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.     

Influence of collagen denaturation on the chemorheological properties of skin,assessed by differential scanning calorimetry and hydrothermal isometric tension measurement

The curves obtained for skin samples of different ages and species by hydrothermal isometric tension (‘HIT’) measurement are compared to those obtained by differential scanning calorimetry (DSC) under the same thermal conditions (for a rise in temperature at a rate of 1.0°C/min). Collagen denaturation, observed by DSC, directly affects the kinetics of the tension variations in the first part of the ‘HIT’ curves, including the early peak due to the presence and destruction of the heat-labile cross-links in the collagen network. The presence of cross-links is in term shown to delay collagen denaturation to an extent which depends in part on their heat-stability. The final part of the ‘HIT’ curves reflecting the effects of heat in the stable polymeric collagen network is no longer affected by collagen denaturation. Thus, both ‘HIT’ and DSC are useful methods to evaluate collagen reticulation in connective tissues.     

Thermal stability and energetics of 15-mer DNA duplex interstrand crosslinked by trans-diamminedichloroplatinum(II)

The effect of the location of the interstrand cross-link formed by trans-diamminedichloroplatinum(II) (transplatin) on the thermal stability and energetics of 15-mer DNA duplex has been investigated. The duplex containing single, site-specific cross-link, thermodynamically equivalent model structures (hairpins) and nonmodified duplexes were characterized by differential scanning calorimetry, temperature-dependent uv absorption, and circular dichroism. The results demonstrate that the formation of the interstrand cross-link of transplatin does not affect pronouncedly thermodynamic stability of DNA: the cross-link induces no marked changes not only in enthalpy, but also in "reduced" (concentration independent) monomolecular transition entropy. These results are consistent with the previous observations that interstrand cross-links of transplatin structurally perturb DNA only to a relatively small extent. On the other hand, constraining the duplex with the interstrand cross-link of transplatin results in a significant increase in thermal stability that is primarily due to entropic effects: the cross-link reduces the molecularity of the oligomer system from bimolecular to monomolecular. Importantly, the position of the interstrand cross-link within the duplex modulates cooperativity of the melting transition of the duplex and consequently its thermal stability.     

Comparative calorimetric studies on the dynamic conformation of plant 5S rRNA. I. Thermal unfolding pattern of lupin seeds and wheat germ 5S rRNAs, also in the presence of magnesium and sperminium cations.

An attempt has been made to correlate differential scanning calorimetry melting profiles of 5S rRNAs from lupin seeds (L.s.) and wheat germ (W.g.) with their structure. It is suggested that the observed differences in thermal unfolding are due to differences in RNA nucleotide sequence and as a consequence in higher order structures. Interesting effects induced by magnesium cation, perprotonated and permethylated sperminium tetracations are discussed. It is suggested that the difference in the stabilizing effect of the three cations results from different mode of their interactions with RNA. "Pure" electrostatic interactions expected for permethylated tetracations are rather weak due to the steric hindrance around each positively charged nitrogen atom. Electrostatic interactions of the other two cations are significantly enhanced by coordination bonding for magnesium and by hydrogen bonding for protonated sperminium cation.     

Thermal denaturation of UV-irradiated wet rat tail tendon collagen

The thermal helix-coil transition of UV irradiated collagen in rat tail tendon has been investigated by differential scanning calorimetry. During UVB irradiation the tendons were immersed in water to keep the collagen fibers in a fully hydrated condition at all times. UV irradiation induced changes in collagen which caused both stabilization and destabilization of the triple helix in fibers. The helix-coil transition for non-irradiated collagen occurred near 64 degrees C, for irradiated 1 and 3 h at 66 and 67 degrees C, respectively. After irradiating for longer times (20-66 h) the helix-coil transition peak occurred at much lower temperatures. The peak was very broad and suggested that collagen was reduced by UV to different polypeptides of different molecular weight and different lower thermal stabilities. It was caused by the disruption of a network of hydrogen-bonded water molecules surrounding the collagen macromolecule.     

Addressing Mechanism of Fibrillization/Aggregation and Its Prevention in Presence of Osmolytes: Spectroscopic and Calorimetric Approach

Understanding the mechanism of protein fibrillization/aggregation and its prevention is the basis of development of therapeutic strategies for amyloidosis. An attempt has been made to understand the nature of interactions of osmolytes L-proline, 4-hydroxy-L-proline, sarcosine and trimethylamine N-oxide with the different stages of fibrillization of hen egg-white lysozyme by using a combination of isothermal titration calorimetry, differential scanning calorimetry, fluorescence spectroscopy, and transmission electron microscopy. Based on thioflavin T fluorescence emission intensities and microscopic images, the nucleation, elongation, and saturation phases of fibrillization have been identified. Isothermal titration calorimetry and differential scanning calorimetry have enabled a quantitative analysis of the nature of interactions of these osmolytes with various conformational states of lysozyme at different stages of fibrillization/aggregation. It is concluded that interaction of the osmolytes with lysozyme fibrils at both the nucleation and elongation stages are important steps in the prevention of fibrillization/aggregation. Identification of the nature of interactions is a key step towards the discovery and synthesis of target oriented potential inhibitors of these associations. This study is a first report in which calorimetry has been used to address interaction of potential inihibitiors with the protein at different stages of fibrillization.     

Thermal and thermodynamic properties of duplex DNA containing site-specific interstrand cross-link of antitumor cisplatin or its clinically ineffective trans isomer

The effect of the single, site-specific interstrand cross-link formed by cisplatin or transplatin on the thermal stability and energetics of a 20-base pair DNA duplex is reported. The cross-linked or unplatinated 20-base pair duplexes were investigated with the aid of differential scanning calorimetry, temperature-dependent UV absorption, and circular dichroism. The cross-link of both platinum isomers increases the thermal stability of the modified duplexes by changing the molecularity of denaturation. The structural perturbation resulting from the interstrand cross-link of cisplatin increases entropy of the duplex and in this way entropically stabilizes the duplex. This entropic cross-link-induced stabilization of the duplex is partially but not completely compensated by the enthalpic destabilization of the duplex. The net result of these enthalpic and entropic effects is that the structural perturbation resulting from the formation of the interstrand cross-link by cisplatin induces a decrease in duplex thermodynamic stability, with this destabilization being enthalpic in origin. By contrast, the interstrand cross-link of transplatin is enthalpically almost neutral with the cross-link-induced destabilization entirely entropic in origin. These differences are consistent with distinct conformational distortions induced by the interstrand cross-links of the two isomers. Importantly, for the duplex cross-linked by cisplatin relative to that cross-linked by transplatin, the compensating enthalpic and entropic effects almost completely offset the difference in cross-link-induced energetic destabilization. It has been proposed that the results of the present work further support the view that the impact of the interstrand cross-links of cisplatin and transplatin on DNA is different for each and might also be associated with the distinctly different antitumor effects of these platinum compounds.     

Peculiarities of DNA-proflavine binding under different concentration ratios

Binding of proflavine to calf thymus DNA is investigated by differential scanning calorimetry and spectrophotometry. It is shown that proflavine interacts with DNA by three binding modes. At high DNA—ligand concentration ratios (P/D), proflavine prefers to intercalate into GC-sites but can also insert into other sites. At low P/D ratios, proflavine interacts with DNA by the external binding mode. The parameters of proflavine-DNA complexation have been calculated from spectrophotometric concentration dependences. Thermodynamic parameters of DNA melting have been calculated from differential scanning calorimetry data.     

Electrical capacitance of lipid bilayer membranes of hydrogenated egg lecithin at the temperature phase transition

Electrical capacitance of the planar bilayer lipid membrane (BLM) formed from hydrogenated egg lecithin (HEL) has been studied during many passages through the phase transition temperature. In contrast to the BLM from individual synthetic phospholipids, membranes from HEL did not demonstrate any capacitance change at the phase transition temperature maximum, as measured by differential scanning calorimeter at 52 degrees C. Instead, two temperatures have been discerned by capacitance records: thickening at 42-43 degrees C and thinning at 57-59 degrees C. The first temperature region is close to the transition temperature of dipalmitoyllecithin, whereas the second is close to that of distearoyllecithin, two main components of the HEL. It was suggested that capacitance measurements were able to reveal a phase separation in the BLM from HEL which was not detected by differential scanning calorimetry. The phase transition of the BLM from the liquid crystal state to the gel state is followed by thickening of the bilayer structure, partly due to a gauche- trans transition of lipid molecules but mainly due to redistribution of the solvent n-decane.     

Cholesteryl oligoethyleneglycol glycosides: fluidizing effect of their embedment into phospholipid bilayers

Glycosides of cholesteryl oligoethyleneglycols have been synthesized and embedded in liposome bilayers. Several methods as steady-state fluorescence polarization, differential scanning calorimetry, zeta potential, and agglutination have been used to describe the physicochemical outcome of the incorporation of these synthetic glycolipids within phospholipid layers. From calorimetry and fluorescence experiments, it is apparent that the glycolipids decrease the transition temperature of the bilayers in a more important extent than cholesterol. Furthermore, the thickness of the aqueous layer fixed around the liposome is independent of the presence of glycolipids, suggesting that their hydrophilic parts (tetraethyleneglycol spacer and sugar moiety) are not completely extended towards the aqueous phase. Nevertheless, an important and specific interaction has been observed between such glycosylated liposomes and vegetal lectins.     

Xanthan and glucomannan mixtures: synergistic interactions and gelation

The synergistic interaction between xanthan and glucomannan in solution and in the gel phase has been studied by circular dichroism spectroscopy and differential scanning calorimetry. The study in solution of the polysaccharidic mixture indicates a preferred stoichiometry of the interaction corresponding to a weight fraction of xanthan around 0.55. This finding is in reasonable agreement with the differential scanning calorimetry measurements carried out on the gel phase. Models from conformational analysis based on these results were formulated in terms of 1:1 and 2:1 Konjac glucomannan/xanthan molecular assemblies. The experimental and calculation results clearly indicate the involvement of the side chains of xanthan and suggest that the ordered portions of the macromolecular complex in solution act in the gel phase as junction zones.     

Structural transitions of carboxymethylated cytochrome c: calorimetric and circular dichroic studies

Circular dichroism and differential scanning calorimetry studies on the unfolding-refolding process of native and carboxymethylated cytochrome c, induced either by temperature or chemical agents, have been performed. The results have shown that the modified protein has a decreased conformational stability with respect to the native state, in agreement with a structure less compact, but still highly folded, which behaves as a thermodynamically stable "intermediate" between native and fully unfolded cytochrome c.     

Reactions of lipid-derived malondialdehyde with collagen.

Malondialdehyde is a product of fatty acid oxidation (e.g. from low density lipoprotein) implicated in the damage of proteins such as collagen in the cardiovascular system (Chio, K. J., and Tappel, A. L. (1969) Biochemistry 8, 2821-2827). Its concentration is raised in diabetic subjects probably as a side effect of increased protein glycation. Collagen has enzyme-catalyzed cross-links formed between its individual molecules that are essential for maintaining the structure and flexibility of the collagen fiber. The cross-link dehydro-hydroxylysinonorleucine reacts irreversibly with 10 mM malondialdehyde at least 3 orders of magnitude faster than glucose reactions with lysine or arginine, such that there is little cross-link left after 1 h at 37 degrees C. Other cross-links and glycated elements of collagen are also vulnerable. Several possible products of malondialdehyde with collagen cross-links are proposed, and the potential involvement of collagenous histidine in these reactions is discussed. We have also isolated Ndelta-(2-pyrimidyl)-L-ornithine from collagenous arginine reacted with malondialdehyde. The yields of this product were considerably higher than those from model reactions, being approximately 2 molecules/collagen molecule after 1 day at 37 degrees C in 10 mM malondialdehyde. Collagenous lysine-derived malondialdehyde products may have been present but were not protected from protein acid hydrolysis by standard reduction techniques, thus resulting in a multitude of fragmented products.     

Cross-linking methionine and amine residues with reactive halogen species

Irreversible cross-links are increasingly being recognized as important posttranslational oxidative protein modifications that contribute to tissue injury during oxidative stress and inflammation. They also have a structural function in extracellular matrix proteins such as collagen IV. Likely contenders for forming such cross-links are the reactive halogen species that are generated by neutrophils and eosinophils, including hypochlorous acid, hypobromous acid, and their related haloamines. Methionine residues are kinetically preferred targets for these oxidants and oxidation can potentially result in sulfilimine (>SN–) bonds with amines. Therefore, we investigated whether oxidation of methionine in the model peptide formyl-Met-Leu-Phe-Lys (fMLFK) produces cross-links with lysine residues, using mass spectrometry to characterize the products. As expected, the sulfoxide was the major product with each reactive halogen species. However, intra- and intermolecular cross-linked products were also formed. Isomers of an intramolecular sulfilimine were readily produced by hypobromous acid and bromamines, with hypochlorous acid forming lesser amounts. The predominant cross-link with chloramines was an intermolecular bond between the sulfur of fMLFK and the amine derived from the chloramine. Reactive halogen species also formed these sulfilimine cross-links in other peptides that contain methionine. We propose that protein cross-links involving methionine and amine residues will form via this mechanism when granulocytes are activated at sites of inflammation. Our results also support the proposal that reactive halogen species generated by the peroxidase peroxidasin could be responsible for the sulfilimine bonds that are integral to the structure of collagen IV.     

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.     

Coil-helix transition of iota-carrageenan as a function of chain regularity

A series of iota-carrageenans containing different amounts of nu-carrageenan (0-23 monomer %) have been prepared from neutrally extracted carrageenan of Eucheuma denticulatum. nu-Carrageenan is the biochemical precursor of iota-carrageenan. The conformational order-disorder transition and rheological properties of these carrageenans were studied using optical rotation, rheometry, size exclusion chromatography coupled to multiangle laser light scattering, and high-sensitivity differential scanning calorimetry. The helix forming capacity of iota-carrageenan turns out to decrease monotonously with increasing amount of nu-units. In contrast, the rheological properties of iota-carrageenan are remarkably enhanced by the presence of a small amount of nu-units, yielding a maximum twofold increase in G' at 3% nu-units. It is concluded that the structure-forming capacity of iota-carrageenan, containing a small amount of nu-carrageenan, is significantly higher than that of pure iota-carrageenan. This phenomenon is explained in terms of the balance between the helical content and the number of cross-links between chains, taking into consideration the fact that nu-units introduce "kinks" in the chain conformation enabling neighboring chains to connect. Increasing amounts of nu-units increase the number of cross-links in the network, resulting in increased gel strength. On the other hand, a reduced length of the helical strands weakens the cross-links between the different chains and, consequently, the gel.     

Thermal Destabilization of Collagen Matrix Hierarchical Structure by Freeze/Thaw

This study aims to characterize and understand the effects of freezing on collagen structures and functionality. Specifically, thermodynamic destabilization of collagen at molecular- and fibril-levels by combination of low temperatures and freezing were experimentally characterized using modulated differential scanning calorimetry. In order to delineate the effects of sub-zero temperature and water-ice phase change, we hypothesized that the extent of destabilization can be determined based on post-thaw heat induced thermal denaturation of collagen. It is found that thermal denaturation temperature of collagen in hydrogel decreases by 1.4–1.6°C after freeze/thaw while no such decrease is observed in the case of molecular solution. The destabilization is predominantly due to ice formation. Exposure to low temperatures in the absence of ice has only minimal effect. Calorimetry measurements combined with morphological examination of collagen matrices by scanning electron microscopy suggest that freezing results in destabilization of collagen fibrils due to expansion of intrafibrillar space by ice formation. This fibril-level damage can be alleviated by use of cryoprotectant DMSO at concentrations as low as 0.5 M. A theoretical model explaining the change in collagen post-thaw thermal stability by freezing-induced fibril expansion is also proposed.     

Observation of unfreezable water in aqueous solution of globule protein by microwave dielectric measurement

A freezing process analyzed by the dielectric method on aqueous solution of albumin has revealed water structure around protein molecule. A relaxation peak due to bound water attached on the protein surface around 100 MHz at room temperature was found. It could be seen commonly in globule proteins. Another peak due to a different kind of unfreezable water was found around 1 GHz at ?6°C. The amount of this water is estimated as 0.36 g water/g protein and in good agreement with that obtained by differential scanning calorimetry and nmr measurements. The water molecules form a shell layer around the protein molecule. © 1995 John Wiley & Sons, Inc.     

Stable, nonreducible cross-links of mature collagen.

During in vivo maturation, and also during in vitro incubation with physiological buffers, native collagen fibers display a progressive increase in tensile strength and insolubility. Paralleling these physiologically important changes is a progressive loss of the reducible cross-links which initially join the triple-chained subunits of collagen fibers. Although there is evidence suggesting that the reducible cross-links are gradually transformed into more stable, nonreducible cross-links during maturation, the nature of the transformation process and the structure of the stable "mature" cross-links has remained a mystery. In order to test the possibility that cross-link transformation involves addition of a nucleophilic amino acid residue to the reducible cross-links, histidine, arginine, glutamate, aspartate, lysine, and hydroxylysine residues were chemically modified, and the effect of each modification procedure on the in vitro transformation of reducible cross-links was ascertained. The results of these experiments indicated that destruction of histidine, arginine, glutamate, and aspartate residues has no measurable effect on the rate and extent of reducible cross-link transformation in hard tissue collagens. In contrast, modification of lysine and hydrocylysine residues with a wide variety of specific reagents completely blocks the transformation of reducible cross-links. Removal of the reversible blocking groups from lysine and hydroxlylysine residues then allows the transformation to proceed normally. These results indicate that collagen maturation involves nucleophilic addition of lysine and/or hydroxylysine residues to the electrophilic double bond of the reducible cross-links, yielding derivatives which are not only more stable but also capable of cross-linking more collagen molecules than their reducible precursors.     

The Role of Cross-Chain Ionic Interactions for the Stability of Collagen Model Peptides

The contribution of ionic interactions to the stability of the collagen triple helix was studied using molecular dynamics (MD) simulations and biophysical methods. To this end, we examined the stability of a host-guest collagen model peptide, Ac-GPOGPOGPYGXOGPOGPO-NH₂, substituting KGE, KGD, EGK, and DGK for the YGX sequence. All-atom, implicit solvent MD simulations show that the fraction of cross-chain ionic interactions formed is different, with the most pronounced in the KGE and KGD sequences, and the least in the DGK sequence. To test whether the fraction of cross-chain ionic interactions correlates with the stability, experimental measurements of thermostability were done using differential scanning calorimetry and circular dichroism spectroscopy. It was found that the melting temperature is very similar for KGE and KGD peptides, whereas the EGK peptide has lower thermostability and the DGK peptide is the least thermostable. A novel, to our knowledge, computational protocol termed temperature-scan MD was applied to estimate the relative stabilities of the peptides from MD simulations. We found an excellent correlation between transition temperatures obtained from temperature-scan MD and those measured experimentally. These results suggest the importance of cross-chain ionic interactions for the stability of collagen triple helix and confirm the utility of MD simulations in predicting interactions and stability in this system.