Observed Melting Temperatures Research Articles

Electrical Resistivity of Cu and Au at High Pressure above 5 GPa: Implications for the Constant Electrical Resistivity Theory along the Melting Curve of the Simple Metals.

The electrical resistivity of solid and liquid Cu and Au were measured at high pressures from 6 up to 12 GPa and temperatures ∼150 K above melting. The resistivity of the metals was also measured as a function of pressure at room temperature. Their resistivity decreased and increased with increasing pressure and temperature, respectively. With increasing pressure at room temperature, we observed a sharp reduction in the magnitude of resistivity at ∼4 GPa in both metals. In comparison with 1 atm data and relatively lower pressure data from previous studies, our measured temperature-dependent resistivity in the solid and liquid states show a similar trend. The observed melting temperatures at various fixed pressure are in reasonable agreement with previous experimental and theoretical studies. Along the melting curve, the present study found the resistivity to be constant within the range of our investigated pressure (6–12 GPa) in agreement with the theoretical prediction. Our results indicate that the invariant resistivity theory could apply to the simple metals but at higher pressure above 5 GPa. These results were discussed in terms of the saturation of the dominant nuclear screening effect caused by the increasing difference in energy level between the Fermi level and the d-band with increasing pressure.

Experiment to Teach Multiple Melting Phenomena in Semicrystalline Polymers Using Differential Scanning Calorimetry

This article describes a laboratory experiment used to investigate the phenomenon of multiple melting in polymers. The experiment is aimed at the level of senior undergraduate chemistry students able to carry out the investigation in a research-style approach, working together in small groups. The experiment highlights characteristic thermal behavioral differences between polymers and small organic molecules. It demonstrates that shifts in observed melting temperature upon heating are typically due to inherent metastability of the polymer system and not to impurities in the sample, for example. Differential scanning calorimetry is used to demonstrate and explore this fundamental yet contemporary subject of polymer melting, using a well-known and commercially available polymer, isotactic polystyrene. Effects of thermal history of the sample, including crystallization temperature and crystallization time, as well as analysis conditions including heating rate, on the melting point of the polymer solid are each investigated. The experiment provides a hands-on example of structure-property relationships in polymer science.

Crystallization of Long-Spaced Precision Polyacetals I: Melting and Recrystallization of Rapidly Formed Crystallites

Rapidly melt-crystallized polyethylenes with acetal groups (−O–CH2–O−) precisely spaced by 12, 18, 19, or 23 methylene backbone carbons exhibit two reorganizations and three melting endotherms on heating at relatively low heating rates. Real-time wide-angle X-ray diffraction (WAXD) experiments further confirm that these transitions are associated with a reorganization to a different crystalline structure. The differential scanning calorimetry endotherms are associated with melting of the initially formed disordered (or hexagonal) phases, followed by melting of Form I and by melting of Form II crystals with increasing temperature. In parallel with these structural changes, the long spacing undergoes a discrete step increase at the polymorphic transitions. Upon the transition from disordered to Form I crystals, the core crystal thickness increases by one repeating unit, whereas the crystal thickness remains basically unchanged during the transition from Form I to Form II crystallites. The effect of even vs odd CH2 spacer on the observed melting temperatures is prevalent for spacer <10 CH2 units. The even or odd number of methylenes between acetals also affects the type and kinetics of packing assembly in layered crystallites. Although a disordered mesomorphic-like structure develops under fast cooling in odd-spaced polyacetals (PA-19, PA-23), the formation of Form I crystals cannot be bypassed in PA-12 and PA-18. Furthermore, some differences are also found in the WAXD patterns of Form II between odd- and even-spaced polyacetals, denoting that staggering of the acetals in the crystallites is affected by the configuration of consecutive acetals with respect to the methylene sequence.

Powder bed fusion of poly(phenylene sulfide) at bed temperatures significantly below melting

In this paper, the authors present evidence of printing poly(phenylene sulfide) (PPS), a high-performance polymer, via powder bed fusion (PBF) using a bed temperature of 230 °C, which is significantly below both its observed melting temperature (Tm ˜ 285 °C) and its observed onset temperature of crystallization (Tc ˜ 255 °C). This contradicts existing material screening guidelines for PBF, which suggest maintaining bed temperature above the observed onset of crystallization. The authors believe that printing PPS at a comparatively-low bed temperature is beneficial for minimizing the occurrence of PPS side-reactions (e.g., chain extension, branching, and crosslinking) during printing and for enabling processing of a high-temperature polymer on “standard” PBF printers, which typically have maximum build temperatures below 250 °C. Existing methods for theoretically determining processing bounds were used to predict a range of energy densities at which PPS can be printed. One combination of process parameter values was selected based on machine constraints imposed by typical PBF machines not designed to print high-temperature polymers and used to fabricate multilayer, complex parts. The presented process parameters result in final part density upwards of 1.18 g/cm3 and ultimate tensile strength and elongation of 62 MPa and 3.3%, respectively. Hypotheses on the generalizability of low-temperature PBF printing of high-performance polymers, and steps towards updating materials and process parameter selection guidelines for PBF, are also presented.

Design of Melting Curve Analysis (MCA) by Real-Time Polymerase Chain Reaction Assay for Rapid Distinction of Staphylococci and Antibiotic Resistance

Background: Staphylococci are important pathogenic bacteria responsible for a range of diseases in humans. Hence, detection of Staphylococcus aureus from coagulase-negative staphylococci (CoNS) is essential in various infections. Objectives: The aim of this study was to design a melting curve analysis (MCA) assay based on Multiplex Real-Time PCR for rapid detection of staphylococci and antibiotic resistance. Methods: The current study used standard strains of positive and negative coagulase staphylococci. As the first step, serial dilutions were prepared with ratios of 108 to 101 cfu/mL based on standard bacterial concentration with 0.5 McFarland and the results were illustrated in dedicated melting curves. Results: All melting curves of gene amplification had an equal melting point. In all dilutions, the observed melting temperatures shown in the melting curves of gene amplification were equal to 83.79°C for ITS-gene, 74.2°C for phop gene, 76.49°C for sap-gene, 78.2°C for mvaA gene, 79.57°C for 16srRNA-gene, 74.83°C for mupA gene, and 76.6°C for mecA gene. Conclusions: The MCA based on real time-PCR for identifying staphylococcal species and antibiotic resistance is a highly effective method with high sensitivity and specificity. © 2019, Archives of Clinical Infectious Diseases.

Decreasing electrical resistivity of gold along the melting boundary up to 5 GPa

ABSTRACTThe electrical resistivity of gold was experimentally measured at high pressures from 2 to 5 GPa and temperatures ∼300 K above melting. The resistivity decreased as a function of pressure and increased as a function of temperature as expected. The temperature dependence of resistivity in the solid and liquid phases are comparable to 1 atm results. The observed melting temperatures at each pressure agree well with previous experimental and theoretical studies. The essential result of this study is that resistivity decreases along the pressure-dependent melting boundary, conflicting with a prediction of invariant behavior as reported in the literature. This result is discussed in terms of the interaction between s and d-bands as both pressure and temperature increase along the melting boundary. The thermal conductivity of gold was calculated from the measured electrical resistivity using the Wiedemann-Franz law. The temperature-induced effect on the thermal conductivity at high temperatures is as expected in both the solid and liquid phase while the pressure-effect shows some variability.

Decreasing electrical resistivity of silver along the melting boundary up to 5 GPa

ABSTRACTThe electrical resistivity of Ag was experimentally measured at high pressures up to 5 GPa and at temperatures up to ∼300 K above melting. The resistivity decreased as a function of pressure and increased as a function of temperature as expected and is in very good agreement with 1 atm data. Observed melting temperatures at high pressures also agree well with previous experimental and theoretical studies. The main finding of this study is that resistivity of Ag decreases along the pressure- and temperature-dependent melting boundary, in conflict with prediction of resistivity invariance. This result is discussed in terms of the dominant contribution of the increasing energy separation between the Fermi level and 4d-band as a function of pressure. Calculated from the resistivity using the Wiedemann–Franz law, the electronic thermal conductivity increased as a function of pressure and decreased as a function of temperature as expected. The decrease in the high pressure thermal conductivity in the liquid phase as a function of temperature contrasts with the behavior of the 1 atm data.

DSC/SAXS analysis of the thickness of lamellae of semicrystalline polymers-restrictions in the case of materials with swollen amorphous phase

Due to the simplicity and speed of the analysis, calorimetric (DSC measurements and Gibbs-Thomson method) or x-ray methods (SAXS measurements and Bragg's law or correlation function method) are the most frequently used in determining the thickness of crystals. This paper revealed that the direct application of the above mentioned methods in order to analyze the lamellar structure of semicrystalline polymers, the amorphous phase of which was swollen, may lead to incorrect results. Swelling of the amorphous phase (without changing the thickness of crystals) leads to a shift of the observed melting temperature towards lower values, what in turn will result in underestimated values of thickness of crystals, in the case of calorimetric method. At the same time, an increase of the value of long period is observed, what in turn will result in overestimated values of thickness of crystals, in the case of classical x-ray method (with the use of value of long period estimated according to the Bragg's law and crystalline volume fraction). We have observed that only the analysis of x-ray data with the use of the correlation function method enables to obtain actual values of the thickness of lamellar crystals of the polymeric materials with swollen amorphous phase. The means for correct analysis are pointed out. The above effects have been presented on the example of model, miscible, non-cocrystallizing semicrystalline polymer/low molecular weight modifier systems.

Fabrication of a mini multi-fixed-point cell for the calibration of industrial platinum resistance thermometers

A mini multi-fixed-point cell (length 118 mm, diameter 33 mm) containing three materials (In–Zn eutectic (mass fraction 3.8% Zn), Sn and Pb) in a single crucible was designed and fabricated for the easy and economical fixed-point calibration of industrial platinum resistance thermometers (IPRTs) for use in industrial temperature measurements. The melting and freezing behaviors of the metals were investigated and the phase transition temperatures were determined using a commercial dry-block calibrator. Results showed that the melting plateaus are generally easy to realize and are reproducible, flatter and of longer duration. On the other hand, the freezing process is generally difficult, especially for Sn, due to the high supercooling required to initiate freezing. The observed melting temperatures at optimum set conditions were 143.11 °C (In–Zn), 231.70 °C (Sn) and 327.15 °C (Pb) with expanded uncertainties (k = 2) of 0.12 °C, 0.10 °C and 0.13 °C, respectively. This multi-fixed-point cell can be treated as a sole reference temperature-generating system. Based on the results, the realization of melting points of the mini multi-fixed-point cell can be recommended for the direct calibration of IPRTs in industrial applications without the need for a reference thermometer.

EpiHRMAssay, in tube and in silico combined approach for the scanning and epityping of heterogeneous DNA methylation.

Reliable and cost-effective assays with adequate sensitivity are required to detect the DNA methylation profile in plants for scientific and industrial purposes. The proposed novel assay, named EpiHRMAssay, allows to quantify the overall methylation status at target loci and to enable high-throughput analyses. It combines in tube High Resolution Melting Analysis on bisulphite-treated templates with the in silico prediction of the melting profile of virtual epialleles using uMELTSM software. The predicted melting temperatures (Tm-s) of a set of epialleles characterized by different numbers of methylated cytosines (#mC) or different mC configurations were obtained and used to build calibration models, enabling the quantification of methylation in unknown samples using only the in tube observed melting temperature (Tm-o). EpiHRMAssay was validated by analysing the promoter region of CMT3, DDM1, and ROS1 genes involved in the regulation of methylation/demethylation processes and chromatin remodelling within a population of peach plants. Results demonstrate that EpiHRMAssay is a sensitive and reliable tool for locus-specific large-scale research and diagnostic contexts of the regulative regions of genes, in a broad range of organisms, including mammals. EpiHRMAssay also provides complementary information for the assessment of heterogeneous methylation and can address an array of biological questions on epigenetic regulation for diversity studies and for large-scale functional genomics.

018 Finite element analysis of heat transfer in a commercially available conduction cryostage

Towards estimating errors in temperature measurement and control caused by non-collocation of specimen and sensor, we developed a finite-element model of heat transfer in a conduction stage cryomicroscope. The model represented a Linkam Scientific Instruments biological cryostage cooled by a liquid nitrogen pump; finite element analysis was performed using COMSOL Multiphysics® software. The computational domain was 2D axisymmetric and comprised the cryostage chamber interior, which was modeled as a 65-mm diameter cylinder filled with nitrogen gas and having a constant-temperature boundary. The silver heating/cooling element was located in the center of the chamber, and a sample (consisting of a water droplet sandwiched between a pair of circular glass coverslips) was in contact with the top surface of the silver block. Heat transfer processes simulated in the model included heat conduction within domains of silver, glass, water, and nitrogen; joule heating in the heating coil embedded within the silver element; and forced convection cooling from nitrogen vapor that is pumped through an interior channel in the silver block. We measured the nitrogen flow rate corresponding to the temperature profiles used in our experiments, and thus were able to determine the value of the Reynolds number to be >10000; for this regime, the coefficient of forced convection could be estimated using the Dittus–Boelter correlation. Another important heat transfer phenomenon in our model was the contact resistance between the top surface of the silver block and the bottom surface of the glass coverslip. To estimate the contact resistance, we performed experiments in which we used the cryomicroscope to warm frozen water droplets from −10 °C until melting was observed. After correcting for effects of droplet size, the measured difference between the expected and observed melting temperatures provided experimental data on the magnitude of thermal gradients within the cryostage. Thus, finite element simulations of the warming experiment were repeated while iteratively adjusting the assumed value of contact resistance, until there was agreement between the predicted and observed temperature differential. In melting experiments with a warming rate of 80 °C/min, the 95% confidence interval for the temperature error at 0 °C was 1.2 ± 0.4 K when data were extrapolated to droplets of negligible diameter; based on these measurements, the contact resistance was determined to be 2.8 K/W. Temperature differentials were also measured in independent melting experiments at warming rates 0.6, 6, and 150 °C/min; the magnitude of the temperature error was found to increase with increased rate of warming, and was well predicted by the finite element model. In simulations of cooling at a rate of 60 °C/min, the magnitude of the predicted temperature error increased over the initial ∼10 s of the cooling process, reaching a maximum of ∼1 K; simulations also showed that the steady state magnitude of the temperature error decreased with decreasing cooling rate. Finite element analysis also demonstrated that significant thermal gradients develop within the portion of the glass coverslip that is situated above the silver block aperture. This gradient was caused in part by heat transfer from the nitrogen gas above the sample. Source of funding: National Science Foundation Grant CBET-0954587, awarded to JOMK. Conflict of interest: None declared. jens.karlsson@villanova.edu

A Periodic Pattern of Evolutionarily Conserved Basic and Acidic Residues Constitutes the Binding Interface of Actin-Tropomyosin

Actin filament cytoskeletal and muscle functions are regulated by actin binding proteins using a variety of mechanisms. A universal actin filament regulator is the protein tropomyosin, which binds end-to-end along the length of the filament. The actin-tropomyosin filament structure is unknown, but there are atomic models in different regulatory states based on electron microscopy reconstructions, computational modeling of actin-tropomyosin, and docking of atomic resolution structures of tropomyosin to actin filament models. Here, we have tested models of the actin-tropomyosin interface in the "closed state" where tropomyosin binds to actin in the absence of myosin or troponin. Using mutagenesis coupled with functional analyses, we determined residues of actin and tropomyosin required for complex formation. The sites of mutations in tropomyosin were based on an evolutionary analysis and revealed a pattern of basic and acidic residues in the first halves of the periodic repeats (periods) in tropomyosin. In periods P1, P4, and P6, basic residues are most important for actin affinity, in contrast to periods P2, P3, P5, and P7, where both basic and acidic residues or predominantly acidic residues contribute to actin affinity. Hydrophobic interactions were found to be relatively less important for actin binding. We mutated actin residues in subdomains 1 and 3 (Asp(25)-Glu(334)-Lys(326)-Lys(328)) that are poised to make electrostatic interactions with the residues in the repeating motif on tropomyosin in the models. Tropomyosin failed to bind mutant actin filaments. Our mutagenesis studies provide the first experimental support for the atomic models of the actin-tropomyosin interface.

Correlation between the variation in observed melting temperatures and structural motifs of the global minima of gallium clusters: An ab initio study

We have investigated the correlation between the variation in the melting temperature and the growth pattern of small positively charged gallium clusters. Significant shift in the melting temperatures was observed for a change of only few atoms in the size of the cluster. Clusters with size between 31-42 atoms melt between 500-600 K whereas those with 46-48 atoms melt around 800 K. Density functional theory based first principles simulations have been carried out on Ga(n)(+) clusters with n = 31, ..., 48. At least 150 geometry optimizations have been performed towards the search for the global minima for each size resulting in about 3000 geometry optimizations. For gallium clusters in this size range, the emergence of spherical structures as the ground state leads to higher melting temperature. The well-separated core and surface shells in these clusters delay isomerization, which results in the enhanced stability of these clusters at elevated temperatures. The observed variation in the melting temperature of these clusters therefore has a structural origin.

Liquid–liquid phase separation in a polyethylene blend monitored by crystallization kinetics and crystal-decorated phase morphologies

A series of polyethylene (PE) blends consisting of a linear high density polyethylene (HDPE) and a linear low density polyethylene (LLDPE) with an octane-chain branch density of 120/1000 carbon was prepared at different concentrations. The two components of this set of blends possessed isorefractive indices, thus, making it difficult to detect their liquid–liquid phase separation via scattering techniques. Above the experimentally observed melting temperature of HDPE, Tm=133°C, this series of blends can be considered to be in the liquid state. The LLDPE crystallization temperature was below 50°C; therefore, above 80°C and below the melting temperature of HDPE, a series of crystalline–amorphous PE blends could be created. A specifically designed two-step isothermal experimental procedure was utilized to monitor the liquid–liquid phase separation of this set of blends. The first step was to quench the system from temperatures of known miscibility and isothermally anneal them at a temperature higher than the equilibrium melting temperature of the HDPE for the purpose of allowing the phase morphology to develop from liquid–liquid phase separation. The second step was to quench the system to a temperature at which the HDPE could rapidly crystallize. The time for developing 50% of the total crystallinity (t1/2) was used to monitor the crystallization kinetics. Because phase separation results in HDPE-rich domains where the crystallization rates are increased, this technique provided an experimental measure to identify the binodal curve of the liquid–liquid phase separation for the system indicated by faster t1/2. The annealing temperature in the first step that exhibits an onset of the decrease in t1/2 is the temperature of the binodal point for that blend composition. In addition, the HDPE-rich domains crystallized to form spherulites which decorate the phase-separated morphology. Therefore, the crystal dispersion indicates whether the phase separation followed a nucleation-and-growth process or a spinodal decomposition process. These crystal-decorated morphologies enabled the spinodal curve to be experimentally determined for the first time in this set of blends.

Electrophoretic Mobility Is a Reporter of Hairpin Structure in Single-Stranded DNA Oligomers

The electrophoretic mobilities of 24 single-stranded DNA oligomers, each containing 26 nucleotide residues, have been measured in polyacrylamide gels and in free solution. The mobilities observed at 20 degrees C differed by approximately 20% in polyacrylamide gels and by approximately 10% in free solution, even though the oligomers contained the same number of bases. Increasing the temperature or adding urea to the solution equalized the mobilities of the oligomers, suggesting that the variable mobilities observed at 20 degrees C are due to the formation of stable secondary structures, most likely hairpins. Thermal melting profiles were measured for eight oligomers in 40 mM Tris acetate buffer. The observed melting temperatures of most oligomers correlated roughly with the mobilities observed at 20 degrees C; however, one oligomer was much more stable than the others. The melting temperatures of four of the oligomers were close to the values predicted by DINAMelt [Markham, N. R., and Zuker, M. (2005) Nucleic Acids Res. 33, W577-W581]; melting temperatures of the other oligomers differed significantly from the predicted values. Thermal melting profiles were also measured for two oligomers as a function of the Tris acetate buffer concentration. The salt concentration dependence of the melting temperatures suggests that 0.15 Tris+ ion per phosphate is released upon denaturation. Because the apparent number of Tris+ ions released is greater than that observed by others for the release of Na+ ions from similar hairpins, the results suggest that DNA hairpins (and, presumably, duplexes) bind more Tris+ ions than Na+ ions in solution.

Detection of Common Disease-Causing Mutations in Mitochondrial DNA (Mitochondrial Encephalomyopathy, Lactic Acidosis with Stroke-Like Episodes MTTL1 3243 A>G and Myoclonic Epilepsy Associated with Ragged-Red Fibers MTTK 8344A>G) by Real-Time Polymerase Chain Reaction

The 3243A>G mutation in the MTTL1 (tRNA(Leu)) gene and the 8344A>G mutation in the MTTK (tRNA(Lys)) gene are the most common mutations found in mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes and myoclonic epilepsy associated with ragged-red fibers, respectively. These mitochondrial DNA mutations are usually detected by conventional polymerase chain reaction followed by restriction enzyme digestion and gel electrophoresis. We developed a LightCycler real-time polymerase chain reaction assay to detect these two mutations based on fluorescence resonance energy transfer technology and melting curve analysis. Primers and fluorescence-labeled hybridization probes were designed so that the sensor probe spans the mutation site. The observed melting temperatures differed in the mutant and wild-type DNA by 9 degrees C for the MTTL1 gene and 6 degrees C for the MTTK gene. This method correctly identified all 10 samples that were 3243A>G mutation-positive, all 4 samples that were 8344A>G mutation-positive, and all 30 samples that were negative for both mutations, as previously identified by traditional gel-based methods. This LightCycler assay is a rapid and reliable technique for molecular diagnosis of these mitochondrial gene mutations.

Thermal and crystallization characteristics of poly(trimethylene terephthalate)/poly(ethylene naphthalate) blends

Poly(trimethylene terephthalate) (PTT)/poly(ethylene naphthalate) (PEN) blends were miscible in the amorphous state in all of the blend compositions studied, as evidenced by a single, composition-dependent glass transition temperature ( T g) observed for each blend composition. The variation in the T g value with the blend composition was well predicted by the Gordon–Taylor equation, with the fitting parameter being 0.57. The cold-crystallization peak temperature decreased with increasing PTT content, while the melt-crystallization peak temperature decreased with increasing amount of the minor component. The subsequent melting behavior after both cold- and melt-crystallization exhibited melting point depression, in which the observed melting temperatures decreased with increasing amount of the minor component. During melt-crystallization, both components in the blends crystallized concurrently just to form their own crystals. The blend with 60% w/w of PTT exhibited the lowest total apparent degree of crystallinity.

Equilibrium Structure and Folding of a Helix-Forming Peptide: Circular Dichroism Measurements and Replica-Exchange Molecular Dynamics Simulations

We have performed experimental measurements and computer simulations of the equilibrium structure and folding of a 21-residue α-helical heteropeptide. Far ultraviolet circular dichroism spectroscopy is used to identify the presence of helical structure and to measure the thermal unfolding curve. The observed melting temperature is 296 K, with a folding enthalpy of −11.6 kcal/mol and entropy of −39.6 cal/(mol K). Our simulations involve 45 ns of replica-exchange molecular dynamics of the peptide, using eight replicas at temperatures between 280 and 450 K, and the program CHARMM with a continuum solvent model. In a 30-ns simulation started from a helical structure, conformational equilibrium at all temperatures was reached after 15 ns. This simulation was used to calculate the peptide melting curve, predicting a folding transition with a melting temperature in the 330–350 K range, enthalpy change of −10 kcal/mol, and entropy change of −30 cal/(mol K). The simulation results were also used to analyze the peptide structural fluctuations and the free-energy surface of helix unfolding. In a separate 15-ns replica-exchange molecular dynamics simulation started from the extended structure, the helical conformation was first attained after ∼2.8 ns, and equilibrium was reached after 10 ns of simulation. These results showed a sequential folding process with a systematic increase in the number of hydrogen bonds until the helical state is reached, and confirmed that the α-helical state is the global free-energy minimum for the peptide at low temperatures.

High pressure-temperature Raman measurements of H2O melting to 22 GPa and 900 K

The melting curve of H(2)O has been measured by in situ Raman spectroscopy in an externally heated diamond anvil cell up to 22 GPa and 900 K. The Raman-active OH-stretching bands and the translational modes of H(2)O as well as optical observations are used to directly and reliably detect melting in ice VII. The observed melting temperatures are higher than previously reported x-ray measurements and significantly lower than recent laser-heating determinations. However, our results are in accord with earlier optical determinations. The frequencies and intensities of the OH-stretching peaks change significantly across the melting line while the translational mode disappears altogether in the liquid phase. The observed OH-stretching bands of liquid water at high pressure are very similar to those obtained in shock-wave Raman measurements.

Studies on the degradation pathway of iron-sulfur centers during unfolding of a hyperstable ferredoxin: cluster dissociation, iron release and protein stability

The ferredoxin from the thermoacidophile Acidianus ambivalens is a representative of the archaeal family of di-cluster [3Fe-4S][4Fe-4S] ferredoxins. Previous studies have shown that these ferredoxins are intrinsically very stable and led to the suggestion that upon protein unfolding the iron-sulfur clusters degraded via linear three-iron sulfur center species, with 610 and 520 nm absorption bands, resembling those observed in purple aconitase. In this work, a kinetic and spectroscopic investigation on the alkaline chemical denaturation of the protein was performed in an attempt to elucidate the degradation pathway of the iron-sulfur centers in respect to protein unfolding events. For this purpose we investigated cluster dissociation, iron release and protein unfolding by complementary biophysical techniques. We found that shortly after initial protein unfolding, iron release proceeds monophasically at a rate comparable to that of cluster degradation, and that no typical EPR features of linear three-iron sulfur centers are observed. Further, it was observed that EDTA prevents formation of the transient bands and that sulfide significantly enhances its intensity and lifetime, even after protein unfolding. Altogether, our data suggest that iron sulfides, which are formed from the release of iron and sulfide resulting from cluster degradation during protein unfolding in alkaline conditions, are in fact responsible for the observed intermediate spectral species, thus disproving the hypothesis suggesting the presence of a linear three-iron center intermediate. Kinetic studies monitored by visible, fluorescence and UV second-derivative spectroscopies have elicited that upon initial perturbation of the tertiary structure the iron-sulfur centers start decomposing and that the presence of EDTA accelerates the process. Also, the presence of EDTA lowers the observed melting temperature in thermal ramp experiments and the midpoint denaturant concentration in equilibrium chemical unfolding experiments, further suggesting that the clusters also play a structural role in the maintenance of the conformation of the folded state.