Dominique Chevalier-Lucia

Inhibitor and substrate binding induced stability of HIV-1 protease against sequential dissociation and unfolding revealed by high pressure spectroscopy and kinetics.

High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of −32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 μM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.

Submicron emulsions processed by ultra-high pressure homogenization

Model oil/water (O/W) emulsions containing (w/w) 15–45% peanut oil plus 4.3% whey proteins or raw whole milk (∼4%, w/w, fat) were processed by ultra-high pressure (HP) homogenization at an inlet temperature of 24 °C using an HP homogenizer with an HP valve, immediately followed by cooling heat exchangers. Mechanical and thermal energies involved in the process were calculated using pressure and temperature values measured at the HP valve inlet and immediate outlet. For all samples, an increase in homogenization pressure decreased the droplet size, leading to submicron fat particles at pressure≥200 MPa and monomodal distributions in the nano-scale range by emulsion recycling once or twice at 200 MPa. Further, microbial inactivation in raw whole milk was enhanced by increasing the homogenization pressure or by recycling milk at 200 MPa. Rheological behavior of model O/W emulsions varied as a function of oil droplet size and oil volume fraction.

High pressure calorimetry at sub-zero temperature: evaluation of the latent heat and frozen water ratio of gelatin gels

Abstract An isothermal differential high-pressure calorimeter was specifically designed and described in this study. After calibration, the apparatus was validated by measuring the latent heat of ice melting. A good agreement was observed between the experimental results and literature data. The latent heat of gelatin gels at two different dry matter contents (2 and 10% w/w) was measured at sub-zero temperatures and high pressure. The main result showed that the latent heat values were influenced by both the melting temperature and the dry matter content. A decrease of the latent heat of melting of the gels with increasing melting pressure (decreasing of the melting temperature) probably indicated a modification in the ratio of frozenable water in these gels under pressure.

UHPH-processed O/W submicron emulsions stabilised with a lipid-based surfactant: physicochemical characteristics and behaviour on in vitro TC7-cell monolayers and ex vivo pig's ear skin.

Submicron O/W emulsions formulated with sesame oil plus a lipid-base surfactant, and with or without retinyl acetate (RAC) as a model hydrophobic biomolecule, were prepared by single-pass homogenisation at ≥ 200 MPa (UHPH) and an initial fluid temperature (Tin) of 24°C. These emulsions were characterised by a monomodal distribution (peak maximum at 260 nm) and a 2-year potential physical stability at ambient temperature. Submicron droplets were investigated in term of (i) physicochemical characteristics (size distribution curves; ζ-potential value), and (ii) impact on TC7-cell monolayers (MTT-assay and cell LDH-leakage). Submicron droplets ± RAC did not affect or increased significantly (p=0.05) TC7-cell metabolic activity after 4-24h of exposure indicating absence of cellular impairment, except when high amounts of droplets were deposed on TC7-cells. Indeed, the lipid-based surfactant deposed alone on TC7-cells at high concentration, induced some significant (p=0.05) cell LDH-leakage, and therefore cell-membrane damage. Cellular uptake experiments revealed a significant (p=0.05) time-dependent internalisation of RAC from submicron droplets, and cellular transformation of RAC into retinol. The turnover of RAC into retinol and therefore RAC bioaccessibility appeared faster for RAC-micelles of similar size-range and prepared at atmospheric pressure with polysorbate 80, than for submicron O/W emulsions. Permeation experiments using pigs ear skin mounted on Franz-type diffusion cells, revealed RAC in dermis-epidermis, in significantly (p=0.05) higher amounts for submicron than coarse pre-emulsions. However, RAC amounts remained low for both emulsion-types and RAC was not detected in the receptor medium of Franz-type diffusion cells.