Effect of pH on collagen flexibility determined from dilute solution viscoelastic measurements

The frequency dependences of the storage and loss shear moduli, G′ and G″ of dilute solutions of collagen at pH 7.4, ionic strength approximately 0.2, were measured at 15.0°C by the Birnboim—Schrag multiple-lumped resonator apparatus. By use of two solvents, water and 50% glycerol, the effective reduced frequency range was extended to cover 2.5 logarithmic decades. The intrinsic viscosity and longest (rotational) relaxation time were considerably smaller than those determined at pH 4.0 in an earlier study. At pH 4.0, the behaviour could be modelled by a rodlike molecule with partial flexibility along its entire length and a persistence length of 161 nm with no loose joints. The behaviour at pH 7.4 corresponds approximately to the expectation for a semiflexible rod with two loose joints near the ends and a similar persistence length (169nm) for the centre segment.     

Separation of l-lysine from dilute aqueous solution using molecular imprinting technique

In the present study, separation of l-lysine from dilute aqueous solution by solid-phase extraction based on molecular imprinting technique using a polar porogen was investigated. l-Lysine imprinted polymer (LLIP) was prepared by free radical solution polymerization of methacrylic acid and ethylene glycol dimethacrylate as functional and cross-linking monomers, in the presence of l-lysine as an imprint molecule, mixture of water and methanol as solvent and AIBN as an initiator. Non-imprinted polymer (NIP) as control was also prepared by the same procedure in the absence of template molecules. LLIP particles were applied to determine the optimum operational condition for l-lysine separation from dilute aqueous solution. In adsorption step, optimum pH and retention time were 7.8 and 90 min, while corresponding values in extraction step were 12 and 50 min, respectively. l and d-Lysine recovery by LLIP at optimum condition were found to be 96 and 58% with corresponding distribution coefficients of 8000 and 460, respectively. The retention capacity of LLIP was 27.26 mg l-lys/g of polymer at optimum condition.     

Crystallization of salt-free chymotrypsinogen and chymotrypsin from solution in dilute ethyl alcohol

Chymotrypsinogen and chymotrypsin crystallize readily from dilute solutions of ethyl alcohol in the absence of salts. The crystals formed in the presence of alcohol differ in appearance from those formed in the presence of ammonium sulfate. Chymotrypsinogen yields well formed polyhedrons instead of fine needles usually produced in ammonium sulfate solution. Chymotrypsin yields fine needles in the presence of alcohol and rhombohedrons in the presence of ammonium sulfate. The enzymatic properties of the crystals formed in the presence of alcohol are identical with those of the crystals isolated in the presence of ammonium sulfate.     

One-dimensional viscoelastic behavior of fibroblast populated collagen matrices

Bio-artificial tissues are being developed as replacements for damaged biologic tissues. Their mechanical properties are critical for load bearing applications. Current testing protocols for bio-artificial tissues vary widely and often do not consider viscoelasticity. Uniaxial stretch tests were performed on fibroblast populated collagen matrices (FPCMs) to determine the influence of specific test protocols on the mechanical behavior. The peak force, hysteresis and shape of the force-stretch curve are affected by the stretch rate, rest period, stretch amplitude and the number and magnitude of preconditioning cycles.     

Modelling canopy photosynthesis using parameters determined from simple non-destructive measurements

Most models for canopy photosynthesis require a large number of parameters as input which have to be determined by means of direct measurements. Such measurements are usually expensive, time consuming and destructive. The objective of the present study was, therefore, to develop a simple but accurate canopy photosynthesis model based on a minimum number of parameters that can be determined non-destructively. The results from previous studies were used to derive an empirical expression which describes the variation in leaf photosynthetic capacity (P_m) as a function of the light distribution in the canopy. The light distribution itself was calculated with a simple model which assumes only three leaf angle classes (0–30°, 30–60° and 60–90°). The leaf area index was determined indirectly from measurements of direct radiation below the canopy. The result was a model for canopy photosynthesis that requires only a few parameters. These parameters are the leaf photosynthetic capacity at the top of the canopy, the relative frequency of leaves in each of the three leaf angle classes, and the fraction of direct radiation below the canopy. Each of these parameters can be determined by means of simple non-destructive measurements. The model was applied to dense stands of two monocotyledonous species: rice (Oryza sativa L.) and pearl millet (Pennisetum americanum (L.) K. Schum.). The rates of canopy photosynthesis thus calculated were compared to those obtained with a more elaborate reference model. The differences between the values obtained with the two models were small. The present photosynthesis model can, therefore, be considered to be a suitable alternative for the more elaborate model. It was further discussed that, since the model is based on purely non-destructive measurements, it will be particularly useful in cases where it is required to estimate canopy photosynthesis at regular intervals over a length of time or in stands of vegetation that cover large areas of land.     

The thickness of monoolein lipid bilayers as determined from reflectance measurements

Measurements of the reflectance of monoolein $\text{n-}\text{alkane}$ and monoolein/squalene lipid bilayers have been made. The total thickness of the bilayer was calculated from the dependence of reflectance on the refractive index of the aqueous salt or sucrose solution surrounding the bilayer. The total thickness was then compared to the thickness of the hydrocarbon chain region as determined from capacitance measurements. From this comparison, we found that the thickness of each polar region of the bilayers in salt solutions was $\text{0.5 ± 0.1}\text{nm}$, independent of the hydrocarbon solvent used. When the aqueous solutions contained sucrose, each polar region was approx. 0.9 nm thick. When $\text{n-}\text{tetradecane}$ and $\text{n-}\text{hexadecane}$ were used as solvents, microlenses of solvent trapped in the monoolein bilayer increased the reflectance. After about one hour, the coalescence of microlenses into larger lenses allowed the reflectance of the bilayer alone to be measured. The use of reflectance to measure the thickness of monoolein bilayers appears to be consistent with other methods and to give useful information about the structure of lipid bilayers.     

Proton flux measurements from tissues in buffered solution

Proton movement across plant cell membranes is part of many important physiological processes. The net proton flux to or from tissues can be determined non-invasively by measuring the proton electrochemical potential gradient in the adjacent solution. In buffered solution, some of the protons crossing the tissue boundary diffuse as proto-nated buffer whose flux is not included in the flux calculated from the proton (hydrogen ion) electrochemical gradient. In this theoretical paper, it is shown how experimenters can calculate the protonated buffer flux from the measured proton flux in solution. The ratio of these two components of total proton flux depends on the pH of the solution and on the concentration and pK of the buffer. For a given concentration of a buffer which has a single pK, the flux ratio rises with pH when the solution pH is lower than the buffer pK. The slope is about 2 on a log₁₀ scale. As the pH increases above the pK, the flux ratio levels off to approach its maximum. With mixed buffers, or one having two or more pK values, the flux ratios are additive: each buffer acts independently based on its concentration and its pK value. Unbuffered solutions always have the buffering effects of water itself and also of carbonates due to carbon dioxide dissolved from the atmosphere. In unbuffered solutions at pH 6, the flux carried by water and carbonate is about 1 % of the measured proton flux. This validates measurements of proton flux from tissues, made by a number of workers, in unbuffered solutions below pH 6.     

Flexibility in the solution structure of human tropoelastin

We investigated the flexibility of full-length tropoelastin in solution by using far- and near-ultraviolet circular dichroism (UV CD) and fluorescence spectroscopy to probe for structural flexibility and residue mobility within secondary and tertiary features of the monomer. Fluorescence spectroscopy revealed the presence of exposed hydrophobicity through the binding of the hydrophobic probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (bis-ANS), which demonstrates that hydrophobic regions form clusters and are not confined to a molecular core. Near-UV CD indicated substantial mobility of aromatic residues. Structural prediction programs (PONDR, DisEMBL, and Globplot version 2.0) estimated 75 +/- 2% disorder in the tertiary structure of tropoelastin on the basis of primary sequence information. A single-site substitution of Trp for Gln (Q513W) at the tropoelastin domain 25-26 interface facilitated fluorescence spectroscopy for revealing that this region is exposed to solvent. Polarization anisotropy demonstrated substantial flexibility of W513 and little change upon denaturation of the monomer with guanidine hydrochloride. Comparable movement was found for native sequence aromatic residues in the presence of glycosaminoglycans and trifluoroethanol. These data prove the intrinsic flexibility of specific residues and adjacent sequences in any native conformation(s) they may take. This study is the first characterization of the level of mobility in defined regions of the full-length tropoelastin monomer and provides direct evidence for regions of flexible structure in tropoelastin.     

The structure of gellan in dilute aqueous solution

A full assignment of high-field nmr spectra of gellan was obtained in dilute aqueous solution by performing a series of selective one-dimensional nmr experiments. The observed nuclear Overhauser effects (NOEs) cannot be interpreted assuming that each sugar residue is intrinsically rigid and in a chair conformation. In fact, the rhamnose residue gives strong NOE contacts coherent only with an equilibrium involving both a chair as well as a boat (or a hemiboat) conformation. Molecular dynamic calculations performed on a heptamer with a central rhamnose support the above finding, and show a structure based on a very stiff single chain in which it is present a flipping of the rhamnose residue. At low temperatures (5-20 degrees C) in very dilute solutions (0.018 mg/mL) nmr spectra show a splitting of the resonance due to the methyl group of rhamnose residue, thus confirming the presence of a slow equilibrium among different conformers.     

Polypeptide-assisted oligomerization of analogs in dilute aqueous solution

Poly (Leu-Lys) was shown to assist the oligomerization of activated nucleotide diphosphates. Short oligomers of pdGp are formed in dilute solution. Activated oligomers can complex to the polypeptide and polymerize to form longer oligomers. Oligomers up to the 18-mer can be obtained under these conditions from 1 × 10^–3 M ImpdGpIm in the absence of a preformed polynucleotide template. The total yield of polymerization remains limited although 50% more pyrophosphate bonds are formed in the presence of polypeptide. However, the elongation effect is more significant since the yield of oligomers longer than the decamer is increased by a factor of 60. The possible prebiotic implications of these experiments are discussed.Abbreviations pdGp the 3,5-bisphosphate of dG - ImpdGpIm the 3,5-bisphosphoimidazolide of dG - KEDTA ethylenediamine tetraacetic acid, potassium salt - Bis-Tris bis(2-hydroxymethyl)iminotris(hydroxymethyl)methane Correspondence to: B. Barbier     

Electrical properties of rabbit corneal endothelium as determined from impedance measurements.

Alternating- and direct-current electrical characteristics of rabbit corneal endothelium were studied under varying experimental conditions. The measurements were performed by sending a 10-microA current (AC or DC) across the tissue layer. Maximal values of transendothelial potential difference and resistance were 1.3 +/- 0.1 mV and 73 +/- 6 omega . cm2, respectively. The short-circuit current was estimated from the potential and resistance values. Impedance loci were obtained for the frequency range 0.5-100 kHz. A capacitive reactance (C = 0.63 +/- 0.02 microF/cm2) was observed in the 100 Hz-100 kHz range. To relate the impedance data to the electrical parameters of the cell membranes, the voltage-divider ratio was determined by sending square pulse across the tissue and measuring voltage responses across the apical and basal membranes with an intracellular microelectrode. The intracellular potential difference was on the average -61 +/- 1 mV, and the voltage-divider ratio was found to be between 0.33 and 4. Impedance data were fit by a computer to an equivalent circuit representing a "lumped" model, and the agreement between the model and the data was satisfactory. The results are discussed in terms of both the morphological characteristics and properties of the fluid transport mechanism across the preparation.     

Sulfhydryl oxidation of reduced insulin in dilute solution

The reduction of insulin by tri-n-butylphosphine followed by air oxidation in dilute solution at pH 9.1 yields A- and B-chain disulfides. A(S-S)2 and B(S-S) have been purified on SP-Sephadex C-25 using a linear gradient of sodium chloride from 0.1 to 0.45 M in 0.5 M acetic acid containing 7 M urea. The overall yield of A(S-S)2 was 70%; and B(S-S), 60%. The A(S-S)2 and B(S-S) had the expected amino acid composition and N-terminal amino acid. The kinetics of reduction and reoxidation of insulin disulfide bonds are discussed.     

Flexibility of type I collagen and mechanical property of connective tissue

The hierarchical organization of the connective tissue, more specifically, the musculoskeletal soft tissue, has been extensively studied. With advancements in experimental methodology, investigation of the structure-function relationship has provided more insight into how the mechanical integrity of the tissue is created. Such information is essential in the linking the macroscopic loading environment of the tissue to the microscopic level of the tissue to be experienced by the cell. The flexibility and elastic modulus of gross connective tissue, the fascicle, the fiber and then the collagen molecule are compared based on the data available in the literature.     

Mixed solvent systems for recovery of ethanol from dilute aqueous solution by liquid-liquid extraction

Distribution coefficients and selectivities of a number of mixed solvent systems have been determined in order to assess their suitability in preferentially extracting ethanol from aqueous solution. The measured values of distribution coefficients and selectivities differ substantially from the values estimated by interpolating between the pure solvents.     

Chain stiffness of heteropolysaccharide from Aeromonas gum in dilute solution by dynamic light scattering

Dynamic light scattering measurements have been made on 15 fractions of aeromonas (A) gum, an extracellular heteropolysaccharide produced by the strain Aeromonas nichidenii, with dimethylsulfoxide containing 0.2M lithium chloride as the solvent at 25 degrees C. Data for the translational diffusion coefficient D covering a molecular weight range from 4.5 x 10(5) to 2.1 x 10(6) and ratios of the z-average radius of gyration (z) (1/2) to the hydrodynamic radius R(H) (calculated with previous (z) data) suggest that the polymer behaves like a semiflexible chain in this solvent similar to the stiffness of cellulose derivatives. Thus the D data are analyzed on the basis of the Yamakawa-Fujii theory for the translational friction coefficient of a wormlike cylinder by coarse-graining the heteropolysaccharide molecule. Excluded-volume effects are taken into account in the quasi-two-parameter scheme, as was done previously for (z) and [eta] (the intrinsic viscosity) of A gum in the same solvent. The molecular weight dependence of R(H) is found to be explained by the perturbed wormlike chain with a persistence length of 10 nm, a linear mass density of 1350 nm(-1), an excluded-volume strength parameter of 1.3 nm, and a chain diameter of 2.8 nm. These parameters are in substantial agreement with those estimated previously from (z) and [eta] data, demonstrating that the solution properties (D, (z), and [eta]) of the heteropolysaccharide are almost quantitatively described by the current theories for wormlike chains in the molecular weight range studied.     

Linear viscoelastic model of a maturing gelatin solution

The linear viscoelastic model proposed in this work considers the viscoelastic nature of maturing gelatin solutions through a relaxation modulus that depends on temperature and maturation. This modulus is defined in the conceptual contexts of the classical rubber elasticity theory and the rheometric gel theory. An analysis of the relationship between the equilibrium elastic modulus and the percolation variable around the gel point is also included yielding a percolation exponent close to 1.7 as expected from previous theoretical predictions. Additionally, a simple kinetic model is proposed to follow the microstructural changes obtained as a consequence of the generation of junction zones, the number of which vary with time during the dynamic rheometric tests used in this work. Thus, the storage and loss moduli are measured at different temperatures and frequencies, during the period of gelatin maturation. The theoretical aspects of the rheological model are presented emphasizing the quantitative changes of rheological parameters with the maturation.     

Persistence length of collagen molecules based on nonlocal viscoelastic model

Persistence length is one of the most interesting properties of a molecular chain, which is used to describe the stiffness of a molecule. The experimentally measured values of the persistence length of the collagen molecule are widely scattered from 14 to 180 nm. Therefore, an alternative approach is highly desirable to predict the persistence length of a molecule and also to explain the experimental results. In this paper, a nonlocal viscoelastic model is developed to obtain the persistence length of the collagen molecules in solvent. A new explicit formula is proposed for the persistence length of the molecule with the consideration of the small-scale effect, viscoelastic properties of the molecule, loading frequency, and viscosity of the solvent. The presented model indicates that there exists a range of molecule lengths in which the persistence length strongly depends on the frequency and spatial mode of applied loads, small-scale effect, and viscoelastic properties of the collagen.