Changes in the physicochemical characteristics of rabbit sclera upon scleral reinforcement

Biochemical analysis and differential scanning calorimetry demonstrated that the connective tissue (capsule) formed around a reinforcing scleroplastic implant is similar to intact sclera, its main component being type I collagen organized in perfect fibrils with cross-linking sufficient for normal thermomechanical properties. DSC also revealed a fraction of collagen with heat-labile ‘immature’ cross-links around implants containing a stimulatory plant product Panaxel, which suggested high synthetic activity of fibroblasts.     

Stability of Collagen During Denaturation

The stability of calf skin collagen (CSC) type I during thermal and chemical denaturation in the presence of glycerol was investigated. Thermal denaturation of type I collagen was performed in the presence of glycerol or in combination with urea and sodium chloride. The denaturation curves obtained in the presence of urea or sodium chloride retained their original shape without glycerol. These curves were shifted upward proportionally to the glycerol concentration in the reaction medium. This means that glycerol and the denaturants act independently. The explanation is based on the difference in the mechanism of their action on the collagen molecule.     

Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides

The thermal unfolding of the catabolite activator protein (CAP) of Escherichia coli and the complexes it forms with adenosine cyclic 3',5'-phosphate (cAMP) and guanosine cyclic 3',5'-phosphate (cGMP) was studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of CAP at pH 7.00 gave an irreversible, symmetrical denaturation curve with a single peak. Distinctly different, more complex DSC curves were obtained for the thermal denaturation of the cAMP-protein and cGMP-protein complexes. The DSC data indicate intermolecular cooperation among CAP dimers, with the extent of oligomerization remaining unchanged during unfolding of the protein. The DSC curves for the thermal denaturation of the cAMP-protein complex and cGMP-protein complex have been resolved into three and two components, respectively, according to the model of independent two-state processes. Analysis of the DSC data suggests two and three independent domains for cGMP-protein and cAMP-protein complexes, respectively, with dissociation of mononucleotide occurring in the second component in both cases during protein denaturation. Furthermore, our studies indicate that the presence of either ligand alters the degree of oligomerization of CAP dimers, cAMP having a greater effect than cGMP.     

Structural characterization of pyrrolic cross-links in collagen using a biotinylated Ehrlich's reagent

The structures of pyrrolic forms of cross-links in collagen have been confirmed by reacting collagen peptides with a biotinylated Ehrlich's reagent. This reagent was synthesized by converting the cyano group of N-methyl-N-cyanoethyl-4-aminobenzaldehyde to a carboxylic acid, followed by conjugation with biotin pentyl-amine. Derivatization of peptides from bone collagen both stabilized the pyrroles and facilitated selective isolation of the pyrrole-containing peptides using a monomeric avidin column. Reactivity of the biotinylated reagent with collagen peptides was similar to that of the standard Ehrlich reagent, but heat denaturation of the tissue before enzyme digestion resulted in the loss of about 50% of the pyrrole cross-links. Identification of a series of peptides by mass spectrometry confirmed the presence of derivatized pyrrole structures combined with between 1 and 16 amino acid residues. Almost all of the pyrrole-containing peptides appeared to be derived from N-terminal telopeptide sequences, and the nonhydroxylated (lysine-derived) form predominated over pyrrole cross-links derived from helical hydroxylysine.     

Effect of thermal denaturation on water–collagen interactions: NMR relaxation and differential scanning calorimetry analysis

The dependence of the proton spin–lattice relaxation rate, and of the enthalpy and temperature of denaturation on water content, were studied by nmr and differential scanning calorimetry (DSC) in native and denatured collagen. Collagen was first heated at four different temperatures ranging from 40 to 70°C. The percentage of denatured collagen induced by these preheating treatments was determined from DSC measurements. The DSC results are discussed in terms of heat‐induced structural changes. A two‐exponential behavior for the spin–lattice relaxation was observed with the appearance of denatured collagen. This was attributed to the presence of a noncollagen protein fraction. The variations in the different longitudinal relaxation rates as a function of the moisture content and of the denatured collagen percentage are described within the multiphase water proton exchange model. This study highlights the complementarity of the information obtained from the two analytical tools used. © 1999 John Wiley & Sons, Inc. Biopoly 50: 690–696, 1999     

Failure of mineralized collagen fibrils: modeling the role of collagen cross-linking

Experimental evidence demonstrates that collagen cross-linking in bone tissue significantly influences its deformation and failure behavior yet difficulties exist in determining the independent biomechanical effects of collagen cross-linking using in vitro and in vivo experiments. The aim of this study is to use a nano-scale composite material model of mineral and collagen to determine the independent roles of enzymatic and non-enzymatic cross-linking on the mechanical behavior of a mineralized collagen fibril. Stress–strain curves were obtained under tensile loading conditions without any collagen cross-links, with only enzymatic cross-links (modeled by cross-linking the end terminal position of each collagen domain), or with only non-enzymatic cross-links (modeled by random placement of cross-links within the collagen–collagen interfaces). Our results show enzymatic collagen cross-links have minimal effect on the predicted stress–strain curve and produce a ductile material that fails through debonding of the mineral–collagen interface. Conversely, non-enzymatic cross-links significantly alter the predicted stress–strain response by inhibiting collagen sliding. This inhibition leads to greater load transfer to the mineral, which minimally affects the predicted stress, increases modulus and decreases post-yield strain and toughness. As a consequence the toughness of bone that has more non-enzymatically mediated collagen cross-links will be drastically reduced.     

Calorimetric study of the mechanism of uncoiling the collagen molecule in the presence of ions

Calorimetric and polarimetric studies of partially denaturated collagen in the presence and absence of one and two valence ions were performed. It is shown that in the presence of salt in partial denaturation of renaturated collagen the denaturation enthalpy is changed upon two steps. Both stages are followed by the conformational alteration. The denaturation enthalpy of the renaturated collagen is less than that of the native one, but in the presence of salts it is rather higher than in its absence. The study made possible to indicate that two-stage alteration of denaturation enthalpy is occurrence of denaturation products with partially reconstructed structure in which the salts take an active part.     

Thermal unfolding of ribonuclease A in phosphate at neutral pH: deviations from the two-state model

The thermal denaturation of ribonuclease A (RNase A) in the presence of phosphate at neutral pH was studied by differential scanning calorimetry (DSC) and a combination of optical spectroscopic techniques to probe the existence of intermediate states. Fourier transform infrared (FTIR) spectra of the amide I' band and far-uv circular dichroism (CD) spectra were used to monitor changes in the secondary structure. Changes in the tertiary structure were monitored by near-uv CD. Spectral bandshape changes with change in temperature were analyzed using factor analysis. The global unfolding curves obtained from DSC confirmed that structural changes occur in the molecule before the main thermal denaturation transition. The analysis of the far-uv CD and FTIR spectra showed that these lower temperature-induced modifications occur in the secondary structure. No pretransition changes in the tertiary structure (near-uv CD) were observed. The initial changes observed in far-uv CD were attributed to the fraying of the helical segments, which would explain the loss of spectral intensity with almost no modification of spectral bandshape. Separate analyses of different regions of the FTIR amide I' band indicate that, in addition to alpha-helix, part of the pretransitional change also occurs in the beta-strands.     

A mechanistic analysis of the increase in the thermal stability of proteins in aqueous carboxylic acid salt solutions

The stability of proteins is known to be affected significantly in the presence of high concentration of salts and is highly pH dependent. Extensive studies have been carried out on the stability of proteins in the presence of simple electrolytes and evaluated in terms of preferential interactions and increase in the surface tension of the medium. We have carried out an in-depth study of the effects of a series of carboxylic acid salts: ethylene diamine tetra acetate, butane tetra carboxylate, propane tricarballylate, citrate, succinate, tartarate, malonate, and gluconate on the thermal stability of five different proteins that vary in their physico-chemical properties: RNase A, cytochrome c, trypsin inhibitor, myoglobin, and lysozyme. Surface tension measurements of aqueous solutions of the salts indicate an increase in the surface tension of the medium that is very strongly correlated with the increase in the thermal stability of proteins. There is also a linear correlation of the increase in thermal stability with the number of carboxylic groups in the salt. Thermal stability has been found to increase by as much as 22 C at 1 M concentration of salt. Such a high thermal stability at identical concentrations has not been reported before. The differences in the heat capacities of denaturation, deltaCp for RNase A, deduced from the transition curves obtained in the presence of varying concentrations of GdmCl and that of carboxylic acid salts as a function of pH, indicate that the nature of the solvent medium and its interactions with the two end states of the protein control the thermodynamics of protein denaturation. Among the physico-chemical properties of proteins, there seems to be an interplay of the hydrophobic and electrostatic interactions that lead to an overall stabilizing effect. Increase in surface free energy of the solvent medium upon addition of the carboxylic acid salts appears to be the dominant factor in governing the thermal stability of proteins.     

Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant.

The stabilities of Bacillus circulans xylanase and a disulfide-bridge-containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second heating cycle, indicating reversible denaturation occurs under those conditions. However, even for these reversible processes, the DSC curves for the wild-type protein showed a scan-rate dependence that was similar to that in the absence of urea. Calorimetric thermograms for the disulfide mutant were significantly less scan-rate dependent in the presence of urea than in the urea-free buffer. The present data show that, just as for irreversible transitions, the apparent transition temperature for the reversible denaturation of proteins can be scan-rate dependent, confirming the prediction of Lepock et al. (Lepock JR, Rithcie KP, Kolios MC, Rodahl AM, Heinz KA, Kruuf J, 1992, Biochemistry 31:12706-12712). The kinetic factors responsible for scan-rate dependence may lead to significant distortions and asymmetry of endotherms, especially at higher scanning rates. This points to the need to check for scan-rate dependence, even in the case of reversible denaturation, before any attempt is made to analyze asymmetric DSC curves by standard thermodynamic procedures. Experiments with the disulfide-bridge-containing mutant indicate that the introduction of the disulfide bond provides additional stabilization of xylanase by changing the rate-limiting step on the thermal denaturation pathway.     

Cross-linking in type IV collagen.

Type IV collagen could not be extracted from human placenta using 6M-urea containing 10mM-dithiothreitol, indicating that the type IV molecule is stabilized within the basement membrane by covalent cross-links. However, various forms of type IV collagen molecule were extractable by means of increasingly severe proteolytic conditions. Type IV collagen tetramers ('spiders') lacking only the C-terminal globular region (NC1) were further digested to the 'long-form' 7S fragment and an assortment of helical rod-like molecules derived from the 'leg' region. These molecules were separated by salt fractionation and examined by rotary-shadowing electron microscopy. Isolation of these fractions from placenta which had been reduced with NaB3H4 revealed that both the 7S (N-terminal) and C-terminal regions contained significant proportions of reducible lysine-derived cross-links. These cross-links were shown to be exclusively the stable oxo-imine hydroxylysino-5-oxonorleucine. The number of the cross-links per molecule was significantly lower than might be expected in order to fully stabilize the molecule. These results suggest that the keto-imine cross-links in type IV collagen have been stabilized in part by conversion into an unknown non-reducible form, although a sensitive immunoassay failed to show the presence of any pyridinoline. Comparison with the fibrous collagen from placenta suggested that the rate of this conversion is much greater in basement-membrane collagen.     

Analysis of elastic and surface tension effects in the lung alveolus using finite element methods

Displacement method finite element theory is used to examine the structural and elastic properties of the constituent network of elastin and collagen of the alveoli that form the mammalian lung. The role of the surface tension of pulmonary surfactant of the lung is also examined using an area-dependent relationship inferred from experimental studies. The pressure-volume (PV) curves of the resulting model are found to compare favourably with measured pressure-volume curves for whole lungs filled with air (surface tension included) and saline (no surface tension effects).     

The thermal behavior of collagen in solution: effect of glycerol and 2-propanol

The thermal stability of different solutions of collagen (Col), collagen mixed with glycerol (Col-G) and collagen mixed with 2-propanol (Col-P) was studied by differential scanning calorimetry (DSC), viscosity and fluorescence. The DSC and viscosity methods showed that glycerol increased the denaturation temperature of collagen about 2°C, while 2-propanol decreased it about 2°C. The values of intrinsic viscosity ([η]) for Col, Col-G and Col-P were 21.67, 20.20 and 24.71 dl/g, respectively. Huggins coefficient (k(H)) increased in the presence of glycerol and decreased in the presence of 2-propanol. It was suggested that glycerol promoted the dissolution of collagen molecular aggregates while 2-propanol enhanced the aggregation. Fluorescence spectra were investigated within the temperature ranging from 15 to 45°C. By comparing the sign of peaks in the two-dimensional (2D) fluorescence correlation maps, the orders of peak response were ～360, ～410>297 nm for Col and Col-G, and 297>～360, ～410 nm for Col-P, respectively. These indicated that the respondences of tyrosine residues, excimer-like species and bityrosine on the perturbation of temperature were different in the presence of glycerol and 2-propanol.     

Differential Scanning Calorimetrie Study of the Thermal Denaturation of Almond β-Glucosidase

The thermal denaturation of almond β-glucosidase [EC 3.2.1.21] was studied by differential scanning calorimetry. The shape of the DSC trace was highly dependent on pH; two peaks were observed between pH 6–8, but only one peak between pH 4–5. All of the DSC curves were resolved into three components according to the model of independent two-state processes, and the thermodynamic parameters for the denaturation were evaluated. The dependence of the shape of DSC curves was accounted for mainly by the rapid changes of denaturation enthalpy and denaturation temperature of the third component in the acidic pH region.     

Flow injection analysis with diode array absorbance detection and dynamic surface tension detection for studying denaturation and surface activity of globular proteins

In this article, a multidimensional dynamic surface tension detector (DSTD), in a parallel configuration with a UV-visible diode array absorbance detector, is presented in a novel flow injection analysis (FIA) application to study the effects of chemical denaturants urea, guanidinium hydrochloride (GdmHCl), and guanidinium thyocyanate (GdmSCN) on the surface activity of globular proteins at the liquid-air interface. The DSTD signal is obtained by measuring the changing pressure across the liquid-air interface of 4-mul drops repeatedly forming at the end of a capillary using FIA. The sensitivity and selectivity of the DSTD signal is related to the surface-active protein concentration in aqueous solution combined with the thermodynamics and kinetics of protein interaction at a liquid-air drop interface. Rapid on-line calibration and measurement of dynamic surface tension is applied, with the surface tension converted into surface pressure results. Continuous surface tension measurement throughout the entire drop growth is achieved, providing insight into kinetic behavior of protein interactive processes at the liquid-air drop interface. Specifically, chemical denaturation of 12 commercial globular proteins-chicken egg albumin, bovine serum albumin, human serum albumin, alpha-lactalbumin (alpha-Lac), myoglobin, cytochrome c, hemoglobin, carbonic anhydrase, alpha-chymotrypsinogen A, beta-lactoglobulin (beta-LG), lysozyme, and glyceraldehyde-3-phosphate-dehydrogenase-is studied in terms of surface pressure (i.e., surface activity) after treatment with increasing concentrations of urea, GdmHCl, and GdmSCN in the 0-8, 0-6, and 0-5 M ranges, respectively. For several of these proteins, the spectroscopic absorbance changes are monitored simultaneously to provide additional information prior to drop formation. Results show that surface pressure of proteins generally increases as the denaturant concentration increases and that effectiveness is GdmSCN > GdmHCl > urea. Protein unfolding curves obtained by plotting surface pressure as a function of denaturant concentration are presented and compared with respect to unfolding curves obtained by using UV absorbance and literature data. Kinetic information relative to the protein adsorption to the air-liquid interface of two proteins, alpha-Lac and beta-LG (chosen as representative proteins for comparison), denatured by the three denaturants is also studied and discussed.     

Structural transition during thermal denaturation of collagen in the solution and film states

Temperature dependent vibrational circular dichroism (VCD) spectra of type I collagen, in solution and film states, have been measured. These spectra obtained for solution sample suggest that the thermal denaturation of collagen results in transition from poly-L-proline II (PPII) to unordered structure. The PPII structure of collagen is identified by the presence of negative VCD couplet in the amide I region, while the formation of unordered structure is indicated by the disappearance of VCD in the amide I region. The temperature dependent spectra obtained for the supported collagen film indicated a biphasic transition, which is believed to be the first vibrational spectroscopic report to support a biphasic transition during thermal denaturation of collagen film. The temperature dependent spectra of collagen films suggest that the thermal stability of collagen structure depends on its state and decreases in the order: supported film > free standing film > solution state. These observations are believed to be significant in the VCD spectroscopic analysis of secondary structures of proteins and peptides.     

Fission of the bilayer lipid tube

The fusion of two black lipid membranes results in the formation of peculiar bilayer lipid tubes (‘cylindrical’) membranes (Neher, E. (1974) Biochim. Biophys. Acta 373, 328–336 and Melikyan, G.B., Abidor, L.G., Chernomordik, L.V. and Chailakhyan, L.M. (1983) Biochim. Biophys. Acta 730, 395–398). The mechanical stability of such tubes has been investigated experimentally and theoretically. With increasing hydrostatic pressure on the outside of the tube the radius of its middle part decreases. After this radius has reached a critical value, which constitutes 0.55 of the radius of the tube base, there occurs a collapse of the tube and its disintegration into two planar bilayers (fission). Expressions are obtained which relate the transmembrane difference of the hydrostatic pressure, causing the collapse, to the geometrical characteristics of the tube (its length and the radius of its base) and to the tension of the lipid bilayer. A method for measuring the membrane tension is proposed on the basis of the phenomenon considered.     

Evaluation of peritoneal tissue by means of differential scanning calorimetry (DSC)

Abdominal surgeries alter the integrity of the peritoneal layer and cause imbalances among immunological, inflammatory and angiogenic mechanisms within the tissue. During laparoscopic procedures a protective function of the peritoneal layer can be disturbed by the gas used to create a pneumoperitoneum. The aim of this study was to characterize peritoneal tissue by means of differential scanning calorimetry (DSC) as a reference for future investigations on the influence of surgical procedures on the physicochemical state of the peritoneum. Thirty-seven patients participated in the study. Patients were divided into three groups according to the type of surgery: group H - patients who underwent hernia repair; group Ch - patients who underwent laparoscopic cholecystectomy; and group C - patients operated due to rectal cancer. It was observed that onset temperature (T(o)), denaturation temperature (T(m)) and change of enthalpy (ΔH) during thermal denaturation of peritoneal collagen in were significantly different for these three groups of patients. The mean values of onset temperature (T(o)) and denaturation temperature (T(m)) in group H were significantly lower, while DH in this group was significantly higher than in the two other groups (Ch and C). This preliminary study does not answer whether the differences in collagen denaturation found in peritoneal tissue from different groups of patients resulted from a different inherent state of the tissue, or from surgical procedures. However, the results suggest that DSC is an appropriate method to study subtle changes in the physicochemical condition of the peritoneum using small samples obtained during surgical procedures.     

The origins and regulation of tissue tension: identification of collagen tension-fixation process in vitro

The absence of a controllable in vitro model of soft tissue remodeling is a major impediment, limiting our understanding of collagen pathologies, tissue repair and engineering. Using 3D fibroblast-collagen lattice model, we have quantified changes in matrix tension and material properties following remodeling by blockade of cell-generated tension with cytochalasin D. This demonstrated a time-dependent shortening of the collagen network, progressively stabilized into a built-in tension within the matrix. This was differentially enhanced by TGFB1 and mechanical loading to give subtle control of the new, remodeled matrix material properties. Through this model, we have been able to identify the 'tension remodeling' process, by which cells control material properties in response to environmental factors.