Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20-60°C) and protein concentration (5-100 µmol L^-1). The time courses of loss of enzyme activity in 100 mmol L^-1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k _a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k _b) and binding (k _-b) as well as the irreversible aggregation of the apo-enzyme (k _agg) were less sensitive to changes in temperature, their activation energy (E _a) being about 52 kJ mol^-1 in comparison with an E _a value of 185 kJ mol^-1 for k _a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5'-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^-1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

Multi-temperature effects on Hill reaction activity of barley chloroplasts

1. 1. The relationship between temperature and Hill reaction activity has been investigated in chloroplasts isolated from barley (Hordeum vulgare L. cv. Abyssinian).

2. 2. An Arrhenius plot of the photoreduction of 2,6-dichlorophenolindophenol (DCIP) showed no change in slope over the temperature range 2–38 °C. The apparent Arrhenius activation energy (E_a) for the reaction was 48.1 kJ/mol.

3. 3. In the presence of an uncoupler of photophosphorylation, methylamine, the E_a for DCIP photoreduction went through a series of changes as the temperature was increased. Changes were found at 9, 20, 29 and 36 °C. The E_a was highest below 9 °C at 63.7 kJ/mol. Between 9 and 20 °C the E_a decreased to 40.4 kJ/mol and again to 20.2 kJ/mol between 20 and 29 °C. Between 29 and 36 °C there was no further increase in activity with increasing temperature. The temperature-induced changes at 9, 20 and 29 °C were reversible. At temperatures above 36 °C (2 min) a thermal and largely irreversible inactivation of the Hill reaction occurred.

4. 4. Temperature-induced changes in E_a were also found when ferricyanide was substituted for DCIP or gramicidin D for methylamine. The addition of an uncoupler of photophosphorylation was not required to demonstrate temperature-induced changes in DCIP photoreduction following the exposure of the chloroplasts to a low concentration of cations.

5. 5. The photoreduction of the lipophilic acceptor, oxidized 2, 3, 5, 6-tetramethyl-p-phenylenediamine, also showed changes in E_a in the absence of an uncoupler.

6. 6. The temperature-induced changes in Hill activity at 9 and 29 °C coincided with temperature-induced changes in the fluidity of chloroplast thylakoid membranes as detected by measurements of electron spin resonance spectra. It is suggested that the temperature-induced changes in the properties and activity of chloroplast membranes are part of a control mechanism for regulation of chloroplast development and photosynthesis by temperature.

Thermal inactivation, denaturation and aggregation of mitochondrial aspartate aminotransferase

A comparative study of thermal denaturation and inactivation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been carried out (10 mM Na phosphate buffer, pH 7.5). Analysis of the data on differential scanning calorimetry shows that thermal denaturation of mAAT follows the kinetics of irreversible reaction of the first order. The kinetics of thermal inactivation of mAAT follows the exponential law. It has been shown that the inactivation rate constant (k_in) is higher than the denaturation rate constant (k_den). The k_in/k_den ratio decreases from 28.8 ± 0.1 to 1.30 ± 0.09 as the temperature increases from 57.5 to 77 °C. The kinetic model explaining the discrepancy between the inactivation and denaturation rates has been proposed. The size of the protein aggregates formed at heating of mAAT at a constant rate (1 °C min^{− 1}) has been characterized by dynamic light scattering.     

Gelation of xanthan with trivalent metal ions

In-situ gelation of semidilute xanthan solutions with trivalent chromium, aluminum or iron ions was studied by rheology and UV-spectroscopy. Measurements of the elastic modulus of xanthan gel cylinders prepared by dialysis against the complexing ion at pH values from 2 to 6 indicate that monomeric species of the ion are ineffective, whereas dimeric or higher oligomeric species are effective in crosslinking the polysaccharide. When chromium was used as the crosslinking species, the dependence of the gelation rate on the ionic concentration followed a power law with a coefficient of 1·7. The gelation time and the gelation rate were found to extrapolate to zero at 1 m Cr for 2·5 mg/ml xanthan. The limiting concentration of xanthan needed for gelation with 5 m Cr(III) at 20°C was estimated as 0·35 mg/ml. This critical xanthan concentration is close to the overlap concentration c^* estimated from the experimentally determined intrinsic viscosity [η] using c^* = 1·4/[η]. An apparent activation energy for crosslinking of xanthan was calculated as E_a = 42 kJ/mol and E_a = 108 kJ/mol for Cr and Al ions, respectively. The fractal dimensionality of xanthan-Cr at the sol-gel transition was estimated as 1·3 applying the Chambon-Winter criterion for gelation, thus indicating that this gelation criterion is applicable also to stiff-chain polysaccharides such as xanthan.     

Changes in denaturation and rheological properties of collagen-hyaluronic acid scaffolds as a result of temperature dependencies

This report describes the effect of temperature on the mechanical viscoelastic properties such as: storage modulus (E′), loss modulus (E″), and loss tangent (tan δ) of the collagen sponges modified with hyaluronic acid (HA). In order to detect collagen–HA copolymer denaturation and to assess its thermal stability, the differential scanning calorimetry (DSC) supplemented by thermogravimetric (TG) measurements was used. The denaturation temperature (T_d) of unmodified collagen samples increased from 69 to 86 °C for cross-linked samples, respectively. These temperature dependencies show remarkable changes in E′ and E″ at selected temperature up to 226 °C for all samples due to the release of loosely and strongly bound water. The influence of HA on the viscoelastic behavior of collagen is manifested by a shift of the tan δ peak associated with the process of decomposition towards higher temperatures resulting in a higher thermo-stability of the modified scaffolds.     

Two-state irreversible thermal denaturation of muscle creatine kinase.

Thermal denaturation of creatine kinase from rabbit skeletal muscle has been studied by differential scanning calorimetry. The excess heat capacity vs. temperature profiles were independent of protein concentration, but strongly temperature scanning rate-dependent. It has been shown that thermal denaturation of creatine kinase satisfies the previously proposed validity criteria for the two-state irreversible model [Kurganov et al., Biophys. Chem.70 (1997) 125]. The energy activation value has been calculated to be 461.0 +/- 0.7 kJ/mol.     

A comparative investigation of the thermal unfolding of pseudoazurin in the Cu(II)-holo and apo form

The contribution of the copper ion to the stability and to the unfolding pathway of pseudoazurin was investigated by a comparative analysis of the thermal unfolding of the Cu(II)-holo and apo form of the protein. The unfolding has been followed by calorimetry, fluorescence, optical density, and electron paramagnetic resonance (EPR) spectroscopy. The thermal transition of Cu(II)-holo pseudoazurin is irreversible and occurs between 60.0 and 67.3 degrees C, depending on the scan rate and technique used. The denaturation pathway of Cu(II)-holo pseudoazurin can be described by the Lumry-Eyring model: N --> U --> [corrected] F; the protein reversibly goes from the native (N) to the unfolded (U) state, and then irreversibly to the final (F) state. The simulation of the experimental calorimetric profiles, according to this model, allowed us to determine the thermodynamic and kinetic parameters of the two steps. The DeltaG value calculated for the Cu(II)-holo pseudoazurin is 39.2 kJ.mol(-1) at 25 degrees C. The sequence of events in the denaturation process of Cu(II)-holo pseudoazurin emergence starts with the disruption of the copper site and the hydrophobic core destabilization followed by the global protein unfolding. According to the EPR findings, the native type-1 copper ion shows type-2 copper features after the denaturation. The removal of the copper ion (apo form) significantly reduces the stability of the protein as evidenced by a DeltaG value of 16.5 kJ.mol(-1) at 25 degrees C. Moreover, the apo Paz unfolding occurs at 41.8 degrees C and is compatible with a two-state reversible process N --> [corrected] U.     

Thermal stability of Artemia HGPRT: effect of substrates on inactivation kinetics

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, E.C. 2.4.2.8) from Artemia cysts exhibits maximum activity at 70°C. Its thermal stability has been examined following enzymatic activity as a function of temperature. Cold-induced renaturation experiments of samples heated at increasing temperatures showed that reversibility of thermal inactivation depends on the incubation time and final temperature. Prolonged incubation of the thermoinactivated enzyme at 0°C did not afford any further increase of the catalytic activity at 37°C. The complex substrate PRPP:Mg protects HGPRT from thermal inactivation. However, incubations with hypoxanthine rendered a less thermostable enzyme at any temperature tested. The irreversible inactivation of HGPRT proceeds in two exponential steps. The analysis of the apparent rate constants for the fast and the slow phases, λ₁ and λ₂ as per the Lumry and Eyring model suggests the existence of more than three states in the thermal denaturation pathway of the free enzyme. In the presence of PRPP:Mg the irreversible process follows a single exponential and proceeds very slowly below 70°C. PRPP:Mg also protects the enzyme from inactivation by NEM and pCMB, suggesting that -SH groups may be in the vicinity of the active site     

Thermal denaturation of porcine pepsin: a study by circular dichroism

Thermal denaturation of porcine pepsin in 10% ethanol was studied by circular dichroism (CD) spectroscopy. It was observed that the process is markedly irreversible. The denaturation unfolding process was strongly dependent on the heating rate, as is expected for an unfolding process kinetically controlled due to the presence of an irreversible reaction. Experimentally, we demonstrate the existence of an unfolded (U) state in equilibrium with the native (N) state. The U state is observed to exist at temperatures lower than 45 degrees C. The van't Hoff enthalpy, DeltaH(vH), was determined from direct estimation of the equilibrium constant at several temperatures (DeltaH(vH)=304.3 kJ/mol). To explain the observed behavior, we have considered a Lumry-Eyring model, which takes into account the presence of the U state in addition to N and denatured (D) states (i.e. N<-->U-->D).     

The effects of temperature and adrenergic agonists on cardiac myocytes of perch (Perca fluviatilis) in cell culture conditions

1. 1. Isolated cardiac myocytes of perch, Perca fluviatilis, were kept in culture conditions for 1–2 months at 12 or 22°C. In the culture most myocytes flattened, lost their spindle-shaped morphology, protruded pseudopod-like branches and many of them started visible contractions in 1–2 weeks and continued beating for several months. Myocytes did not divide in the sparse cell population used. Typical intracellular structures could be seen in electron micrographs still after 1–2 months, but the sarcoplasmic organization became gradually more irregular in the culture.

2. 2. Beat rates showed linear temperature relationship on the Arrhenius plot. Myocytes cultivated at 22°C showed higher frequencies and slightly less dependence on temperature than myocytes cultivated at 12°C (apparent activation energies (E_a) 86 and 107 kJ/mol, respectively).

3. 3. Temperature dependence of frequencies was related to the presence of added serum or adrenergic agonists: β-adrenergic agonists increased the frequencies and rendered the cells less dependent on temperature; apparent activation energy was 43 kJ/mol for isoprenaline or adrenaline and 108 kJ/mol for noradrenaline and control group.

4. 4. Heat tolerance was greater in myocytes cultivated at 22°C than in myocytes cultivated at 12°C, and the change in tolerance appeared in 12 h after the alteration of culture temperature and the increased tolerance was persistent after that.

5. 5. It is suggested, that the processes of quick heat-hardening and of slower but persistent heat resistance acclimation developed in these cells in culture conditions but not the capacity acclimation, which seems to be dependent on adrenergic regulation of beat rate.

Ricochet inter-ring haptotropic rearrangement of σ-methyl-(η-indenyl) chromium tricarbonyls. Experimental kinetic and theoretical DFT study

σ-Methyl-(η⁵-indenyl) chromium tricarbonyl (III) rearranges quantitatively into η⁶-1-endo-methylindene) chromium tricarbonyl (IV) in C₆D₆ solution at 30–60°C. Methyl group attachment to the positions 2 or 3 of indenyl ligand in (III) has no influence on the activation parameters of this ricochet inter-ring haptotropic rearrangement (ΔG^#=23.6 kcal mol⁻¹; ΔH^#=18.9±0.2 kcal mol⁻¹; ΔS^#=−18.6±0.2 cal K⁻¹ mol⁻¹). (IV) undergoes further irreversible isomerization at 60–120° into (ν⁶-3-methylindene) chromium tricarbonyl (V) with a higher activation barrier (ΔG^#=28.5±0.1 kcal mol⁻¹) via two consecutive [1,5]-sigmatropic hydrogen shifts. The mechanisms of both rearrangements have been studied in detail using density functional theory (DFT) calculations with extended basis sets. Calculations show that the rearrangement (III) → (IV) proceeds in two steps. Methyl group migration from chromium into position 1 of the indenyl ligand is the rate-determining step leading to the formation of the 16-electron intermediate (VII). The calculated activation barrier (E_a=19.6 kcal mol⁻¹) is in good agreement with the experimental one. Further rearrangement (VII) → (V) proceeds via a trimethylenemethane-type transition state (XVIII) with an activation barrier 11.8 kcal mol⁻¹. The coordination of the chromium tricarbonyl group at the six-membered ring has only minor influence on the kinetic parameters of the hydrogen [1,5]-sigmatropic shift in indene.     

Equilibrium and kinetic studies on reversible and irreversible denaturation of micrococcal nuclease

The effect of pH and temperature on the thermal denaturation of micrococcal nuclease wer4e investigated. The ranges employed were between pH3.30 and pH9.70 and between 10 degrees C and 85 degrees C, respectively. The reversible denaturation involved in the whole process was clearly discriminated from the irreversible one. The former took place with a large enthalpy change of 384 kJ mol(-1) at pH 9.70, where the enzyme exhibited it s maximum activity. The latter probably led to aggregation because the successive long incubation after complete deactivation caused precipitation. A reasonable scheme explaining the process involving both denaturations was proposed and the kinetic on the irreversible deactivation was performed. It was revealed that the irreversible deactivation involved two types of reactions whose activation energies were relatively small: 22.2 kJ mol(-1) and 18.8kJ mol(-1). The presence of sucrose suppressed the reversible denaturation without significant influence on enthalpy change, whereas it affected little the irreversible deactivation kinetically. The effects of pH change and addition of sucrose on the denaturation were discussed thermodynamically, especially in terms of the entropy change. (c) 1994 John Wiley & Sons, Inc.     

The effect of copper/zinc replacement on the folding free energy of wild type and Cys3Ala/Cys26Ala azurin

The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation experiments have shown that, in both cases, the thermal transitions of the zinc derivative of azurins can be depicted in terms of the classical Lumry–Eyring model, NUF, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temperature than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temperature. Comparison of the thermodynamic data shows that the presence of Zn in the active site stabilises the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temperature of Zn azurin has increased by 4 °C, while the unfolding free energy, ΔG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the observed ΔG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the reference state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilising effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn derivative, which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, E_a, as derived from the kinetic stability analysis.     

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.     

Polyvinylferrocenium immobilized enzyme electrode for choline analysis

The response characteristics of a new enzyme electrode for determining choline are reported. The enzyme electrode consists of a polyvinylferrocenium perchlorate coated Pt surface onto which the enzyme, choline oxidase, is attached. Choline oxidase catalyzes the oxidation of choline to betaine, producing H₂O₂. Current due to H₂O₂ oxidation catalyzed by polyvinylferrocenium centers was measured. The effects of choline concentration, the amount of enzyme immobilized and the operating pH and temperature on the response of the enzyme electrode were studied. The effects of interferents were also investigated. The response time was found to be 60–70 s and the upper limit of the linear working portion was found to be 1.2 mM choline concentration. The minimum substrate concentration that produced detectable current was 4.0×10⁻⁶ M choline concentration. The steady-state current of this enzyme electrode was reproducible within ±4.6% of relative error. The apparent Michaelis–Menten constant (K_Mapp) and the activation energy, E_a, of this immobilized enzyme system were found to be 2.32 mM and 38.91 kJ/mol, respectively.     

Dynamic changes in sweat rates and evaporation rates through clothing during hot exposure

Dynamic changes in local sweat rates (S_w) and local evaporation rates from clothing (E_cl) have been observed during hot exposure. Four young male subjects wearing a cotton T-shirt and half shorts were exposed to 40 °C/50% for 1 h following exposure to 28 °C/50% for 30 min. Amount of water absorbed in clothing (M_sw), clothing surface temperatures (T_cl), local heat flow rates, skin temperatures, body weight, rectal temperature, S_w and E_cl were continuously measured. Upon exposure to the heat, decrease in heat gain to the skin was observed as opposed to increase in S_w, E_cl, M_sw and heat gain to the clothing surface.     

Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the transition to the denatured state with residual structure

Glucose oxidase (GOX; beta-d-glucose:oxygen oxidoreductase) from Aspergillus niger is a dimeric flavoprotein with a molecular mass of 80 kDa/monomer. Thermal denaturation of glucose oxidase has been studied by absorbance, circular dichroism spectroscopy, viscosimetry, and differential scanning calorimetry. Thermal transition of this homodimeric enzyme is irreversible and, surprisingly, independent of GOX concentration (0.2-5.1 mg/ml). It has an apparent transition temperature of 55.8 +/- 1.2 degrees C and an activation energy of approximately 280 kJ/mol, calculated from the Lumry-Eyring model. The thermally denatured state of GOX after recooling has the following characteristics. (i) It retains approximately 70% of the native secondary structure ellipticity; (ii) it has a relatively low intrinsic viscosity, 7.5 ml/g; (iii) it binds ANS; (iv) it has a low Stern-Volmer constant of tryptophan quenching; and (v) it forms defined oligomeric (dimers, trimers, tetramers) structures. It is significantly different from chemically denatured (6.67 m GdmHCl) GOX. Both the thermal and the chemical denaturation of GOX cause dissociation of the flavin cofactor; however, only the chemical denaturation is accompanied by dissociation of the homodimeric GOX into monomers. The transition temperature is independent of the protein concentration, and the properties of the thermally denatured protein indicate that thermally denatured GOX is a compact structure, a form of molten globule-like apoenzyme. GOX is thus an exceptional example of a relatively unstable mesophilic dimeric enzyme with residual structure in its thermally denatured state.     

A computational formula for heat influx in animal experiments

1. 1.The bahavioural paradigm in which cold-exposed animals can work for pulses of infrared radiation has been extensively used in the literature, but a formula to calculate the amount of heat obtained has not been advanced.

2. 2.This paper describes a computational formula for heat influx in rats: E = 3.64 · 10⁻⁶ · n · d · I · M^0.6 where E is heat influx (kJ), n is number of rewards, d is reward duration (sec), I is irradiance (mW/cm²), and M is body mass (g).

Differential scanning calorimetry and X-ray diffraction study of tendon collagen thermal denaturation

Differential scanning calorimetry, high and small angle X-ray diffraction analyses have been carried out on air-dried and rehydrated rat tail tendon collagen in order to test the reversibility of collagen thermal denaturation. The mean enthalpy values calculated for the denaturation process of air-dried and rehydrated samples are ΔH_D = 9.0 ± 0.8 cal/g and ΔH_D = 11.9 ±0.7 cal/g respectively, while the denaturation temperatures are T_D = 112 ± 1°C and T_D = 51 ± 1°C. Partial reversibility of the coiled coil—random coil process can be obtained by storing the samples in air or more rapidly by equilibration in water. After denaturation air-dried collagen fibres recover not only their molecular structure but also their characteristic fibrillar structure. The latter does not greatly influence the mean experimental enthalpy values.     

Modeling of irreversible thermal protein denaturation at varying temperature. II. The complete kinetic model of Lumry and Eyring.

The model for thermal denaturation of proteins involving consecutive reversible and irreversible steps (Lumry and Eyring model) has been analyzed. The most general case, when equilibrium in the first step is established slowly in comparison with the rate of the second step and the heat effect value for the second step is either greater than or less than zero, has been considered. The theoretical dependences of excess heat capacity on temperature have been constructed. The variation of the shape of the theoretical curves with varied values of the enthalpy change for the second step, Arrhenius equation parameters for both steps, and the scanning rate has been studied.