Thermal denaturation of sheared and unsheared chromatin by absorption and circular dichroism measurements.

Thermal denaturation of chromatin is observed by simultaneously monitoring absorption and circular dichroism at 276 nm as functions of temperature. Either observation indicates that sheared chromatins shows less thermal stability than native chromatin. The temperature-dependent ellipticities at 276 nm of these chromatins show features not seen in the absorption curves: the ellipticity of unsheared chromatin increases with temperature, while this increase is abolished or greatly reduced in the same chromatin after shearing. After its first thermal transition (prior to the helix-coli transition) the unsheared chromatin achieves the same ellipticity as sheared chromatin.     

Thermal properties of ethylene glycol aqueous solutions

Preventing ice crystallization by transforming liquids into an amorphous state, vitrification can be considered as the most suitable technique allowing complex tissues, and organs cryopreservation. This process requires the use of rapid cooling rates in the presence of cryoprotective solutions highly concentrated in antifreeze compounds, such as polyalcohols. Many of them have already been intensively studied. Their glass forming tendency and the stability of their amorphous state would make vitrification a reality if their biological toxicity did not reduce their usable concentrations often below the concentrations necessary to vitrify organs under achievable thermal conditions. Fortunately, it has been shown that mixtures of cryoprotectants tend to reduce the global toxicity of cryoprotective solutions and various efficient combinations have been proposed containing ethanediol. This work reports on the thermal properties of aqueous solutions with 40, 43, 45, 48, and 50% (w/w) of this compound measured by differential scanning calorimetry. The glass forming tendency and the stability of the amorphous state are evaluated as a function of concentration. They are given by the critical cooling rates v(ccr)above which ice crystallization is avoided, and the critical warming rates v(cwr) necessary to prevent ice crystallization in the supercooled liquid state during rewarming. Those critical rates are calculated using the same semi-empirical model as previously. This work shows a strong decrease of averaged critical cooling and warming rates when ethanediol concentration increases, V(ccr) and V(cwr) = 1.08 x 10 (10) K/min for 40% (w/w) whereas V(ccr) = 11 and V(cwr) = 853 K/min for 50% (w/w). Those results are compared with the corresponding properties of other dialcohols obtained by the same method. Ethylene glycol efficiency is between those of 1,2-propanediol and 1,3-propanediol.     

Thermal stability of collagen fibers in ethylene glycol

The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.     

Urea denaturation of chromatin periodic structure.

Isolated chicken erythrocyte nuclei dispersed in urea solutions (0-5.0 M) have been examined in terms of their low-angle X-ray diffraction and electron microscopic properties. At high urea concentrations, the characteristic low-angle X-ray reflections of chromatin are absent, and the spheroid chromatin particles (v bodies) are markedly perturbed. This lability of chromatin periodic structure to high concentrations of urea is consistent with previous hydrodynamic and spectroscopic studies.     

Thermal denaturation profiles of deoxypolynucleotide-ligand complexes: semiempirical studies

We have semiempirically studied the thermal denaturation profiles of complexes formed between double strand polynucleotides and pure stabilizer nonspecific binding ligands. By using the McGhee model (J. D. McGhee, (1976) Biopolymers 15, 1345-1375) we have found a simple, analytical relationship between the melting temperature (Tm) and the Kh (intrinsic association constant), nh (apparent site size), and wh (cooperativity constant) values of the interaction. The validity of this approach strongly depends on the sigma value (sigma being the nucleation parameter of the DNA). Through the equation so obtained it is possible to calculate the Kh, nh, and wh values from the melting temperature of three experimental thermal denaturation profiles at different r (ligand/polynucleotide ratio) values. The method has been checked by studying the thermal denaturation profiles of daunomycin-poly(d(A-T)).poly(d(A-T)) complexes in two different salt concentrations. The results so obtained are compared with those previously described using other techniques. The applicability of the method here developed is discussed in relation with both the nature of the ligands and the value of the nucleation parameter (sigma).     

Thermal denaturation and regeneration of japanese-radish peroxidase.

Thermal denaturation of Japanese-radish peroxidase [EC 1.11.1.7] was investigated with respect to its spectrophotometric properties and effect on the enzymatic activity. Inactivation of the peroxidase occurred at temperatures higher than 60degrees and involved three processes, i.e., dissociation of protohemin from the holoperoxidase, a conformation change in the apperoxidase, and the modification or degradation of protohemin. The splitting process of protohemin from holoperoxidase as followed by the change in the absorption spectrum at high temperatures coincided with the degrease in the activity, and it was found to be at least biphasic. The regeneration of peroxidase on cooling to room temperature was essentially reversible at neutral pH, while at pH 5 and pH 9 these processes were irreversible. The irreversibility at acidic pH was mainly due to an irreversible change in the conformation of the apoenzyme. The difference spectrum of heat-treated apoperoxidase exhibited a denaturation blueshift with negative maxima at 287 and 294 nm, and the total protein fluorescence quantum yield. qprotein, increased by 20% compared to that of the untreated apoenzyme. On the other hand, the irreversibility at alkaline pH was largely attributable to the modification of protohemin. Apoperoxidase was more resistnat to heat denaturation but the modification or degradation of protohemin in heated enzyme was greater at alkaline pH than at acidic pH. The pyridine-ferrohemochrome spectrum of peroxidase exhibited slight shifts of the maxima of the alpha-band to shorter wavelength on heat treatment, and the paper chromatogram showed the presence of a new derivative other than protohemin. The modified product is probably (2(4)-vinyl-4(2)-hydroxyethyldeuterohemin.     

Thermal denaturation of nucleosomal core particles.

Thermal denaturation of very homogeneous preparations of core particles from chicken erythrocyte chromatin is studied by several techniques. The change in absorbance, which is very closely paralleled by changes in heat capacity, which is very closely paralleled by changes in heat capacity, is a biphasic process with inflexions at 60 degrees C and 74 degrees C. In contrast, isolated DNA of the same length denatures in a single transition around 44 degrees C. Monitoring the circular dichroism of the cores during thermal denaturation reveals biphasic changes in the secondary structure of the DNA, preceding the base unstacking by 10 degrees C in the first and 3 degrees C in the second phase. However, measurable alterations in the secondary structure of the histones are confined to the second phase with a melting temperature at 71 degrees C. Increase in the ionic strength of the buffer from 1 mM to 10 mM leads to almost monophasic melting curves as measured by absorbance and CD, while not causing any measurable conformational changes at room temperature. The melting of core particles is interpreted as a denaturation of about 40 base pairs in the first phase, followed by a massive breakdown of the native structure of a tight histone-DNA complex, which frees the remaining 100 base pairs for unstacking.