Amino acid transport in thermophiles: characterization of an arginine-binding protein from Thermotoga maritima. 3. Conformational dynamics and stability.

Arginine-binding protein from Thermotoga maritima (TmArgBP) is a 27.7 kDa protein possessing the typical two domain structure of the periplasmic binding protein family. The protein is characterized by high specificity and affinity for binding a single molecule of l-arginine. In this work, the effect of temperature and/or guanidine hydrochloride on structure and stability of the protein in the absence and in the presence of l-arginine has been investigated by differential scanning calorimetry, far-UV circular dichroism and intrinsic tryptophan phosphorescence and fluorescence. The results revealed that TmArgBP undergoes an irreversible one-step thermal unfolding process in a cooperative mode. The TmArgBP melting temperature was recorded at 115 °C. The presence of l-arginine did not change the protein secondary structure content as well as the intrinsic phosphorescence and fluorescence protein properties, even if it increases the structural stability of the protein. The obtained results are discussed in combination with a detailed inspection of the three-dimensional structure of the protein.

Thermal unfolding of monomeric and dimeric beta-lactoglobulins.

The thermal stabilities of dimeric bovine beta-lactoglobulin and monomeric equine beta-lactoglobulin were investigated at neutral pH by means of differential scanning calorimetry, circular dichroism, tryptophan fluorescence, and by binding of an hydrophobic probe. Differential scanning calorimetry showed the presence of two structural domains with different thermal stabilities in both proteins. Thermodynamic analysis of the calorimetric signal revealed that the two domains unfold independently according to a mechanism where an equilibrium step is followed by an irreversible transition. The spectroscopic data supported this model and allowed recognition of the structural regions corresponding to the more thermally stable domain. The differences in thermal stability between the two proteins can be primarily ascribed to the properties of the less stable domain.

Study of diffusion through LDPE film of Di-n-butyl phthalate.

The apparent diffusion coefficients (D) across a PE-LD film of Di-n-butyl phthalate (DnBP) dissolved in isooctane and ethanol, were calculated in different ways and compared. DnBP, a common plasticizer of plastics and printing inks, may be a possible contaminant of flexible packaging and PE-LD is the most common food-contacting polymer. To perform diffusion measurements, small plastic sachets filled with solutions of the contaminant were used at three different temperatures. The D coefficients of the two solvents and dissolved DnBP were calculated using the lag time method, a formula taking into account the molar weight and absolute temperature, and an equation based on the initial concentration and the amounts migrated after subsequent times. The results demonstrated great solvent interference in the plasticizer diffusion across polyethylene; in particular as far as isooctane is concerned, a remarkable co-diffusion of both the plasticizer and the non-polar solvent must be assumed. Isooctane quickly penetrates PE-LD but ethanol also diffuses across PE-LD, although at a greatly reduced speed. These facts must be considered for realistic prediction of migration or effectiveness of the functional barrier.