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Crystal structures together with enthalpies and temperatures of fusion of two substituted amino acids, N-acetylsarcosinamide (NASarA) and N-acetyl-L-isoleucinamide (NAIA), were determined by single crystal X-ray analysis and differential scanning calorimetry, respectively. The results were compared with those of some analogous amino acid derivatives previously studied. The detailed knowledge of crystallographic parameters is undoubtedly useful for discussing the thermodynamic results and rationalizing the fusion behaviour, owing to the rather poor knowledge of the molecular interactions occurring in the melt.

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The heats of dilution in water of binary and ternary solutions of the two enantiomeric forms ofN-acetylalanineamide have been measured at 25°. The excess enthalpies, expressed as virial expansion series, permit evaluation of the pairwise self and cross enthalpic coefficients. As for the chiral forms of some monosaccharides, the cross coefficient for the interaction between theD andL forms ofN-acetylalanineamide is slightly but significantly different from the corresponding self coefficient. A weak, water-mediated chiral recognition can be assumed to exist between pairs of amide molecules.

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Journal of Thermal Analysis and Calorimetry
Authors: G. Barone, C. Giancola, T. H. Lilley, C. A. Mattia, and R. Puliti

Enthalpies and temperatures of fusion or transition for four substituted dipeptides (Nacetylamides of glycyl-L-alanine (NAGAA),L-alanyl-L-alanine (NAA2A),L-prolyl-glycine (NAPGA) andL-leucyl-L-proline monohydrate (NALPA·H2O)) were determined by differential scanning calorimetry and the entropies of fusion derived. The results obtained have been compared with those of the corresponding substituted aminoacids and some of their racemic crystalline mixtures. The enthalpies and entropies of fusion of some substituted aminoacids have been redetermined. The results are discussed in comparison with crystal structural data, which has been reported in the literature or determined recently by some of the authors. Rationalization of the fusion parameters was attempted mainly on the basis of the number of intramolecular hydrogen bonds and the packing densities in the crystals.

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Journal of Thermal Analysis and Calorimetry
Authors: C. Giancola, A. Buono, G. Barone, L. De Napoli, D. Montesarchio, D. Palomba, and G. Piccialli

Abstract  

In this work we report a thermodynamic characterization of stability and melting behaviour of two 24-mer DNA triplexes. The third strand, that binds the Watson-Crick double helix with Hoogsteen hydrogen bonds, contains 3′-3′ phosphodiester junction that determines the polarity inversion. The target double helix is composed of adjacent and alternate fragments of oligopurine-oligopyrimidine tracts. The two helices differ from the substitution of the cytosine, involved in the junction, with the thymine. Calorimetric data reported here provide a quantitative measure of the influence of pH and base modification on the stability of a DNA triplex.

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Abstract  

It is presented a study concerning the influence of guanidinium chloride (GuHCl) and urea on thermal stability of Bovine Pancreatic Ribonuclease A (RNAase A) at differentpH values. As expected, at increasing the denaturant concentration, the protein thermostability decreases. This is shown by a decrease of both the thermodynamic parameters, temperature and heat effect, characterising the denaturation process. In order to analyse the calorimetric curves we adopt a statistical thermodynamic approach. The individual one-dimensional DSC profiles have been expanded into another dimension by varying the GuHCl concentration, so that a heat capacity surface is defined for eachpH. By means of the ICARUS program, developed in our laboratory, we accomplish a two dimensional deconvolution of the experimental data linking the binding equilibrium to the denaturation process. This analysis provides a well founded and complete statistical thermodynamic characterisation of denaturation process of RNAase A in the presence of GuHCl and allows to calculate the thermodynamic parameters associated to the binding of denaturant molecule.

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Journal of Thermal Analysis and Calorimetry
Authors: G. Barone, F. Catanzano, P. Del Vecchio, D. Fessas, C. Giancola, and G. Graziano

Abstract  

In this study we try to re-analyze thepH dependence of thermal stability of small globular proteins. From the thermodynamic point of view a long series of calorimetric and spectroscopic investigations has shown that the decreased stability in very acidic conditions can be ascribed to entropic effects. The same conclusion is reached, from a microscopic point of view, by assuming that a binding of protons on equal and noninteracting sites takes place as a consequence of unfolding process. By linking the conformational unfolding equilibrium to the proton binding equilibrium, a model is developed that is able to describe the dependence on thepH of the thermal denaturation processes of small globular protiens. The application of the model to hen lysozyme and T4 lysozyme correctly accounts for the experimental results.

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A new software package (THESEUS) has been assembled for the analysis of the DSC data, Concerning the thermal denaturation of biological macromolecules. The system is useful to obtain accurate physico-chemical information, bypassing the casual and systematic errors, very common in these experiments. It can also be used for handling data from other instruments and methodologies giving thermodynamic, spectroscopic or other kind of data as a function of temperature. Because many of the researches in this field are of exploratory nature and continuously new unfolding mechanisms are described or hypothesized in the current literature, we have written and assembled this powerful and flexible program of general applicability, in order to put the operator in a position to control each step of the calculation procedure and use his own experience for choosing the better way to solve unexpected problems.

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Journal of Thermal Analysis and Calorimetry
Authors: G. Barone, P. Del Vecchio, D. Fessas, C. Giancola, G. Graziano, and A. Riccio

Abstract  

DSC measurements have been accomplished in aqueous solutions of bovine pancreatic ribonuclease A (RNAase A) in the presence of subsaturating amounts of 3′ cytidine monophosphate (3′ CMP) and 2′ cytidine monophosphate (2′ CMP) atpH 5.0 and 5.5. In these conditions the experimental profiles do not conform to a one-step unfolding process. It can be emphasized, as a general phenomenon, that a strong linkage between the temperature-induced protein unfolding and the ligand binding, when the ligand is less than the saturation level, causes marked distortions from a two-state transition. A purely equilibrium thermodynamic analysis gives a correct account of this behaviour and allows to simulate calorimetric curves. It is thus possible to obtain, in an indirect manner, information about the thermodynamic parameters concerning the binding process, namely the association constant and the binding enthalpy. The values ofKb and Δb H for 3′CMP and 2′CMP, so determined, are consistent with the literature data.

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Journal of Thermal Analysis and Calorimetry
Authors: G. Barone, S. Capasso, P. Del Vecchio, C. De Sena, D. Fessas, C. Giancola, G. Graziano, and P. Tramonti

Abstract  

In a previous paper, we report a preliminary DSC study on bovine (BSA) and human (HSA) serum albumins. However, at accurate HPLC analysis the commercial proteins show three peaks: Fraction V-I, probably globulins (as declared by the producers), Fraction V-II (about 15–18% of the product) and Fraction V-III that represents pure BSA or HSA. A hypothesis is that the Fraction II is a covalent dimer, or trimer or a mixture of both, generated during the scalf-life of the commercial product. Denaturation enthalpies of the purified Fraction V-III and Fraction V-II of BSA, have been determined calorimetrically, at changing thepH, and the results of both compared with those obtained on the untreated protein. Few calorimetric experiments have been also carried on a BSA monomer derivative with sulphidril group protected. Computer program have been developed for the deconvolution of exo- and endothermic effects and for the analysis of thermal denaturation profiles.

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Journal of Thermal Analysis and Calorimetry
Authors: G. Barone, P. Del Vecchio, D. Fessas, C. Giancola, G. Graziano, P. Pucci, A. Riccio, and M. Ruoppolo

The thermal denaturation of microbial Ribonuclease T1 (RNAase T1) as a function ofpH, was studied by means of DSC microcalorimetry. The midpoint denaturation temperatures, enthalpy changes and heat capacity changes of Ribonuclease T1 were compared with those obtained for pancreatic Ribonuclease A (RNAase A). It was found that the microbial T1 protein undergoes a more complex conformational transition than the simple two-state transition shown by Ribonuclease A. The hypothesis of the presence of a ‘molten globule’ form is discussed. The conformational stability of RNAase T1 is lower than that of RNAase A at highpH values. Indeed, the maximum stability of RNAase T1 occurs atpH ≈ 5, whereas that of RNAase A occurs atpH ≈ 8. AtpH=3.7 an irreversible aggregation phenomenon was indicated by the existence of a reproducible exothermic peak. The conformational transition of RNAase T1 is reversible in the range ofpH 4.5–7.0, whereas it becomes irreversible atpH≥8.0 as for RNAase A.

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