Decreased collagen stability as a response to the loss of structural integrity of thyroid cartilage

The thermal behavior, birefringence properties, and the biochemical composition of thyroid cartilage tissues have been studied. The hyaline cartilage, which was visualized as a quasi-isotropic medium, was composed of type II collagen, which did not denature at temperatures up to 100 degrees C. However, in hyaline cartilage digested by trypsin, the denaturation of collagen occured at 60 degrees C. Collagen fibers in the perichondrium were composed of type I and II collagen and formed a highly organized anisotropic structure (birefringence about 4.75 x 10(-3)) with a melting temperature of about 65 degrees C. The temperature of collagen denaturation in perichondrium in the whole system perichondrium-hyaline cartilage increased up to 75 degrees C, indicating the immobilization of perichondrium collagen by the extracellular matrix of the hyaline constituent.     

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple‐helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ～60‐80°C for hydrated samples. We report high temperature Raman spectra for freeze‐dried cartilage samples that show an increase in laser‐excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H₂O is retained within the freeze‐dried tissue. This is confirmed by thermogravimetric analysis that show 5‐7 wt% H₂O remaining within freeze‐dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re‐hydration following recovery indicate that the capacity of the denatured collagen to re‐absorb water is reduced. Our results are important for revealing the presence of bound H₂O within the collagen component of connective tissue even after freeze‐drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

Transformation of the dermal collagen framework under laser heating

The aim of this study was to compare between the changes undergone by the dermal collagen framework when heated by IR laser radiation and by traditional means and to reveal the specific features of the dermal matrix modification under moderate IR laser irradiation. Rabbit skin specimens were heated to 50°C, 55°C, 60°C and 65°C in a calorimeter furnace and with a 1.68‐μm fiber Raman laser. The proportion of the degraded collagen macromolecules was determined by differential scanning calorimetry. Changes in the architectonics of the collagen framework were revealed by using standard, phase‐contrast, polarization optical and scanning electron microscopy techniques. The collagen denaturation and dermal matrix amorphization temperature in the case of laser heating proved to be lower by 10°C than that for heating in the calorimeter furnace. The IR laser treatment of the skin was found to cause a specific low‐temperature (45°C‐50°C) transformation of its collagen framework, with some collagen macromolecules remaining intact. The transformation reduces to the splitting of collagen bundles and distortion of the course of collagen fibers. The denaturation of collagen macromolecules in the case of traditional heating takes its course in a threshold manner, so that their pre‐denaturation morphological changes are insignificant.     

Double thermal transitions of type I collagen in acidic solution

Contributed equally to this work. To further understand the origin of the double thermal transitions of collagen in acidic solution induced by heating, the denaturation of acidic soluble collagen was investigated by micro-differential scanning calorimeter (micro-DSC), circular dichroism (CD), dynamic laser light scattering (DLLS), transmission electron microscopy (TEM), and two-dimensional (2D) synchronous fluorescence spectrum. Micro-DSC experiments revealed that the collagen exhibited double thermal transitions, which were located within 31–37?°C (minor thermal transition, T _s?～?33?°C) and 37–55?°C (major thermal transition, T _m?～?40?°C), respectively. The CD spectra suggested that the thermal denaturation of collagen resulted in transition from polyproline II type structure to unordered structure. The DLLS results showed that there were mainly two kinds of collagen fibrillar aggregates with different sizes in acidic solution and the larger fibrillar aggregates (T _p2?=?40?°C) had better heat resistance than the smaller one (T _p1?=?33?°C). TEM revealed that the depolymerization of collagen fibrils occurred and the periodic cross-striations of collagen gradually disappeared with increasing temperature. The 2D fluorescence correlation spectra were also applied to investigate the thermal responses of tyrosine and phenylalanine residues at the molecular level. Finally, we could draw the conclusion that (1) the minor thermal transition was mainly due to the defibrillation of the smaller collagen fibrillar aggregates and the unfolding of a little part of triple helices; (2) the major thermal transition primarily arose from the defibrillation of the larger collagen fibrillar aggregates and the complete denaturation of the majority part of triple helices.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

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.     

Electric properties of macromolecules. IX. Dipole moment, polarizability, and optical anisotropy factor of collagen in solution from electric birefringence

The electric birefringence of collagen solutions has been measured over a wide range of field strength with the pulse technique. The soluble collagen was from rat tail tendon. The solvent used was dilute acetic acid. Very pronounced saturation of the electric birefringence was observed, permitting calculation of the optical anisotropy factor. The Kerr constant was determined by extrapolation to zero field strength. From the dependence on field strength of the birefringence, the permanent dipole moment and the anisotropy of polarizability were separately determined. The contribution of the former to the Kerr constant was found to be twice as large as that of the latter. The same conclusion was obtained from the initial slope of the rise curves of the birefringence at low fields. The permanent dipole moment was 1.5 × 10⁴ Debye, and the anisotropy of polarizability was about 3 × 10^?15 cm.³. The magnitude of the latter indicates that the ion atmosphere polarization is important. Effects of added salt and thermal denaturation on the electric birefringence were explored.     

USE OF ULTRASONIC SPECTROSCOPY AND VISCOSIMETRY FOR THE CHARACTERIZATION OF CHICKEN SKIN COLLAGEN IN COMPARISON WITH COLLAGENS FROM OTHER ANIMAL TISSUES

Use of traditional sources of collagen such as pork, bovine, and carp has some limitations. Chicken skin can be valuable alternative. In this work collagen was isolated from chicken skin using a modified procedure. Molecular properties of chicken collagen were analyzed and compared to collagen from other animal skins. Acid-soluble collagen type I was obtained with a yield of 25% and water content around 67%. Viscosimetry and ultrasonic spectroscopy were newly used for molecular characterization. By ultrasonic attenuation measurements, a pre-aggregation phase in the interval from 20°C to 27°C was observed, which is a proof of disaggregation and liquefaction. From 40°C upward, the liquefaction process finishes and aggregation continues. In a bovine sample this phenomenon starts at 40°C, in chicken at 50°C, and continues until 70°C. By viscosimetry, the denaturation temperature was confirmed as 40°C for bovine and 50°C for chicken collagen. Chicken collagen has a two times higher lysine level than bovine, which provides molecular stability side-chain interactions. With regard to higher thermal stability and favorable amino acid composition, waste chicken skin has the potential to be an excellent alternative source of raw collagen with applications in the food industry and biomedicine.     

Comparative analysis of the structure and thermal stability of sea urchin peristome and rat tail tendon collagen

We have purified collagen from two distinct sources; the vertebrate, rat tail tendon and an invertebrate, sea urchin adult tissue, the peristome. The collagenous nature of the purification products was confirmed by amino acid compositional analysis. Both preparations had high contents of glycine and proline residues and hydroxyproline was also present. The total pyrrolidine (proline+hydroxyproline) content decreased from 17.9 mole% in rat tail collagen to 12.9 mole% in peristome collagen. Distinctly different circular dichroic spectra were measured for these collagens. Analyses of spectra, measured as a function of temperature, revealed distinct thermal denaturation profiles. The melting temperature for rat tail collagen was 38.5 degrees C, while the corresponding value for peristome collagen was significantly lower at 27 degrees C. A similar thermal denaturation profile was obtained for rat tail collagen in digestion experiments using a 41-kDa gelatinase activity, isolated from sea urchin eggs. These results identify structural differences between a typical, vertebrate type I fibrillar collagen and an echinoderm collagen which serves as a constituent of a mutable connective tissue. These differences may relate to the functional roles played by collagen in these distinctly different tissues.     

Dynamic Light Scattering Studies on Hydrodynamic Properties of Fibrinogen-Fibronectin Complex

Abstract

A high molecular weight ‘cryogel’ was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37°C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20°C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20°C in 0.05 M Tris- HCl(pH7.4) containing 0.5 M NaCl was estimated as 8.5× 10^?8 cm²s^?1. The complex did not dissociate over the temperature range from 20 to 37°C. The diffusion coefficients of the complex decreased significantly at 12°C and 40°C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40°C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15°C, but not at near- freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.     

Reversing-pulse electric birefringence of poly(γ-benzyl-L-glutamate). III. The temperature dependence of the transient and steady-state behavior in cyclohexanone

The reversing-pulse electric birefringence (RPEB) technique was applied to the study of the temperature effect on the electrooptical and hydrodynamic properties of a fractionated [Glu(OBzl)]_n sample, which is molecularly dissolved in cyclohexanone. The aim was to develop a standard analytical method for thermal denaturation and temperature-induced conformational transitions. The field-on reverse and steady-state signal, and the field-off decay signal, were measured at 535 nm and at a constant low field strength (ca. 3 kV/cm) over a wide temperature range (5–90°C). The steady-state birefringence and the relaxation time in the decay process were also measured at two constant temperatures (5 and 70°C) over a wide field strength range (E ≤ 20 kV/cm). By the combination of these two different sets of RPEB measurements, the unwanted effect of the high pulse field on polymer conformation at elevated temperatures could be minimized. Together with the density and viscosity of cyclohexanone measured between 5 and 95°C, the following quantities could be evaluated: the weight-average permanent dipole moment and polarizability anisotropy, the reduced optical anisotropy factor (Δg/n), the weight-average length, and the degree of polydispersity. All these quantities, except for Δg/n, were found to be almost independent of temperature (5–90°C) and concentration (1.54–4.27 mM).     

Hydroxyproline content determines the denaturation temperature of chick tendon collagen

A series of nine procollagen samples in which the hydroxyproline content varied from <1% to 44% of the total imino acids was prepared by incubating embryonic chick tendon cells with varying concentrations of α,α′-dipyridyl, an inhibitor of proline hydroxylase. The thermal stability of these procollagen preparations was then investigated by using pepsin digestion at different temperatures as an enzymatic probe of conformation. Using this technique, the denaturation temperature of the procollagen was found to be directly proportional to the hydroxyproline content. A denaturation temperature of 23.5 °C was found for the unhydroxylated procollagen and 37.9 °C for fully hydroxylated procollagen. These results suggest that hydroxyproline is crucial to the thermal stability of the collagen triple helix. They also imply that unhydroxylated molecules are not triple helical within the cell at 37 °C and that triple helix formation may be necessary for normal secretion.     

Dynamic mechanical and rheo-optical studies of collagen and gelatin

The frequency dependence of dynamic mechanical properties of rat tail tendon (RTT), enzyme-solubilized collagen membranes (ESC), AKM-23 dialysis membranes, and gelatin film have been measured at 110, 11, and 3.5 Hz from - 160 to 220°C. RTT and AKM-23 are devoid of a rubbery region; there are as many as six mechanical loss transitions. Gelatin and ESC membranes behave as rubbery materials above room temperature; only three tan δE peaks can be resolved for these materials. Strain birefringence was used to measure the crystalline and amorphous contribution of orientation induced by strain. Both the birefringence and the strain optical coefficient are sensitive to the amount of water in a sample. The effect of chemical swelling agents and of annealing on birefringence are described. Stress relaxation data on gelatin film were analyzed with the rubber elasticity theory to give the average number of chains per unit volume, the specific polarizability, the stress-birefringence ratio and the average molecular weight between hydrogen bonds were calculated. The intrinsic amorphous birefringence for “wet” gelatin film is 1.25 × 10^?2; it is estimated to be about 6 × 10^?2 for “dry” gelatin film.     

Calorimetry of rat tail tendon collagen before and after denaturation: the heat of fusion of its absorbed water

The specific heat, of rat tail tendon at various water contents was measured as a function of temperature. The resulting graphs showed peaks arising from the melting, near 50°C, of helical material in the collagen, and from the melting of absorbed water in the range -40°C to 0°C. The heat of melting of helical material was 11.7 cal per gram of dry tendon. Determination of the heat and temperature of fusion of the absorbed water allowed resolution of the water into four states in the case of tendon before denaturation, and three states after denaturation. The four states are (1) water not freezable on cooling to - 70°C, (2) freezable water with-both heat and temperature of fusion different from the values for ordinary water, (3) freezable water with the heat of fusion of ordinary water, but a different temperature of fusion, and (4) water not distinguished from ordinary water. The fourth state was absent in denatured tendon. The results are discussed in terms of increasing size of clusters of absorbed water molecules.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

Preparation and Some Properties of Type I Collagen from Fish Scales

Soluble collagen from fish (sardine) scales was yielded at about 5% with 0.5 m acetic acid after demineralization with EDTA, while a great portion of the collagen remained insoluble. The solubility of this insoluble collagen was about 20% at 45°C (denaturation temperature of soluble collagen) for 24 h. The remaining 80% of the insoluble collagen was denatured in the form of insoluble gelatin, and that may be an interesting food material.     

Fourier transform IR spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies

The ir spectra of lathyritic rat skin collagen and calf skin gelatin solutions at a variety of temperatures were obtained using Fourier transform ir spectroscopy and a 9-reflection, 2-pass ZnSe prism sample cell. The spectra were then deconvolved (based on Kauppinnen's method) and the behavior of the amide I band at ～ 1650 cm^?1 observed in detail. Throughout the temperature range studied (4–50°C), three component absorption peaks within the amide I band (at 1633, 1643, and 1660 cm^?1) are common to the spectra irrespective of the degree of triple helix content of the sample. Changes in the relative intensities of these component peaks are, however, conformationally dependent. During denaturation of the triple helix, the dominant 1660-cm^?1 component in the native collagen spectrum diminishes and the 1633-cm^?1 peak becomes relatively intensified. The inherently strong basicity of the carbonyl group of the proline residues together with the frequent occurrence of this imino acid in the X position of the Gly-X-Y triplet of collagen largely accounts for the ?30-cm^?1 shift of the amide I band during denaturation. Temperature and conformationally dependent changes in the fine structure of the amide I band from dilute solutions of collagen can be monitored in a reproducible and quantitative fashion.     

Heat stabilization dependence on redox state of cytochrome cd1 oxidase from Pseudomonas aeruginosa

The irreversible thermal denaturation of cytochrome cd₁ oxidase from $\text{P.}\text{aeruginosa}$ as a function of the oxidation-reduction states of its hemes was observed with a differential scanning calorimeter. Upon full reduction of the four hemes, the apparent denaturation temperature decreases by about 10° and the denaturation enthalpy decreases slightly: oxidized, 5.9 cal/gm; reduced, 5.4 cal/gm. At pH 7.5, the first order rate constants for denaturation at 90°C are: reduced, 33 × 10^?3s^?1; oxidized, 3 × 10^?3s^?1. Thus, oxidation of the hemes reuults in heat stabilization of the cytochrome oxidase. The activation energy for denaturation of fully reduced oxidase, 53 kcal/mol, is less than that for fully oxidized protein (73 kcal/mol).     

Temperature-correlated force and structure development in elastomeric polypeptides: the Ile1 analog of the polypentapeptide of elastin

The Ile¹ analog of the polypentapeptide of elastin, (L · Ile¹-L · Pro²-Gly³-L · Val⁴-Gly⁵)_n, abbreviated as Ile¹-PPP, was synthesized with n > 100 to determine the effect of the increased hydrophobicity of the pentamer resulting from Val¹ replacement by Ile¹ on the previously characterized inverse temperature transition of the polypentapeptide of elastin (PPP). Ile¹-PPP, dissolved in water at 4°C, was found to aggregate, forming a viscoelastic coacervate on raising the temperature. The onset of aggregation was 8°C for Ile¹-PPP, as compared to 24°C for PPP. Characterization by CD demonstrated an increase in intramolecular order on raising the temperature from 8°C to 25°C, and demonstrated similar conformations for PPP and Ile¹-PPP before and after their respective transitions. The CD-characterized transition also occurred at a temperature some 15°C lower than that of PPP. By means of 20-Mrad γ-irradiation cross-linking of the Ile¹-PPP coacervate, an elastomeric matrix was formed with an elastic modulus, similar to that of 20-Mrad cross-linked PPP. The temperature dependence of elastomeric force of cross-linked Ile¹-PPP showed an abrupt increase from essentially zero at 8°C to three-quarters of full force at 10°C and essentially full force by 20–25°C. This development of elastomeric force for the more hydrophobic Ile¹-PPP matrix, which parallels the increase in intramolecular order characterized by the CD studies, also occurs at a temperature some 15°C lower than that for the PPP matrix. Thus, in these elastomeric polypeptides, development of elastomeric force is coupled to an inverse temperature transition, the temperature of which depends inversely on the hydrophobicity of the constituent pentamer. It appears that a series of elastomeric polypeptide biomaterials are possible in which the temperature over which elastomeric force develops can be varied.