Thermodynamic and structural analysis of homodimeric proteins: Model of β-lactoglobulin
ABSTRACT The energetics of protein homo-oligomerization was analyzed in detail with the application of a general thermodynamic model. We have studied the thermodynamic aspects of protein-protein interaction employing β-lactoglobulin A from bovine milk at pH=6.7 where the protein is mainly in its dimeric form. We performed differential calorimetric scans at different total protein concentration and the resulting thermograms were analyzed with the thermodynamic model for oligomeric proteins previously developed. The thermodynamic model employed, allowed the prediction of the sign of the enthalpy of dimerization, the analysis of complex calorimetric profiles without transitions baselines subtraction and the obtainment of the thermodynamic parameters from the unfolding and the association processes and the compared with association parameters obtained with Isothermal Titration Calorimetry performed at different temperatures. The dissociation and unfolding reactions were also monitored by Fourier-transform infrared spectroscopy and the results indicated that the dimer of β-lactoglobulin (N(2)) reversibly dissociates into monomeric units (N) which are structurally distinguishable by changes in their infrared absorbance spectra upon heating. Hence, it is proposed that β-lactoglobulin follows the conformational path induced by temperature:N(2)⇌2N⇌2D. The general model was validated with these results indicating that it can be employed in the study of the thermodynamics of other homo-oligomeric protein systems.
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ABSTRACT: Iindex of published papers on thermology or temperature measurement Voluime 4: 2011 to 2013
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ABSTRACT: About 40% of the marketed drugs and 70-90% of new drug candidates are insoluble in water and therefore poorly bioavailable, which significantly compromises their therapeutic effects. A Formulation of nanosuspensions achieved by reducing the pure drug particle size down to seb-micron range is one of the most promising approaches to overcome the insolubility. However, the nanosuspension formulations are subject to instability because of nucleation and particle growth. Therefore, a stabilizer is needed to be incorporated into the nanosuspension formulation during the preparation process to suppress the aggregation of drug particles. β-LG, a globular protein, is broken by heat-induced denaturation and its hydrophobic area is exposed, allowing it to associate with organic particles. PTX, an insoluble drug, is widely used for the clinical treatment of human cancer. However, this drug's clinical application is greatly limited by intrinsic defects, including poor solubility, adverse side effects and poor tumor penetration. In this study, we prepared β-LG-stabilized PTX nanosuspensions (PTX-NS) by coating the protein onto nanoscaled drug particles, investigate the stabilization effect of β-LG on PTX-NS and evaluate its in vitro and in vivo performance. PTX-NS with a diameter of approximately 200 nm was easily prepared. β-LG produced significantly stabilized-effect on PTX-NS via the interaction between the hydrophobic area of the protein and the hydrophobic surface of the drug particles, resulting in a conformational change of the protein, the loss of both secondary and tertiary structures, and the transition of Trp residues to a less hydrophobic condition. Importantly, unlike other conventional nanoparticles, PTX-NS could directly translocate across the membrane into the cytosol in an energy-independent manner, without entrapment within the endosomal-lysosomal system. Moreover, compared with Taxol, PTX-NS increased AUC and Cmax by 26 and 16-fold, respectively, and prolonged T1/2 by 314-fold. As expected, PTX-NS had better in vitro and in vivo antitumor activity compared to PTX alone. Additionally, β-LG is cyto- and bio-compatible, and PTX-NS is not toxic to healthy tissues. In conclusion, the present study has suggested the high potency of globular proteins, such as β-LG, as novel biomaterials for nanosuspension platform to improve the drug delivery for disease treatment.Molecular Pharmaceutics 03/2015; 12(5). DOI:10.1021/mp5008037 · 4.79 Impact Factor
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ABSTRACT: Published papers on thermology or temperature measurement, between 2011 and 2012,