Thermal stability and secondary structure of feruloylated Rapana thomasiana hemocyanin

Journal of Thermal Analysis and Calorimetry - This is the first report on the conjugation of a hemocyanin from Rapana thomasiana (RtH) with ferulic acid, which itself exhibits an immunostimulatory...     

Thermal stability and folding kinetics analysis of disordered protein,securin

Lacking a stable tertiary structure, intrinsically disordered proteins (IDPs) possess particular functions in cell regulation, signaling, and controlling pathways. The study of their unique structural features, thermal stabilities, and folding kinetics is intriguing. In this study, an identified IDP, securin, was used as a model protein. By using a quasi-static five-step (on-path) folding process, the function of securin was restored and analyzed by isothermal titration calorimetry. Fluorescence spectroscopy and particle size analysis indicated that securin possessed a compact hydrophobic core and particle size. The glass transition of securin was characterized using differential scanning microcalorimetry. Furthermore, the folding/unfolding rates (k_obs) of securin were undetectable, implying that the folding/unfolding rate is very fast and that the conformation of securin is sensitive to solvent environmental change. Therefore, securin may fold properly under specific physiological conditions. In summary, the thermal glass transition behavior and undetectable k_obs of folding/unfolding reactions may be two of the indices of IDP.     

Structure and stability of arthropodan hemocyanin Limulus polyphemus

In the hemolymph of many arthropodan species, respiratory copper proteins of high molecular weight, termed hemocyanins (Hcs) are dissolved. In this communication, we report on the protein stability of different hemocyanin species (Crustacea and Chelicerata) using fluorescence spectroscopy. Five to seven major electrophoretically separable protein chains (structural subunits) were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography from different hemocyanins with very high sequence homology of the active site regions binding copper ions (CuA and CuB), and especially the relative sequence positions of histidine (His) and tryptophan (Trp) residues of these protein segments are in all cases identical. The conformational stabilities of the native dodecameric aggregates and their isolated structural subunits towards various denaturants (pH and guanidine hydrochloride (Gdn.HCl)) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby both, the oxy- and apo-forms of the protein are considered. These two classes of Crustacea and Chelicerata Hcs have the similar Trp-fluorescence quantum yields, but different values of lambda(max) emission (about 325 and 337 nm, respectively). Differences in the quantum yields are observed of the oxy- and apo-forms, which must be attributed to the fluorescence quenching effect of the two copper ions (CuA and CuB) in the active site. The position of emission maximum indicates tryptophan side chains are situated in a non-polar environment. Denaturation studies of Hcs by Gdn.HCl indicate that the denaturation process consists of two steps: dissociation of the native molecule into its structural subunits and denaturation of the subunits at concentrations >1.5M Gdn.HCl. Two steps of denaturation are also observed after keeping the protein in buffer solutions at different pH values with different pH-stability for holo-oxy and apo-Hc forms.     

Thermal analysis of protein behavior

The measurement and significance of heats associated with conformational transitions in solvated proteins and their synthetic analogues are discussed. An outline is given of the principles underlying several calorimetric and non-calorimetric techniques. Some new quantitative results relating to the denaturation of the protein lysozyme, obtained with a modified commercial differential scanning calorimeter, are presented.     

Thermal denaturation of mixtures of human serum proteins

Differential scanning calorimetry (DSC) has been employed to study the thermal denaturation processes of the main protein fractions of blood serum. These processes have been compared for albumins (nondefatted (HSA) and fatty acid free (HSAf)), α,β-globulins, γ-globulins, and their mixtures in aqueous (pH 6.5) and buffer (pH 7.2) solutions. The results have indicated that α,β-globulins inhibit γ-globulins’ aggregation in buffer solutions. The decrease of stability of HSA and HSAf aqueous solutions has been observed in the presence of γ-globulins. The mixtures of albumins and γ-globulins have revealed the tendency to ready aggregation in water. Moreover, the results have suggested that neither γ-globulins nor albumins severely change the stability of α,β-globulins.     

Salt-specific stability and denaturation of a short salt-bridge-forming alpha-helix

The structure of a single alanine-based Ace-AEAAAKEAAAKA-Nme peptide in explicit aqueous electrolyte solutions (NaCl, KCl, NaI, and KF) at large salt concentrations (3-4 M) is investigated using approximately 1 mus molecular dynamics (MD) computer simulations. The peptide displays 71% alpha-helical structure without salt and destabilizes with the addition of NaCl in agreement with experiments of a somewhat longer version. It is mainly stabilized by direct and indirect (" i + 4")EK salt bridges between the Lys and Glu side chains and a concomitant backbone shielding mechanism. NaI is found to be a stronger denaturant than NaCl, while the potassium salts hardly show influence. Investigation of the molecular structures reveals that consistent with recent experiments Na (+) has a much stronger affinity to side chain carboxylates and backbone carbonyls than K (+), thereby weakening salt bridges and secondary structure hydrogen bonds. At the same time, the large I (-) has a considerable affinity to the nonpolar alanine in line with recent observations of a large propensity of I (-) to adsorb to simple hydrophobes, and thereby "assists" Na (+) in its destabilizing action. In the denatured states of the peptide, novel long-lived (10-20 ns) "loop" configurations are observed in which single Na (+) ions and water molecules are hydrogen-bonded to multiple backbone carbonyls. In an attempt to analyze the denaturation behavior within the preferential interaction formalism, we find indeed that for the strongest denaturant, NaI, the protein is least hydrated. Additionally, a possible indication for protein denaturation might be a preferential solvation of the peptide backbone by the destabilizing cosolute (sodium). The mechanisms found in this work may be of general importance to understand salt effects on protein secondary structure stability.     

Thermal denaturation of collagen analyzed by isoconversional method

An isoconversional method is proposed to be used for evaluating activation energy of protein denaturation. Applied to DSC data on collagen denaturation, the method yields an activation energy that decreases throughout the process. The Lumry-Eyring model gives an explanation for this decrease and affords estimates for the enthalpy of the reversible step and the activation energy of the irreversible step of denaturation. The reversible unfolding is detectable by multi-frequency temperature-modulated DSC.     

Thermal stability and degradation products analysis of benzocyclobutene-terminated imide polymers

Benzocyclobutene-terminated imides were prepared and fully characterized with ¹H NMR, MS, and FT-IR. The thermal degradation of polymers was investigated by using thermogravimetric analyzer (TGA) and high-resolution pyrolysis-gas chromatography–mass spectrometry (HR-Py-GC–MS). TGA showed that thermal degradation of the polymer was a single-stage process in N₂, whereas a three-stage degradation in air atmosphere. The major involved products were found to be CO₂, naphthalene and naphthalene derivatives. Degradation mechanism of the polymer was suggested and the relationship between structures of the polymer and degradation products was also discussed.     

Cold denaturation of yeast frataxin offers the clue to understand the effect of alcohols on protein stability

Although alcohols are well-known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. In this paper, we present a study of the effects of alcohols on protein stability using Yfh1, the yeast ortholog of the human protein frataxin. Exploiting the unusual property of this protein of undergoing cold denaturation around 0 degrees C without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol, ethanol, and methanol. In all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. On the basis of simple thermodynamic considerations, we are able to interpret the literature on the effects of alcohols on proteins and to generalize our findings. We suggest that alcohols, at low concentration and physiological pH, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. Our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation.     

Thermal denaturation of ribonuclease T1 a DSC study

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 atpH8.0 as for RNAase A.This work was financed by the National Research Council (C.N.R.-Rome) and by Ministry of University and Scientific and Technological Research.     

Atomistic mechanism of protein denaturation by urea

Effects of urea on protein stability have been studied from all-atom molecular dynamics simulations of ubiquitin, G311 protein, and immunoglobulin binding domain (B1) of streptococcal protein G (GB1) in water and 8 M aqueous urea solution. The mechanism of the change in the solvent environment and the early events in protein unfolding by urea have been identified with emphasis on the change in the interactions of hydrophilic and hydrophobic parts of the protein by calculating the potential of mean force (PMF). Urea replaces the protein-protein and protein-water contacts by forming stronger contacts with the protein, which is indicated by the longer survival times of the protein-urea hydrogen bonds.     

Thermal denaturation of bovine serum albumin and its oligomers and derivativespH dependence

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.     

Phytochemical analysis and antidiabetic efficacy of Morus rubra

Morus rubra (red mulberry) is a folk medicine used in management of diabetes. The aim of this article to investigate the antidiabetic activity of hydro alcoholic leaf extracts of Morus rubra in the sprague dawley male rats (SD) model. Authentication of plant material, total Phenolic contents, total flavonoid contents, HPLC analysis, in-vitro antioxidant activity, acute toxicity, in-vivo oral glucose tolerance test, in-vivo STZ induced antidiabetic activity along with biochemical parameters (triglycerides, low-density lipoprotein, high-density lipoprotein, total cholesterol, serum glutamate phosphotransferase, serum glutamate oxaloacetic acid transferase, urea and creatinine). The total phenolic contents 60.32 ​± ​0.80 ​mg/g GAE (gallic acid equivalent) were found higher in ethanolic extract. The total flavonoids 16 ​± ​0.42 ​mg/g QE (quercetin equivalent) were found in petroleum ether extract. M. rubra leaf extracts were tested by HPLC for qualitative and quantitative analysis. 100 ​mg of the dried powder of Morus leaves was dissolve in methanol: water (80:20, v/v) for analysis. Chromatograms of HPLC show the presence of chlorogenic acid 0.55 mg/gm, quercetin 1.90 ​mg/g, isoquercetin 0.68 mg/gm and rutin 0.10 mg/gm. IC₅₀ (Inhibitory concentration) value has been found higher in chloroform extract (92.89 ​μg/ml). Metformin drug is used for positive control, which causes 14.6% improvement in glucose tolerance while M. rubra has been found 11.4. % at the dose of 300 ​mg/kg body wt in normal rats. In STZ treated diabetic rats, it reduces 13.0% blood glucose level while metformin reduces 21.9% up to dosing level of 300 ​mg/kg body wt. It produces reduction in biochemical parameters like, total cholesterol (TC), triglycerides (TG), SGPT, LDL, urea, creatinine and SGOT while HDL has been increased. Results show the hydro-alcoholic leaves extract of Morus rubra exhibited antidiabetic, antioxidant and hypolipidemic properties. This suggests that M. rubra has great potential to develop novel antidiabetic drug to manage this disastrous disease.     

An electrospray-ionization mass spectrometry analysis of the pH-dependent dissociation and denaturation processes of a heterodimeric protein

Electrospray ionization mass spectrometry (ESI-MS) was applied to the analysis of the dissociation and denaturation processes of a heterodimeric yeast killer toxin SMKT. The two distinct subunits of SMKT noncovalently associate under acidic conditions, but become dissociated and denatured under neutral and basic conditions. In order to understand the unique pH-dependent denaturation mechanism of this protein, a pH titration was performed by utilizing ESI-MS. The molecular ions of the heterodimer which possesses the highly ordered structure, were mainly observed below pH 4.6. However, the two subunits immediately dissociated at this pH. The spectra measured with various settings of the mass spectrometer indirectly demonstrated that the pH-dependent dissociation occurs in the liquid phase. The current result as well as the three-dimensional structure of SMKT suggest that the deprotonation of a specific carboxyl group triggers a cooperative dissociation process of this protein. In conclusion, the pH titration of a protein by ESI-MS is particularly effective, when the unfolding process or the biological function of the protein is related to the interaction with other molecules.     

Thermal analysis and longtime stability of amorphous Co100−xBx alloys

Crystallization behaviour of amorphous Co_100?xB_x alloys (17 <x ≤ 40) has been investigated by differential thermal analysis (DTA) and dynamic temperature X-ray diffraction (DTXD) methods in the freshly prepared state and at a period of about eight years after preparation. The crystallization temperatures lie in the range 670 K–760 K. An average decrease of about 1.25 K/year over a period of eight years has been observed to take place in the crystallization temperatures of these materials. The value of heat of crystallization (ΔH _cr) and activation energy lie in the range 2.3 kJ/g-at – 5.9 kJ/g-at and 2.1 eV – 2.4 eV, respectively. The phases obtained at crystallization temperatures during DTXD analysis have been discussed.     

Thermal denaturation produced degenerative proteins and interfered with MS for proteins dissolved in lysis buffer in proteomic analysis

In 1-DE, proteins were traditionally mixed with the standard Laemmli buffer and boiled for several minutes. Recently, proteins dissolved in lysis buffer were used to produce better-resolved 2-DE gels, but thermal denaturation procedure still remained in some proteomic analysis. To determine the detailed effects of thermal denaturation on SDS-PAGE and MS, both 1-DE and 2-DE were performed using proteins heated at 100°C for different periods of time, and 17 protein bands/spots were positively identified by MALDI TOF/TOF MS/MS. Protein profiles on both 1-DE and 2-DE gels changed obviously and more polydisperse bands/spots were observed with increased heating time for over-heated samples. Based on these observations, an alternative protein marker-producing method was designed by directly dissolving protein standards without BSA into lysis buffer. This new kind of protein marker could be stored at room temperature for a long time, thus was more convenient for using and shipping. The identification of 17 proteins via MS and comparison of their identities revealed MASCOT-searched scores, number of both matched peptides, total searched peptides and sequence coverage became progressively lower with increasing denaturation intensity, probably due to the interference of thermal denaturation on trypsin cleavage efficiency and produced redundant modified peptides. Therefore, it was concluded that thermal denaturation not only changed the protein profiles and produced more polydisperse protein bands/spots, but also heavily interfered with the subsequent MS analysis, hence not recommended in future proteomic analysis for proteins dissolved in lysis buffer.     

Thermal stability of ketoprofen

The purpose of this investigation is to calculate the kinetic parameters and the kinetic model for the studied process. The results are further used to predict the system’s behaviour in various circumstances. A kinetic study regarding the ketoprofen—involving active substance’s thermal decomposition—was performed under isothermal conditions and in a nitrogen atmosphere, for the temperature steps: 260; 265; 270; 275; and 280 °C. The thermogravimetry/derivative thermogravimetry data were processed by three differential methods: isothermal–isoconversional, Friedman’s isothermal–isoconversional, and isothermal model-fittings. The obtained results are in good accordance with those obtained under non-isothermal conditions of a previous study, and confirm the necessity for the kinetic parameters to be determined, under different thermal conditions, by the adequate calculation methods.     

Thermal stability of Peroxynitrates

Peroxynitrates are thermally unstable intermediates (at ambient temperatures) in the atmospheric degradation of hydrocarbons. In this work, thermal lifetimes of nine peroxynitrates have been measured as a function of temperature and, for two of them, also, as a function of total pressure. In the presence of excess NO, relative concentrations of the peroxynitrates were followed in a 420 I reaction chamber as a function of time by means of longpath IR absorption using a Fourier transform spectrometer. Original data on the unimolecular decomposition rate constants are presented for the peroxynitrates RO₂NO₂ with R = C₆H₁₁, CH₃C(O)CH₂, C₆H₅CH₂, CH₂I, CH₃C(O)OC(H)CH₃, C₆H₅OCH₂, (CH₃)₂NC(O), C₆H₅OC(O), and C₂H₅C(O). Thermal lifetimes at room temperature and atmospheric pressure are very short (in the order of seconds) for substituted methyl peroxynitrates (i.e., R'CH₂O₂NO₂) but rather long for substituted formyl peroxynitrates (i.e., R″C(O)O₂NO₂). Kinetic data from this and previous work from our laboratory are used to derive structure‐stability relationships which allow an estimate of the thermal lifetimes of peroxynitrates from readily available ¹³C n.m.r. shift data. ©1999 John Wiley & Sons, Inc. Int J Chem Kinet 31: 127–144, 1999