Galina G. Zhadan

Comparative calorimetric study of non-amyloidogenic and amyloidogenic variants of the homotetrameric protein transthyretin.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant hereditary type of amyloidosis involving amino acid substitutions in transthyretin (TTR). V30M-TTR is the most frequent variant, and L55P-TTR is the variant associated with the most aggressive form of FAP. The thermal stability of the wild-type, V30M-TTR, L55P-TTR and a non-amyloidogenic variant, T119M-TTR, was studied by high-sensitivity differential scanning calorimetry (DSC). The thermal unfolding of TTR is a spontaneous reversible process involving a highly co-operative transition between folded tetramers and unfolded monomers. All variants of transthyretin are very stable to the thermal unfolding that occurs at very high temperatures, most probably because of their oligomeric structure. The data presented in this work indicated that for the homotetrameric form of the wild-type TTR and its variants, the order of stability is as follows: wild-type TTR approximately > T119M-TTR > L55P-TTR > V30M-TTR, which does not correlate with their known amyloidogenic potential.

Applications of scanning microcalorimetry in biophysics and biochemistry

Abstract Scanning calorimetry is a very powerful and convenient technique for studying temperature-induced conformational transitions in biological systems. The present paper reviews recent applications of the microcalorimetry method in biochemistry and biophysics.

Dynamic properties of Newcastle Disease Virus envelope and their relations with viral hemagglutinin-neuraminidase membrane glycoprotein.

The lipid composition of Newcastle Disease Virus (NDV) Clone-30 strain shows a low lipid/protein ratio, a high cholesterol/phospholipid molar ratio, and major phospholipids being qualitatively different to other NDV strains. The major fatty acyl constituents are palmitic, stearic, oleic, and linoleic acids; cerebrosides, sulfatides and two kinds of gangliosides are also found in the NDV membrane. It is reported for the first time in NDV that phospholipid classes are asymmetrically distributed over the two leaflets of the membrane: 60 +/- 4.5% of the phosphatidylcholine and 70 +/- 5.0% of the sphingomyelin are in the outer monolayer. Intact viral membranes and reconstituted NDV envelopes showed similar dynamic properties. Hemagglutinin-neuraminidase (HN) and fusion (F) proteins of NDV membrane affect the lipid thermotropic behaviour in reconstituted proteoliposomes made up of a single class of phospholipids. It is shown that the lipid composition is more important than the bulk membrane fluidity/order for both sialidase (neuraminidase) and hemagglutinating HN activities. Sialidase and hemagglutinating activities requires the presence of definite phospholipids (phosphatidylethanolamine) in its environment.

Thermal stability of peroxidase from Chamaerops excelsa palm tree at pH 3

The structural stability of a peroxidase, a dimeric protein from palm tree Chamaerops excelsa leaves (CEP), has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism and steady-state tryptophan fluorescence at pH 3. The thermally induced denaturation of CEP at this pH value is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, leading to the conclusion that in solution CEP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of CEP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N-->kD, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate.

Thermostability of cardosin A from Cynara cardunculus L.

The structural stability of cardosin A, a plant aspartic proteinase (AP) from Cynara cardunculusL., has been investigated by high-sensitivity differential scanning calorimetry, intrinsic fluorescence and circular dichroism spectroscopy, and enzymatic activity assays. Even though the thermal denaturation of cardosin A is partially irreversible, valid thermodynamic data can be obtained within a wide pH region. Also, although cardosin A is a heterodimeric enzyme its thermal denaturation occurs without simultaneous dissociation to unfolded monomers. Moreover, in the 3–7 pH region the excess heat capacity can be deconvoluted into two components corresponding to two elementary two-state transitions, suggesting that the two polypeptide chains of cardosin A unfold independently. Detailed thermodynamic and structural investigations of cardosin A at pH = 5.0, at which value the enzyme demonstrates maximum stability and enzymatic activity, revealed that after thermal denaturation the polypeptide chains of this protein retain most of their secondary structure motifs and are not completely hydrated.

pH‐dependent thermal transitions of lentil lectin

The thermal stability of lentil lectin in the 5.0–10.0 pH range was studied by high‐sensitivity differential scanning calorimetry and infrared spectroscopy. The thermally induced transitions for protein were irreversible and strongly dependent upon the scan rate at all pH values, suggesting that the denaturation is under kinetic control. It is shown that process of lentil lectin denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, , where k is a first‐order kinetic constant that changes with temperature, as given by the Arrhenius equation, N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.

Purification and structural stability of white Spanish broom (Cytisus multiflorus) peroxidase

New plant peroxidase has been isolated to homogeneity from the white Spanish broom Cytisus multiflorus. The enzyme purification steps included homogenization, NH(4)SO(4) precipitation, extraction of broom colored compounds and consecutive chromatography on Phenyl-Sepharose, HiTrap™ SP HP and Superdex-75 and 200. The novel peroxidase was characterized as having a molecular weight of 50 ± 3 kDa. Steady-state tryptophan fluorescence and far-UV circular dichroism (CD) studies, together with enzymatic assays, were carried out to monitor the structural stability of C. multiflorus peroxidase (CMP) at pH 7.0. Thus changes in far-UV CD corresponded to changes in the overall secondary structure of enzyme, while changes in intrinsic tryptophan fluorescence emission corresponded to changes in the tertiary structure of the enzyme. It is shown that the process of CMP denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, N ⟶ kD, where k is a first-order kinetic constant that changes with temperature following the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.

Protein involvement in thermally induced structural transitions of pig erythrocyte ghosts

Thermal transitions in pig erythrocyte ghosts were studied by differential scanning calorimetry and thermal gel analysis (TGA). Heating of the suspension of pig erythrocyte ghosts induced at least six thermodynamically irreversible transitions. Each of these transitions is believed to be due to a localized structural transition induced by thermal stress. Using TGA and covalent attachment of the anionic transport inhibitor regions in the thermograms corresponding to the heat sorption of some proteins of the pig erythrocyte ghosts were identified.

Biosorption of Heavy Metals from Acid Mine Drainages onto Pig Bristles, Poultry Feathers and Crustacean Shells Industrial Biowastes

Normal 0 21 false false false MicrosoftInternetExplorer4 The removal of metals ions from aqueous solutions plays an important role in water pollution control. In this study, a biosorption process for the bioremediation of heavy metal-contaminated acid mine drainages, located in Western Spain, has been developed. The process is based on the physico-chemical properties for the adsorption, ion exchange, and complexation of metal ions by biopolymers keratin and chitin from different industrial biowastes such as pig bristles, poultry feathers and crustacean shells. The selectivity for metals, the first order kinetics and yields of the corresponding biosorption processes of uranium and other metals polluting such acid mine drainages by such biosorbents are described. The biowaste rich in keratin (pig bristles) seems to show a higher biosorption capacity than that of bioresidues rich in chitin (crustacean shells). Moreover, factors such as the lower contamination by metals of acid waters, the lower influent water volume/biosorbent mass ratio, the configuration of the packed-bed reactor and the partial hydrolysis of keratin increase both the capacity and the rate of the process of metal biosorption onto the biosorbent.

Thermal stability of matrix protein from Newcastle disease virus.

The thermal stability of the matrix protein (M protein) of Newcastle disease virus (NDV) has been investigated using high-sensitivity differential scanning calorimetry (DSC) at pH 7.4. The thermal folding/unfolding of M protein at this pH value is a reversible process involving a highly cooperative transition between folded and unfolded monomers with a transition temperature (Tm) of 63 °C, an unfolding enthalpy, ΔH(Tm), of 340 kcal mol(-1), and the difference in heat capacity between the native and denatured states of the protein, ΔCp, of 5.1 kcal K(-1) mol(-1). The heat capacity of the native state of the protein is in good agreement with the values calculated using a structure-based parameterization, whereas the calculated values for the hypothetical fully-unfolded state of the protein is higher than those determined experimentally. This difference between the heat capacity of denatured M protein and the heat capacity expected for an unstructured polypeptide of the same sequence, together with the data derived from the heat-induced changes in the steady-state fluorescence of the protein, indicates that the polypeptide chain maintains a significant amount of residual structure after thermal denaturation.