scanning transmission electron microscope

The water concentration in biological cells plays a predominant role in cellular life. Using electron energy loss spectroscopy, the feasibility to measure the water content in cells has already been demonstrated. In this paper, we present an upgrade of water measurement in hydrated cryosections by spectrum imaging mode in a medium-voltage scanning transmission electron microscope. The electron energy loss spectra are recorded in spectrum imaging mode in a 2ⁿ×2ⁿ pixels array. Each spectrum is processed in order to determine the water mass content in the corresponding pixel. Then a parametric image is obtained in which grey levels are related to water concentration. In this image, it is possible to recognize the different subcellular compartments. By averaging the water concentration over the relevant pixels, we can determine the water mass content in the concerned subcellular compartment. As an example, we present water mass content measurement at subcellular level in rat hepatocytes.     

Enzyme cascade converting cyclohexanol into ε-caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli

The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity V_MAX(app) for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml⁻¹. For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a V_MAX(app) of 491 mU ml⁻¹ was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD_578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.     

Structure and Hypotensive Effect of Flavonoid Glycosides in Kinkan (Fortunella japonica) Peelings

An attempt was made to isolate the hypotensive substances from a hot water extract of kinkan. Eight flavonoid glycosides were isolated by repeated chromatography and by gel filtration after extracting with n-butanol and treating with lead subacetate. Their structures were established to be 6,8-di-C-glucosylapigenin (1), 3,6-di-C-glucosylacacetin (2), 2″-O-α-l-rhamnosyl-4′-O-methyl-vitexin (3), 2″-O-α-l-rhamnosyl-4′-O-methylisovitexin (4), 2″-O-α-l-rhamnosylvitexin (5), 2″-O-α-l-rhamnosylorientin (6), 2″-O-α-l-rhamnosyl-4′-O-methylorientin (7) and ponicilin (8) by UV. MS, ¹-NMR and ¹³C-NMR spectroscopy, and by sugar analysis. Each component was intravenously injected in SHR-SP (0.5 ~ 1.0 mg/100 g of body weight), 1, 2, 5 and 6 were found to lower the rat blood pressure.

Among these compounds, 2, 3, 4, 6 and 7 were new flavone glycosides.