Yannick Guinet

Evidence of a two-stage thermal denaturation process in lysozyme: A Raman scattering and differential scanning calorimetry investigation

Raman spectroscopy (in the low-frequency range and the amide I band region) and modulated differential scanning calorimetry investigations have been used to analyze temperature-induced structural changes in lysozyme dissolved in 1H2O and 2H2O in the thermal denaturation process. Low-frequency Raman data reveal a change in tertiary structure without concomitant unfolding of the secondary structure. Calorimetric data show that this structural change is responsible for the configurational entropy change associated with the strong-to-fragile liquid transition and correspond to about 1/3 of the native-denaturated transition enthalpy. This is the first stage of the thermal denaturation which is a precursor of the secondary structure change and is determined to be strongly dependent on the stability of the hydrogen-bond network in water. Low-frequency Raman spectroscopy provides information on the flexibility of the tertiary structure (in the native state and the transient folding state) in relation to the fragility of the mixture. The unfolding of the secondary structure appears as a consequence of the change in the tertiary structure and independent of the solvent. Protein conformational stability is directly dependent on the stability of the native tertiary structure. The structural transformation of tertiary structure can be detected through the enhanced 1H/2H exchange inhibited in native proteins. Taking into account similar features reported in the literature observed for different proteins it can be considered that the two-stage transformation observed in lysozyme dissolved in water is a general mechanism for the thermal denaturation of proteins.

The contribution of Raman spectroscopy to the analysis of phase transformations in pharmaceutical compounds

We show in this paper the contribution of the whole Raman spectrum including the phonon spectrum, to detect, identify and characterize polymorphic forms of molecular compounds, and study their stability and transformation. Obtaining these kinds of information is important in the area of pharmaceutical compounds. Two different polymorphic systems are analyzed through investigations in indomethacin and caffeine exposed to variable environmental conditions and various stresses, as possibly throughout the production cycle of the active pharmaceutical ingredient. It is shown the capability of the low-frequency Raman spectroscopy to reveal disorder in the crystalline state, to detect small amorphous or crystalline material, and to elucidate ambiguous polymorphic or polyamorphic situations.

Thermostabilization Mechanism of Bovine Serum Albumin by Trehalose

Thermal denaturation of bovine serum albumin (BSA) is analyzed from differential scanning calorimetry (DSC) and Raman spectroscopy investigations. DSC curves exhibit a marked dependence on protein concentration. BSA thermal denaturation becomes broader and bimodal, and the temperature of denaturation increases with increasing protein concentration. Raman scattering investigations simultaneously carried out in the low-frequency range (10-350 cm(-1)) and in the amide I band region (1500-1800 cm(-1)) indicate that the denaturation process is described as a biphasic process independent of protein concentration. The dependence of the protein stability upon the protein concentration can be interpreted from the coupling of protein and solvent dynamics. The confrontation of previous results obtained from Raman investigations on lysozyme (LYS) and the present study of BSA brings out significant information on protein dynamics and the coupling of protein and hydration-water dynamics in relation with the solvent accessible surface area. Contrary to LYS, the modification of the dynamics of hydration water by the protein is clearly observed on BSA. The influence of trehalose on the protein dynamics was analyzed. We found that trehalose reduces the dynamic fluctuations of polar side chains at the protein-solvent interface. The mechanism of thermostabilization by trehalose is related to the reduction of the exposure of hydrophobic groups of BSA to the water molecules, and to a strengthening of intermolecular O-H interactions in the hydrogen-bond network of water, leading to the stabilization of the tertiary structure.

Using the low-frequency Raman spectroscopy to analyze the crystallization of amorphous indomethacin

This paper gives a detailed analysis of the low-frequency Raman spectrum (LFRS) in the 5-250cm(-1) region, corresponding to collective vibrations, in the crystalline forms and in the amorphous state of indomethacin (IMC). This study points out the high sensitivity of the LFRS to detect, identify and evaluate the first traces of crystallization in comparison with high-frequency regions where internal vibration bands are detected. This analysis reveals that amorphous IMC prepared by cryogrinding instantaneously partially crystallizes at room temperature in the stable gamma phase, well below T(g)=43 degrees C. A method based on the treatment of the LFRS to determine precise and very low volume of crystallized material within amorphous matrix is described and used to analyze the crystallization kinetics of ground amorphous IMC powder. This study demonstrates that Raman spectroscopy is also a well-adapted technique to point out small amount of amorphous state in crystalline matrix. Crystallization of ground IMC powder was also analyzed by isothermal microcalorimetry experiments, which is one of the most widely used methods to analyze isothermal crystallization and to evaluate crystallinity.

Low- and High-Frequency Raman Investigations on Caffeine: Polymorphism, Disorder and Phase Transformation

Raman investigations are carried out both in crystalline forms of caffeine and during the isothermal transformation of the orientationally disordered form I into the stable form II at 363 K. The time dependence of the Raman spectrum exhibits no significant change in the intramolecular regime (above 100 cm(-1)), resembling the spectrum of the liquid state. By contrast, significant changes are observed below 100 cm(-1), and the low-frequency spectra of forms I and II are observed to be different from that of the liquid. The temperature dependence of the 5-600 cm(-1) spectrum gives information on the static disorder through the analysis of collective motions, while information on dynamic disorder are obtained from the study of the 555 cm(-1) band corresponding to internal vibrations in the pyrimidine ring. This analysis indubitably reveals that form II is also orientationally disordered with a local molecular arrangement that mimics that in form I and the liquid state. The comparison of the low-frequency spectra recorded in theophylline and form II of caffeine allows one to describe the stable form of caffeine from the packing arrangement of anhydrous theophylline with the consideration of reorientational molecular disorder.

Analysis of the Mechanism of Lysozyme Pressure Denaturation from Raman Spectroscopy Investigations, and Comparison with Thermal Denaturation

Pressure denaturation of lysozyme dissolved in H(2)O and D(2)O was analyzed using Raman investigations in a wide frequency range. The simultaneous analysis of regions corresponding to the molecular fingerprint of the protein (500-1800 cm(-1)), and the low- (50-450 cm(-1)) and high- (2600-3800 cm(-1)) frequency spectra, allow us to probe protein denaturation and the organization of water molecules. The pressure- and heat-induced transformations are compared. Both pressure- and heat-denatured states are obtained through an intermediate state characterized by intact secondary structure and enhanced water penetration in the tertiary structure. As a consequence of a weaker penetration upon pressurizing, it was found that the pressure-denatured state was partially unfolded compared with the heat-denatured state. The mechanism of pressure denaturation was related to the disruption of the hydrogen-bond network of water onto a set of clusters characterized by strengthened O - H interactions, inducing a hardening of protein dynamics. The mechanism is opposite to that observed upon heating, i.e., the softening of the hydrogen bond network of water inducing a softer protein dynamics. The analysis of the intramolecular O-H stretching reveals that pressurizing lysozyme aqueous solution favors the development of low-density water from the protein surface to the bulk, contrasting to the compression of pure water leading to crystallization of high-density ice-VI.

Thermal Denaturation of Beta-Lactoglobulin and Stabilization Mechanism by Trehalose Analyzed from Raman Spectroscopy Investigations

The thermal denaturation process of beta-lactoglobulin has been analyzed in the 20-100 degrees C temperature range by Raman spectroscopy experiments simultaneously performed in the region of amide modes (800-1800 cm(-1)) and in the low-frequency range (10-350 cm(-1)). The analysis of amide modes reveals a two-step thermal denaturation process in the investigated temperature range. The first step corresponds to the dissociation of dimers associated with an increase of flexibility of the tertiary structure. In the second step, large conformational changes are detected in the secondary structure and described as a loss of alpha-helix structures and a concomitant formation of beta-sheets. Raman investigations in the low-frequency range provide important information on the origin of the denaturation process through the analysis of the solvent dynamics and its coupling with that of the protein. The softening of the tetrahedral structure of water induces the dissociation of dimers and makes the tertiary structure softer, leading to the water penetration in the protein interior. The methodology based on Raman investigations of amide modes and in the low-frequency region was used to analyze the mechanism of beta-lactoglobulin thermostabilization by trehalose. The main effect of trehalose is determined to be related to its capabilities to distort the tetrahedral organization of water molecules.

Process induced transformations during tablet manufacturing: Phase transition analysis of caffeine using DSC and low frequency micro-Raman spectroscopy

The phase transition of a model API, caffeine Form I, was studied during tableting process monitored with an instrumented press. The formulation used had a plastic flow behavior according to the Heckel model in the compression pressure range of 70-170 MPa. The quantitative methods of analysis used were Differential Scanning Calorimetry (DSC) and low frequency Micro Raman Spectroscopy (MRS) which was used for the first time for the mapping of polymorphs in tablets. They brought complementary contributions since MRS is a microscopic spectral analysis with a spatial resolution of 5 μm(3) and DSC takes into account a macroscopic fraction (10mg) of the tablet. Phase transitions were present at the surfaces, borders and center of the tablets. Whatever the pressure applied during the compression process, the transition degree of caffeine Form I toward Form II was almost constant. MRS provided higher transition degrees (50-60%) than DSC (20-35%). MRS revealed that caffeine Form I particles were partially transformed in all parts of the tablets at a microscopic scale. Moreover, tablet surfaces showed local higher transition degree compared to the other parts.

Polymorphic Transformation of Anhydrous Caffeine upon Grinding and Hydrostatic Pressurizing Analyzed by Low-Frequency Raman Spectroscopy

Low-frequency Raman investigations were carried out upon pressurizing and grinding both crystalline forms of anhydrous caffeine at room temperature. These investigations have led to the detection of metastable states under stress. Upon moderated hydrostatic compression, only form I transform into a metastable state characterized by a Raman band-shape resembling that of form II. Above 2 GPa, both pressurized forms convert into an identical disordered state, suggesting a pressure-induced amorphization. In contrast to hydrostatic compression, grinding induces transformation of each phase into the other, leading to an intermediate state only stabilized under long enough grinding. The origin of these metastable states induced by stress was related to the disordered nature of both crystalline forms of caffeine and the stability conditions at room temperature of form I.

Evidence for transient kinetics of nucleation as responsible for the isothermal transformation of supercooled liquid into the glacial state of triphenyl phosphite

The first-order transformation of the supercooled liquid into the glacial state of triphenyl phosphite was investigated by differential scanning calorimetry (DSC) using two different thermal procedures. In the first procedure the transformation was analyzed by heating run DSC experiments. The glaciation process was interpreted as an aborted crystallization because of a high nucleation rate in a temperature range where the crystal growth is low. This relative separation between the nucleation- and growth-rate curves suggests that the glacial state can be described as a supercooled liquid–nanocrystalline mixed phase, characterized by a high-density nucleation which frustrates further crystallization. In a second procedure, DSC experiments were carried out during isothermal transformations of the supercooled liquid into the glacial state. The sigmoidal shapes of DSC isotherms are interpreted as transient kinetics of nucleation rather than nucleation and growth processes.