Scan Rate Dependence Research Articles

Time-Dependence of Molecular Mobility during Structural Relaxation and its Impact on Organic Amorphous Solids: An Investigation Based on a Calorimetric Approach

To develop a calorimetry-based model for estimating the time-dependence of molecular mobility during the isothermal relaxation of amorphous organic compounds below their glass transition temperature (Tg). The time-dependent enthalpy relaxation times of amorphous sorbitol, indomethacin, trehalose and sucrose were estimated based on the nonlinear Adam-Gibbs equation. Fragility was determined from the scanning rate dependence of Tg. Time evolution of the fictive temperature was determined from Tg, the heat capacity of the amorphous and crystalline forms, and from the enthalpy relaxation data. Relaxation time changes significantly upon annealing for all compounds studied. The magnitude of the increase in relaxation time does not depend on any one parameter but on four parameters: Tg, fragility, and the crystal-liquid and glass-liquid heat capacity differences. The obtained mobility data for indomethacin and sucrose, both stored at Tg-16 K, correlated much better with their different crystallization tendencies than did the Kohlrausch-Williams-Watts (KWW) equation. The observed changes in relaxation time help explain and address the limitations of the KWW approach. Due consideration of the time-dependence of molecular mobility upon storage is a key element for improving the understanding necessary for stabilizing amorphous formulations.

Kinetic study on the irreversible thermal denaturation of Schistosoma japonicum glutathione s-transferase

The thermal unfolding pathway of the Schistosoma japonicum glutathione S-transferase (Sj26GST) was previously interpreted by applying equilibrium thermodynamics and a reversible two-state model (Kaplan et al., (1997) Protein Science, 6, 399–406), though weak support for this interpretation was provided. In our study, thermal denaturation of Sj26GST has been re-examined by differential scanning calorimetry in the pH range of 6.5–8.5 and in the presence of the substrate and S-hexylglutathione. Calorimetric traces were found to be irreversible and highly scan-rate dependent. Thermogram shapes, as well as their scan-rate dependence, can be globally explained by assuming that thermal denaturation takes place according to one irreversible step described by a first-order kinetic constant that changes with temperature, as given by an Arrhenius equation. On the basis of this model, values for the rate constant as a function of temperature and the activation energy have been determined. Data also indicate that binding of GSH or S-hexylglutathione just exert a very little stabilising effect on the dimeric structure of the molecule.

Critical points in the Bragg glass phase of a weakly pinned crystal of Ca3Rh4Sn13

New experimental data are presented on the scan rate dependence of the magnetization hysteresis width ΔM(H) (∞ critical current densityJ c(H)) in isothermalMH scans in a weakly pinned single crystal of Ca3Rh4Sn13, which displays second magnetization peak (SMP) anomaly as distinct from the peak effect (PE). We observe an interesting modulation in the field dependence of a parameter which purports to measure the dynamical annealing of the disordered bundles of vortices injected through the sample edges towards the destined equilibrium vortex state at a givenH. These data, in conjunction with the earlier observations made while studying the thermomagnetic history dependence inJ c(H) in the tracing of the minor hysteresis loops, imply that the partially disordered state heals towards the more ordered state between the peak field of the SMP anomaly and the onset field of the PE. The vortex phase diagram in the given crystal of Ca3Rh4Sn13 has been updated in the context of the notion of the phase coexistence of the ordered and disordered regions between the onset field of the SMP anomaly and the spinodal line located just prior to the irreversibility line. A multi-critical point and a critical point in the (H,T) region of the Bragg glass phase have been marked in this phase diagram and the observed behavior is discussed in the light of recent data on multi-critical point in the vortex phase diagram in a single crystal of Nb.

Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T m ≈ 100 °C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry–Eyring model, N ⇔ U ⇒ F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed.

Comparative thermal stabilities of recombinant adenoviruses and hexon protein

Differential scanning calorimetry was used to identify the thermal stability profile of the replication deficient and protein IX deleted recombinant adenovirus type 5 that contains the p53 transgene (rAd/p53) in phosphate buffered saline (vPBS) or 10% glycerol (TRIS/phosphate buffer). The wildtype adenovirus (Ad/WT) and purified hexon protein (major capsid protein) were also evaluated in 10% glycerol (TRIS/phosphate buffer) as controls. The thermal profile of rAd/p53 revealed three endothermic transitions ( T 1, T 2 and T 3) occurring between 25 °C and 90 °C. T 1, which occurred at 46.7 °C in vPBS and 49.4 °C in TRIS/PO 4 10% glycerol buffer, was irreversible following repeated scanning and attributed to the degradation of the intact vector. The latter two endothermic transitions, T 2 and T 3, occurring at 69 °C and 78 °C, respectively, corresponded with the two transitions of purified hexon in temperature and amount of heat absorbed. The thermal profile of Ad/WT revealed four endothermic transitions at 51.5 °C ( T 1), 70.5 °C ( T 2A), 73.6 °C ( T 2B), and 77.4 °C ( T 3). The higher temperature of degradation as well as additional transition was attributed to the presence of protein IX associated with the hexon. The positions and excess molar heat capacities of the intact rAds were found to be affected by pH, glycerol, vector concentration and the presence or absence of protein IX in the capsid. Irreversibility of T 1 implied that the degradation of the intact virus may follow first-order kinetics. The thermal scan rate dependence of T 1 further confirmed that degradation of the intact virus may be first-order. The apparent activation energies for the degradation of the intact vectors were determined from the scan rate dependence of T 1 and shown to be affected by protein IX in the capsid and solution conditions. Analysis of rAd samples incubated at 45 °C by Field Emission Electron Microscopy (FESEM) confirmed that loss of single particles was first-order. Although aggregates were observed in the samples, degradation appeared to be the dominant reaction leading to disappearance of single virions from the aqueous matrix. Based on thermal and FESEM analysis, an empirical model was proposed that accounted for the disappearance of single rAd particles. At or near T 1, degradation of rAd particles followed a unidirectional, pseudo-first order reaction. However, at lower temperatures, disappearance of single virions resulted from competing irreversible degradation and aggregation reactions.

Scan-Rate-Dependent Melting Transitions of Interleukin-1 Receptor (Type II): Elucidation of Meaningful Thermodynamic and Kinetic Parameters of Aggregation Acquired from DSC Simulations

The role of thermal unfolding as it pertains to thermodynamic properties of proteins and their stability has been the subject of study for more than 50 years. Moreover, exactly how the unfolding properties of a given protein system may influence the kinetics of aggregation has not been fully characterized. In the study of recombinant human Interleukin-1 receptor type II (rhuIL-1R(II)) aggregation, data obtained from size exclusion chromatography and differential scanning calorimetry (DSC) were used to model the thermodynamic and kinetic properties of irreversible denaturation. A break from linearity in the initial aggregation rates as a function of 1/T was observed in the vicinity of the melting transition temperature (T(m) approximately 53.5 degrees C), suggesting significant involvement of protein unfolding in the reaction pathway. A scan-rate dependence in the DSC experiment testifies to the nonequilibrium influences of the aggregation process. A mechanistic model was developed to extract meaningful thermodynamic and kinetic parameters from an irreversibly denatured process. The model was used to simulate how unfolding properties could be used to predict aggregation rates at different temperatures above and below the T(m) and to account for concentration dependence of reaction rates. The model was shown to uniquely identify the thermodynamic parameters DeltaC(P) (1.3 +/- 0.7 kcal/mol-K), DeltaH(m) (74.3 +/- 6.8 kcal/mol), and T(m) with reasonable variances.

Kinetic Stabilization and Fusion of Apolipoprotein A-2:DMPC Disks: Comparison with apoA-1 and apoC-1

Denaturation studies of high-density lipoproteins (HDL) containing human apolipoprotein A-2 (apoA-2) and dimyristoyl phosphatidylcholine indicate kinetic stabilization. Circular dichroism (CD) and light-scattering melting curves show hysteresis and scan rate dependence, indicating thermodynamically irreversible transition with high activation energy E a. CD and light-scattering data suggest that protein unfolding triggers HDL fusion. Electron microscopy, gel electrophoresis, and differential scanning calorimetry show that such fusion involves lipid vesicle formation and dissociation of monomolecular lipid-poor protein. Arrhenius analysis reveals two kinetic phases, a slower phase with E a,slow = 60 kcal/mol and a faster phase with E a,fast = 22 kcal/mol. Only the fast phase is observed upon repetitive heating, suggesting that lipid-poor protein and protein-containing vesicles have lower kinetic stability than the disks. Comparison of the unfolding rates and the melting data recorded by differential scanning calorimetry, CD, and light scattering indicates the rank order for the kinetic disk stability, apoA-1 > apoA-2 > apoC-1, that correlates with protein size rather than hydrophobicity. This contrasts with the tighter association of apoA-2 than apoA-1 with mature HDL, suggesting different molecular determinants for stabilization of model discoidal and plasma spherical HDL. Different effects of apoA-2 and apoA-1 on HDL fusion and stability may reflect different metabolic properties of apoA-2 and/or apoA-1-containing HDL.

Determination of the basic timescale in kinetic Monte Carlo simulations by comparison with cyclic-voltammetry experiments

While kinetic Monte Carlo simulations can provide long-time simulations of the dynamics of physical and chemical systems, it is not yet possible in general to identify the inverse Monte Carlo attempt frequency with a physical timescale in any but the simplest systems. Here we demonstrate such an identification by comparing simulations with experimental data. Using a dynamic lattice-gas model for the electrosorption of Br on Ag(1 0 0), we measure the scan-rate dependence of the separation between positive- and negative-going peaks in cyclic voltammetry and compare simulated and experimental peak separations. By adjusting the Monte Carlo attempt frequency, good agreement between simulated and experimental peak separations is achieved. It is also found that the uniqueness of the determination depends on the relative values of the adsorption/desorption and diffusion free-energy barriers.

Nonmonotonic temperature dependence of heat capacity through the glass transition within a kinetic model.

The heat capacity of a supercooled liquid subjected to a temperature cycle through its glass transition is studied within a kinetic model. In this model, the beta process is assumed to be thermally activated and described by a two-level system. The alpha process is described as a beta relaxation mediated cooperative transition in a double well. The overshoot of the heat capacity during the heating scan is well reproduced and is shown to be directly related to delayed energy relaxation in the double well. In addition, the calculated scan rate dependencies of the glass transition temperature T(g) and the limiting fictive temperature T(f) (L) show qualitative agreement with the known results. Heterogeneity is found to significantly reduce the overshoot of heat capacity. Furthermore, the frequency dependent heat capacity has been calculated within the present framework and found to be rather similar to the experimentally observed behavior of supercooled liquids.

Heat-induced denaturation and aggregation of ovalbumin at neutral pH described by irreversible first-order kinetics.

The heat-induced denaturation kinetics of two different sources of ovalbumin at pH 7 was studied by chromatography and differential scanning calorimetry. The kinetics was found to be independent of protein concentration and salt concentration, but was strongly dependent on temperature. For highly pure ovalbumin, the decrease in nondenatured native protein showed first-order dependence. The activation energy obtained with different techniques varied between 430 and 490 kJ*mole(-1). First-order behavior was studied in detail using differential scanning calorimetry. The calorimetric traces were irreversible and highly scan rate-dependent. The shape of the thermograms as well as the scan rate dependence can be explained by assuming that the thermal denaturation takes place according to a simplified kinetic process where N is the native state, D is denatured (or another final state) and k a first-order kinetic constant that changes with temperature, according to the Arrhenius equation. A kinetic model for the temperature-induced denaturation and aggregation of ovalbumin is presented. Commercially obtained ovalbumin was found to contain an intermediate-stable fraction (IS) of about 20% that was unable to form aggregates. The denaturation of this fraction did not satisfy first-order kinetics.

A differential scanning calorimetric study on the irreversible thermal unfolding of concanavalin A

A differential scanning calorimetry study on the thermal denaturation of concanavalin A at pH 5.2 where it exists in the dimeric form was carried out. The calorimetric transitions were observed to be irreversible and the transition temperature of the protein increased with increasing scan rate, indicating that the thermal denaturation process is under kinetic control. The thermal unfolding, and its scan rate dependence could be explained according to the kinetic scheme N 2 → k 2 U with k as first-order kinetic constant whose change with temperature is given by the Arrhenius equation. Using this model, rate constant as a function of temperature and activation energy of the process have been calculated. The average activation energy of the kinetic process using different approaches is 129±10 kJ mol −1. The differential scanning calorimetric results on transition temperatures and calorimetric enthalpies supported by intrinsic fluorescence indicate that the irreversibility in the calorimetric transitions of concanavalin A includes a combination of post-transition aggregation, chain separation and loss of cofactor.

Heme and pH-dependent stability of an anionic horseradish peroxidase

Horseradish peroxidase A1 thermal stability was studied by steady-state fluorescence, circular dichroism and differential scanning calorimetry at pH values of 4, 7 and 10. Changes in the intrinsic protein probes, tryptophan fluorescence, secondary structure, and heme group environment are not coincident. The T m values measured from the visible CD data are higher than those measured from Trp fluorescence and far-UV CD data at all pH values showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. However ejection of the heme group leads to an irreversible unfolding behavior at pH 4, while at pH 7 and 10 refolding is still observed. This is putatively correlated with the titration state of the heme pocket. Thermal transitions of HRPA1 showed scan rate dependence at the three pH values, showing that the denaturation process was kinetically controlled. The denaturation process was interpreted in terms of the classic scheme, N↔U→D and fitted to far-UV CD ellipticity. A good agreement was obtained between the experimental and theoretical T m values and percentages of irreversibility. However the equilibrium between N and U is probably more complex than just a two-state process as revealed by the multiple T m values.

An in Situ Time-Dependent Study of CO Oxidation on Pt(111) in Aqueous Solution by Voltammetry and Sum Frequency Generation

In situ time-dependent measurements using voltammetry and sum frequency generation (SFG) were carried out to study CO oxidation on a Pt(111) electrode. The scan-rate dependence of CO oxidation was measured by changing the scan rate from 50 to 0.5 mV/s. While the basic shape of the CO stripping curves was unaffected by sweep rate, the oxidation potential of CO was lowered by 0.1 V, from 0.84 to 0.74 V. The measurements of SFG spectra and CO stripping were carried out simultaneously to clarify the discrepancy between SFG and CO stripping measurements reported in earlier SFG studies. Our results showed that the SFG peak attributed to the CO stretch vanished immediately after the CO stripping current reached its maximum value. In the preignition potential region, the frequency shift of the CO stretch observed by SFG agrees with that observed by IR spectroscopy. Additionally, in the ignition potential region, a significant blue shift of CO stretching frequency was observed. For the first time, a 360% enhancement of SFG intensity coinciding with the red shift of stretching frequency was observed in the preignition potential region. While it was not observed by IR spectroscopy, it shows similar behavior to limited Raman spectroscopy measurements obtained from a roughened Pt surface.

Hydrodynamic Study of Ion Transfer at the Liquid/Liquid Interface: the Channel Flow Cell

A hydrodynamic system based on the channel flow cell for voltammetric detection of ions at the liquid/liquid interface is reported. The current response for tetraethylammonium ion transfer across a membrane-supported liquid/liquid interface is shown to be consistent with existing theory for both the flow rate and voltage scan rate dependence of such processes, with no calibration factors or other adjustable parameters required. The analytical utility of such a device is discussed with specific regard to in situ measurements in flow systems.

L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study.

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.

Potential scan rate dependence of underpotential and bulk depositions of lead on screen-printed silver electrodes

This is the first report on studies of the underpotential deposition of Pb (upd-Pb) on a disposable screen-printed silver electrode (AgSPE, non-single crystalline surface). The dependence of upd-Pb and bulk Pb deposition (bulk-Pb) on potential scan rate ( ν) was demonstrated for the AgSPE in pH 3 KNO 3/HNO 3 solutions. At ν=20 mV s −1, the extent of formation of reversible upd-Pb was ∼15 times less than that of irreversible bulk-Pb. However, for ν>100 mV s −1, only the upd-Pb was observed. Scanning electron microscopic (SEM) analysis speculated 3D-nucleation ( hcp) of bulk-Pb on the AgSPE at low scan rate ( ν=1 mV s −1). On the other hand, at a faster scan rate ( ν=300 mV s −1), no marked crystallites were obtained. By interpretation with the CV results, the later case was proved to be formation and complete stripping of the upd-Pb on the AgSPE and this property was further utilized for analytical application. The nearly reversible behavior of the upd-Pb process at the disposable AgSPE is a judicious choice for the effective stripping analysis of Pb by square-wave voltammetry.

Surface Molecular Motion of Monodisperse α,ω-Diamino-Terminated and α,ω-Dicarboxy-Terminated Polystyrenes

Surface glass transition behaviors of monodisperse α,ω-diamino-terminated and α,ω-dicarboxy-terminated polystyrenes (α,ω-PS(NH2)2 and α,ω-PS(COOH)2) were studied by scanning force microscopy and were compared with the results of proton-terminated polystyrene (PS−H). All surface glass transition temperatures, Tgs, of PS−H, α,ω-PS(NH2)2, and α,ω-PS(COOH)2 were discernibly lower than each corresponding bulk glass transition temperature, Tgb. However, the magnitude of Tgs was strongly dependent on the chemical structure of chain end groups, because the surface concentration of chain ends varied with the surface free energy difference between the main chain part and the chain end portion, via the surface segregation or surface depletion of chain ends. This result makes it clear that chain end chemistry is one of determining factors on the magnitude of Tgs. On the basis of the time−temperature superposition principle applied to the scanning rate dependence of lateral force as a function of temperature, the appa...

The electrooxidation of sulfur-containing compounds at boron-doped diamond electrode

The electrooxidation of homocysteine, glutathione (GSH), 2-mercapto ethanesulfonic acid and cephalexin at boron-doped diamond (BDD) electrodes and glassy carbon (GC) electrodes was investigated by cyclic voltammetry. The oxidation of these sulfur-containing compounds exhibited well-defined irreversible responses. This preliminary study has shown that BDD has better sensitivity than GC. Concentration dependence has been studied and indicated the promise of using BDD electrodes for quantitative determination. All of the compounds displayed recognizable oxidation peaks at BDD electrodes at millimolar concentration levels. Scan rate dependence of GSH has been examined. It was shown that there was no adsorption on the surface of the BDD electrode for the low concentration.

Thermal destabilization of rhodopsin and opsin by proteolytic cleavage in bovine rod outer segment disk membranes.

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein.

Electron transfer reaction of poly(ethylene oxide)-modified azurin in poly(ethylene oxide) oligomers

The native azurin (Az) was chemically modified with activated poly(ethylene oxide) (PEO) to solubilize in the PEO oligomers. PEO 2000 (average molecular weight of 2000)-modified Az (PEO 2000-Az) was dissolved without denaturation in PEO 200 (average molecular weight of 200). The electronic absorption spectrometry exhibited no detectable conformational change of PEO 2000-Az in phosphate buffer. The electron paramagnetic resonance (EPR) spectrum of PEO 2000-Az is also quite similar to that of native Az in water. This result confirms that the chemical modification with PEO 2000 does not cause any structural change around the copper site. Moreover, since no changes in the EPR spectrum are observed in PEO 200, the copper coordination geometry must be maintained even in PEO 200. The redox activity of PEO 2000-Az in PEO 200 (containing 0.5 mol dm −3 NaClO 4) was investigated by cyclic voltammetry. When we use the PEO 350-SH (α-methoxy-ω-mercapto-poly(ethylene oxide); PEO with average molecular weight of 350) modified gold electrode as a working electrode, the redox response was observed in PEO 200. The result of the measurements for the scan rate dependence suggests that PEO 2000-Az could be immobilized on the PEO 350-SH modified electrode.