Synchrotron Small-angle X-ray Scattering Data Research Articles

Pore Structure Characterization of Coal by Synchrotron Small-Angle X-ray Scattering and Transmission Electron Microscopy

Coal is a porous medium with complex pore structures. The characteristics of the pore structure play an important role in various aspects of coal use, including extraction of methane from coal seams, CO2 sequestration in coal, and water purification by activated carbon. To describe comprehensively the pore structure of coal, we apply transmission electron microscopy (TEM) and synchrotron small-angle X-ray scattering (SAXS) measurements to six coal samples from medium to high rank. The positive deviation of SAXS data from Porod’s law was observed. The positive deviation correction of SAXS data was carried out to quantitatively obtain the pore size distribution and specific surface area. We find that the pore size distribution is independent of the coal rank but varies with the vitrinite content; pores in vitrinite-rich coals are smaller than those in vitrinite-poor coals for the same rank. Channel-like and interconnected pores are observed for both high- and low-volatile bituminous coals. Among all coal sa...

Structural analysis of influenza A virus matrix protein M1 and its self-assemblies at low pH.

Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.

Nature of molecular network in thermal shrinkage behavior of oriented high‐density polyethylene

ABSTRACTShrinkage and structural evolution of oriented high‐density polyethylene on heating were investigated by a combination of thermomechanical analysis (TMA) and synchrotron small angle X‐ray scattering (SAXS) techniques. Under varying load conditions, TMA study was performed to record the continuous length changes as a function of temperature. The value of shrinkage without any load could be evaluated by a linear extrapolation method, which eliminated the influence of the required tension by traditional TMA approach. In addition, the apparent modulus of network was used to describe the nature of entangled molecular network in detail during the shrinkage process. Importantly, it was found that the apparent modulus decreased gradually with increasing temperature. Furthermore, the SAXS data provided a direct evidence for the variation trend of shrinkage stress obtained by the tensile testing stage, and the results confirmed that the shrinkage force mainly originates from interfibrillar networks. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014, 52, 368–376

Phase transformation and precipitation of an Al–Cu alloy during non-isothermal heating studied by in situ small-angle and wide-angle scattering

Understanding the classic precipitation sequence of Al–Cu alloys, solid solution → Guinier–Preston (GP) zones → θ″ → θ′ → stable θ, is of academic importance. In situ synchrotron small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) techniques were employed simultaneously to study the temperature-dependent behavior of various intermediate precipitation steps in the non-isothermal heating of Al–5.4 wt%Cu alloy. This study quantitatively demonstrates the concurrent evolution of the lattice structure, volume fraction (growth and dissolution) and structural growth in the thickness and length directions with temperature for various intermediate (metastable) precipitates for the first time. The detailed phase transformation mechanism and structural evolution in the precipitation sequence (for GP zones, θ″, θ′ and θ phases) can then be resolved. Our data analysis also considered the concurrent existence of multiple precipitates in the precipitation sequence. Moreover, the evolutional behavior of the orientation of precipitates in each precipitation step can be concurrently revealed. Different SAXS analysis models were proposed to successfully interpret the SAXS data. The new information presented by the SAXS/WAXS approach provides insight into the phase transformation mechanism of precipitation in Al–Cu alloys.

Comparative Characterization of Apo-, Reconstituted- and In Vivo-Folded forms of a Durum Wheat Metallothionein

Durum wheat metallothionein (dMT), a plant type 1 metallothionein, with a long “hinge” region between metal coordinating cysteine clusters, is efficient cadmium (Cd) chelator. In this work, biophysical features of purified recombinant holo-dMT, its demetallated form (apo-dMT) and the reconstituted Cd5-dMT are compared to obtain insight into the structure and metal binding features of this protein. Results show that the purified holo-dMT is polydisperse and has 5.3±0.5 Cd2+ ions per molecule. Demetallation followed by size exclusion chromatography yields homogeneous apo-dMT which can be reconstituted with Cd2+. Synchrotron small angle X-ray scattering (SAXS) demonstrates that apo-dMT, at pH 2.0, is flexible and extended in solution. According to UV-vis, CD and native-PAGE data conformation of apo-dMT is sensitive to pH changes in the range 2.0 to 8.0. Reconstitution of the apo-protein at pH 8.0, with Cd2+ appears to take place in two phases during which first the monomer is folded to accommodate 5 Cd2+ ions and then reorganization into oligomeric forms allows incorporation of further metal ions. SAXS data indicate that holo-dMT has limited flexibility in structure, but its conformation is significantly more compact than that of apo-dMT. Results of UV-vis and circular dichroism spectroscopy show that the in vitro folded protein is structurally different from the purified holo-dMT with the same number of Cd2+ ions.This work was supported by Sabanci University Internal Research Fund Project No. IACF08-00514 and by the bilateral program TUBITAK-Julich Research Center projects TBAG-U-155 and TBAG-U-157.

Trilayer-cubic core–shell structure of PbS/EuS nanocrystals revealed by the combination of the synchrotron small-angle X-ray scattering method and energy-dispersive X-ray spectroscopy

Multilayer nanostructure analysis has been carried out for core-shell PbS/EuS nanocrystals by the combination of the synchrotron small-angle X-ray scattering (SAXS) method and energy-dispersive X-ray spectroscopy (EDS). SAXS patterns of a dilute hexane solution of the PbS/EuS nanocrystals were measured at a high signal-to-noise ratio by using synchrotron radiation. Initial structure models used for SAXS data analyses were obtained from EDS images of the particle where Eu and O atoms are localized only in the shell part of a quasi-cubic particle and, on the other hand, Pb and S atoms in the core. By simulating the intensity of SAXS using multilayer-cubic models with various core-shell electron density distributions, it was found that the particle structure was explained approximately as a trilayer-cubic model having the PbS core and the EuS/Eu2O3 shell. The diagonal length of the cubic particle was ca. 9.4 nm and the estimated thicknesses of EuS and Eu2O3 layers in the shell were ca. 1.9 and 2.9 nm at maximum, respectively.

Effect of hard segment content and carbon-based nanostructures on the kinetics of flexible polyurethane nanocomposite foams

Reactive flexible polyurethane (PU) foams were synthesized with two contents of hard segments (HS) and filled with multi-walled carbon nanotubes (MWCNTs), functionalized MWCNTs (f-MWCNTs) and functionalized graphene sheets (FGS). The effect of the HS content and the carbon nanofillers on the kinetics of polymerization and the kinetics of phase-separation have been studied by Fourier transform infrared spectroscopy (FT-IR) and synchrotron small-angle X-ray scattering (SAXS). A slow down on the rate of polymerization and on the development of the polymer structure due to the increase of the HS content and the inclusion of the nanoparticles was observed. Therefore, this work demonstrates that there is a relationship between the kinetics of polymerization and the kinetics of phase separation in flexible PU nanocomposite foams. SAXS data was used to generate 3D microstructures of PU nanocomposite foams and the phase-separated morphology was observed by atomic force microscopy (AFM).

Unusual Structural Morphology of Dendrimer/CdS Nanocomposites Revealed by Synchrotron X-ray Scattering

Low-resolution structure of CdS nanoparticles (NPs) grown in the presence of the third-generation rigid polyphenylenepyridyl dendrimers (PPPDs) is analyzed by synchrotron small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). The combination of rigidity of the PPPDs and a high local concentration of chelating nitrogens strongly interacting with growing CdS NPs yields anisometric particles instead of conventional spherical ones. The scattering data from the free PPPDs and from the composite CdS/PPPD NPs are interpreted in terms of 3-D models revealing a peculiar morphology of the nanocomposite whereby the PPPDs enclose the CdS NPs like a “flattened ball-in-hands”. The sizes of the CdS NPs found by SAXS are in good agreement with the TEM data. The presented approach to elucidate particle morphology should open the ways of detailed characterization of the modern composite materials from the SAXS data.

Real time synchrotron SAXS and WAXS investigations on temperature related deformation and transitions of β-iPP with uniaxial stretching

In-situ synchrotron small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) were carried out to investigate the uniaxial drawing-induced deformation and structure transitions of β form isotactic polypropylene (iPP) at varying temperatures (30 °C, 60 °C, 80 °C, 100 °C and 120 °C). The WAXS results indicated that the initial strain for the strain-induced β–α transformation decreases with the tensile temperature according to the engineering stress–strain curves. The SAXS data showed that the long period increased along the direction perpendicular to the tensile force and changed little along the tensile direction with increasing strain in the elastic deformation stage before the yield point. The analysis of the obtained scattering results indicated that the angle between parent and daughter lamella rotates from initial 40° or 140° to close to 90° accounts for the lateral expansion of the samples with tension, which matches the essential auxetical behavior. A structure deformation and transition mechanism was proposed for β form iPP with uniaxial drawing. The initialization of the crystalline structure transition is after the yield point, then the mechanical loading-induced β–α transition seems to be a gradual process with lamella slippage and breaking which triggers the β–α polymorphic transition.

Characterization of Bupivacaine-Loaded Formulations Based on Liquid Crystalline phases and Microemulsions: The Effect of Lipid Composition

This report details the structural characterization and the in vitro drug-release properties of different local anesthetic bupivacaine (BUP)-loaded inverted-type liquid crystalline phases and microemulsions. The effects of variations in the lipid composition and/or BUP concentration on the self-assembled nanostructures were investigated in the presence of the commercial distilled glycerol monooleate Myverol 18-99K (GMO) and medium-chain triglycerides (MCT). Synchrotron small-angle X-ray scattering (SAXS) and rotating dialysis cell model were used to characterize the BUP formulations and to investigate the in vitro BUP release profiles, respectively. The evaluation of SAXS data for the BUP-loaded GMO/MCT formulations indicates the structural transition of inverted-type bicontinuous cubic phase of the symmetry Pn3m → inverted-type hexagonal (H(2)) phase → inverted-type microemulsion (L(2)) with increasing MCT content (0-40 wt %). In the absence of MCT, the solubilization of BUP induces the transition of Pn3m → H(2) at pH 7.4; whereas a transition of Pn3m → (Pn3m + H(2)) is detected as the hydration is achieved at pH 6.0. To mimic the drug release and transport from in situ formed self-assembled systems after subcutaneous administration, the release experiments were performed by injecting low viscous stimulus-responsive precursors to a buffer in the dialysis cell leaving the surface area between the self-assembled system and the release medium variable. Our results suggest that the pH-dependent variations in the lipidic partition coefficient, K(l/w), between the liquid crystalline nanostructures and the surrounding buffer solution are significantly affecting BUP release rates. Thus, a first step toward understanding of the drug-release mechanism of this drug-delivery class has been undertaken tackling the influence of drug ionization as well as the type of the self-assembled nanostructure and its release kinetics under pharmaceutically relevant conditions.

Phase Transitions in Prequenched Mesomorphic Isotactic Polypropylene during Heating and Annealing Processes As Revealed by Simultaneous Synchrotron SAXS and WAXD Technique

Time-resolved simultaneous synchrotron small-angle X-ray scattering (SAXS) and wide-angle X-ray diffraction (WAXD) technique was used to investigate the phase transitions in prequenched mesomorphic isotactic polypropylene (iPP) samples during heating and annealing processes, respectively. For the heating process, it is shown that the mesomorphic-to-monoclinic phase transition is relatively faster for the mesomorphic iPP sample obtained with the high quenching rate than that with the low quenching rate. For the former, the stability of α-monoclinic crystals formed during heating is relatively higher. As for the annealing process, WAXD and SAXS data illustrate that the higher the annealing temperature (T(a)), the earlier the mesomorphic-to-monoclinic phase transition occurs. Namely, T(a) controls the phase transition rate. Both heating and annealing processes show that the increase of content of α-monoclinic crystal phase is mainly at the expense of the mesomorphic phase, with the content of amorphous phase almost invariable. The isothermal crystallization kinetics for the prequenched mesomorphic iPP sample was analyzed through the Avrami equation, revealing a two-dimensional crystal growth under the diffusion-limited mechanism.

Study of Microstructure in Polyethylene Terephthalate(<i>PET</i>) Fiber Using SAXS

An experimental method was developed in this work to study the PET fibers by synchrotron small-angle X-ray scattering (SAXS). Compared with the traditional measurement in air, the new method measured in the glycerin is helpful to eliminate edge scattering arising from the fiber edge. The results showed that intensity from edge scattering is so high as to cover the microstructure information. PET fiber with a diameter of 25 μm was measured by the new experimental method, and a lobed shape can be seen clearly on the meridian which didn't emerge in traditional measurement. The SAXS data were evaluated to extract parameters of lamellar structure and microvoids. In addition, the effect of fiber diameter on edge scattering was also investigated.

The role of calcium in membrane condensation and spontaneous curvature variations in model lipidic systems

In this study, the dynamical behaviour of calcium-induced disordered to well-ordered structural transitions has been investigated by time-resolved synchrotron small-angle X-ray scattering (SAXS) in the milliseconds to seconds range. The in situ monitoring of the formed non-equilibrium self-assembled structures was achieved by the successful combination of synchrotron SAXS with stopped flow measurements. The effect of the rapid mixing of aqueous dispersions of dioleoylphosphatidyalglycerol (DOPG)/monoolein (MO) with low concentrations of Ca(2+) ions is reported. Under static conditions and in the absence of Ca(2+) ions, the evaluation of SAXS data for DOPG/MO aqueous dispersions prepared with three different DOPG/MO molar ratios indicates the formation of either a sponge-like L(3) phase or uncorrelated bilayers. Clearly, the lipid composition plays a vital role in modulating the structural behaviour of these aqueous dispersions in the absence and also in the presence of Ca(2+) ions. The rapid-mixing experiments revealed that the fast and strong interactions of Ca(2+) ions with the negatively charged DOPG/MO membranes triggers the transformation from the L(3) phase or the uncorrelated bilayers to the well-ordered dehydrated L(α) phase or to inverted type bicontinuous cubic phases, V(2), with either a symmetry of Pn3m or Im3m. Additionally, we recently reported (A. Yaghmur, P. Laggner, B. Sartori and M. Rappolt, PLoS ONE, 2008, 3, e2072) that low concentrations of Ca(2+) ions trigger the formation of the inverted type hexagonal (H(2)) phase in DOPG/MO aqueous dispersions with a molar DOPG/MO ratio of 30/70. These are also temperature-sensitive structural transitions. Intriguingly, the strong association of Ca(2+) ions with the negatively charged DOPG/MO membranes leads to fast re-organization of the two lipids and simultaneously induces fast tuning of the curvature.

Encapsulation of a Hydrophobic Drug into a Polymer-Micelle Core Explored with Synchrotron SAXS

Synchrotron small-angle X-ray scattering (SAXS) at the SPring-8 40B2 and 45XU beamlines was carried out on aqueous solutions of (PEG-P(Asp(Bzl))): partially benzyl-esterified poly(ethylene glycol)-block-poly(aspartic acid) with LE540 loaded up to 8.3 wt %, where LE540 is a very hydrophobic retinoid antagonist drug. The scattering profiles showed characteristic features for core-shell spherical micelles, confirming that P(Asp(Bzl)) forms a hydrophobic core and PEG forms a hydrophilic shell. Before the addition of LE540, a diffraction peak was observed around q = 4 nm(-1), where q is the magnitude of the scattering vector. This peak can be attributed to ordering between alpha-helices made of P(Asp(Bzl)), the so-called nonspecific hexatic arrangement. The P(Asp(Bzl)) helices disappeared as LE540 was added. This result can be interpreted by assuming a uniform distribution of LE540 in the core. By use of a core-shell spherical micelle model, the SAXS data could be well fitted for all of the samples. The analysis indicated that the core radius increases sigmoidally from 5.9 to 6.9 nm upon addition of LE540 whereas the shell radius stayed at 12.5-12.8 nm. The aggregation number that is the average number of PEG-P(Asp(Bzl))'s consisting of one micelle slightly increased from 145 to 182.

Synchrotron investigation on the sheared structure evolution of syndiotactic polypropylene crystallization process

The final structure of molten syndiotactic polypropylene (sPP) sheared under different conditions was investigated by synchrotron small-angle x-ray scattering (SAXS) and wide-angle x-ray diffraction (WAXD) techniques to elucidate the shear effects on sPP crystalline structure. The results obtained from the WAXD show that there is no variation on crystalline form but a little difference on the orientation of the 200 reflection. The SAXS data indicate that the lamellar thickness and long period have not been affected by shear but the lamellar orientation is dependent on shear. The experimental data of sPP crystallization from sheared melt may indicate a mesophase structure that is crucial to the shear effects on the final polymer multiscale crystalline structures.

Intrinsic structural disorder of mouse proNGF

The unprocessed precursor of the Nerve Growth Factor (NGF), proNGF, has additional functions, besides its initially described role as a chaperone for NGF folding. The precursor protein endows apoptotic and/or neurotrophic properties, in contrast to the mature part. The structural and molecular basis for such distinct activities are presently unknown. Aiming to gain insights into the specific molecular interactions that govern rm-proNGF biological activities versus those of its mature counterpart, a structural study by synchrotron small angle X-ray scattering (SAXS) in solution was carried out. The different binding properties of the two proteins were investigated by surface plasmon resonance (SPR) using, as structural probes, a panel of anti-NGF antibodies and the soluble forms of TrkA and p75(NTR) receptors. SAXS measurements revealed the rm-proNGF to be dimeric and anisometric, with the propeptide domain being intrinsically unstructured. Ab initio reconstructions assuming twofold symmetry generated two types of structural models, a globular "crab-like" and an elongated shape that resulted in equally good fits of the scattering data. A novel method accounting for possible coexistence of different conformations contributing to the experimental scattering pattern, with no symmetry constraints, suggests the "crab-like" to be a more likely proNGF conformation. To exploit the potential of chemical stabilizers affecting the existing conformational protein populations, SAXS data were also collected in the presence of ammonium sulphate. An increase of the proNGF compactness was observed. SPR data pinpoints that the propeptide of proNGF may act as an intrinsically unstructured protein domain, characterized by a molecular promiscuity in the interaction/binding to multiple partners (TrkA and p75(NTR) receptors and a panel of neutralizing anti-NGF antibodies) depending on the physiological conditions of the cell. These data provide a first insight into the structural basis for the selectivity of mouse short proNGF, versus NGF, towards its binding partners.

Agglomeration of proteins in acoustically levitated droplets

An ultrasonic trap (acoustic levitator) was used as an analytical tool to allow container-free handling of proteins in small sample volumes. This trap was combined for the first time with synchrotron small-angle X-ray scattering (SAXS) for structure analysis of biological macromolecules in a solution. The microfocus beamline at BESSY was used as a source of intense X-ray radiation. Apoferritin (APO) was used as a model protein, and its aggregation behavior in a levitator was followed from a diluted solution to the solid state. Different stages of APO agglomeration were observed without solid container walls, which may influence aggregation behavior and produce a parasitic scattering background. Starting with a volume of 5 microL we analyzed the concentration dependence of APO structure factors in the range from 5 to 1,200 mg/mL (solid protein). The solution was stirred automatically due to convection inside the droplet caused by the ultrasonic field. SAXS data recording of APO was performed in time intervals of 60 s during an aggregation experiment of 30 to 60 min.

Amorphous-Phase Miscibility and Crystal Phases in Blends of Polymorphic Poly(hexamethylene terephthalate) with Monomorphic Poly(pentamethylene terephthalate)

Annealing or heat scan-induced lamellar thickening and factors influencing crystal unit cells in polymorphic poly(hexamethylene terephthalate) (PHT) were probed using polarized-light optical microscopy (POM), differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WAXD), synchrotron small-angle X-ray scattering (SAXS). The DSC and WAXD results show that post-scanning or annealing (up to 140 °C) on 110 °C-crystallized PHT neither transform crystal cell types nor does thin lamella into thick lamella. Rapid lamellar thickening in PHT could take place during DSC scanning, which was proven by comparing SAXS data. Tmax (final temperature of heating); within a range of 180 up to 220 °C) does not influence the polymorphism or multiple melting peaks in PHT. All evidence suggests that kinetic factors are less influential on the polymorphism in PHT. Further, polymorphic poly(hexamethylene terephthalate) (PHT) was blended with monomorphic poly(pentamethylene terephthalate) (PPT) to form a crystalline/crystalline blend system. The semicrystalline PPT and PHT are miscible in the melt state or quenched amorphous phase. The miscibility, via weak intermolecular interactions, in the amorphous phase of the PHT/PPT blends exerts almost no influence on the crystalline domains, where PPT does not interfere with the formation of α or β crystal forms in PHT, and vise versa, PHT does not interfere with the sole α-crystal form in PPT.

On the nature of the second-order optical nonlinearity of nanoinhomogeneous glasses in the Li2O-Nb2O5-SiO2 system

The submicroscopic structure of lithium niobium silicate glasses of the compositions 2xLiNbO3 · (1 − x)(Li2O · 2SiO2) (x = 0.40, 0.45, 0.50) and 30Li2O · 25Nb2O5 · 45SiO2 in the initial state and after heat treatment for different times at temperatures in the vicinity of the glass transition point T g are investigated using X-ray powder diffraction, small-angle neutron scattering (SANS), synchrotron small-angle X-ray scattering (SAXS), and electron microscopy. A nanostructure with inhomogeneities ∼40 A in size is formed in glasses at the initial stages of phase separation at temperatures in the range 600–670°C. This structure is responsible for the appearance of the second-order optical nonlinearity. The SANS, SAXS, and electron microscopic data on the inhomogeneity size are in good agreement with each other. According to the X-ray diffraction, SANS, and SAXS data, the ordering of the glass structure and the difference between the density of inhomogeneities and the density of the matrix increase in the course of heat treatment. At the initial stage of amorphous phase separation, the glass decomposes into regions enriched in SiO2 and regions with an increased content of lithium and niobium. An increase in the temperature or time of heat treatment results in the precipitation of LiNbO3 ferroelectric crystals. The results obtained allow us, for the first time, to make the inference that nanoscale changes in the glass structure lead to considerable changes (by one order of magnitude and more) in the quadratic optical nonlinearity, which can be controlled by heat treatment. The origin of the second-order optical nonlinearity is associated with both the nanosized modulations of the polarizability due to the inhomogeneous glass structure and the polarity of structural nanoinhomogeneities from which the LiNbO3 phase precipitates at the later stages of phase separation.

Met-myoglobin Association in Dilute Solution during Pressure-Induced Denaturation: an Analysis at pH 4.5 by High-Pressure Small-Angle X-ray Scattering

In this paper, we report on the original global fit procedure of synchrotron small-angle X-ray scattering (SAXS) data applied to a model protein, met-myoglobin, in dilute solution during temperature- and pressure-induced denaturation processes at pH 4.5. Starting from the thermodynamic description of the protein unfolding pathway developed by Hawley (Hawley, S. A. Biochemistry 1971, 10, 2436), we have developed a new method for analyzing the set of SAXS curves using a global fitting procedure, which allows us to derive the form factor of all the met-myoglobin species present in the solution, their aggregation state, and the set of thermodynamic parameters, with their p and T dependence. This method also overcomes a reasonably poor quality of the experimental data, and it is found to be very powerful in analyzing SAXS data. SAXS experiments were performed at four different temperatures from hydrostatic pressures up to about 2000 bar. As a result, the presence of an intermediate, partially unfolded, dimeric state of met-myoglobin that forms during denaturation has been evidenced. The obtained parameters were then used to derive the met-myoglobin p, T phase diagram that fully agrees with the corresponding phase diagram obtained by spectroscopic measurements.