Mikrokalorymetryczne badania przemian konformacyjnych albuminy poddanej działaniu wybranych czynników fizykochemicznych

Abstract

Thermal unfolding of albumin in aqueous solutions proceeding under different physico-chemical conditions has been investigated using differential scanning calorimetry (DSC). The dependence of the observed conformational restructuring on the kind of albumin (human, bovine), its form (fatty acid free and nondefatted), the properties of solvent (water, ethanol solutions), ionic strength, pH, protein concentration and experimental conditions has been discussed in this work. The endothermic unfolding transition has been shown to be modified by time changes and changes induced by such environmental factors as radio frequency radiation or UV radiation. Considering the process of albumin thermal unfolding within the equilibrium thermodynamics, a deconvolution of DSC traces have been performed using the appropriate mathematical models. Structurally independent subunits revealed during thermal denaturation of albumin has been found pH dependent. At pH range corresponding to the N form of albumin these subunits could be correlated with three albumin domains for human albumins and nondefatted bovine albumin. Under the same conditions two subunits have been revealed for fatty acid free bovine albumin: C-terminal fragment containing domain III and the greater part of domain II and the N-terminal fragment containing domain I and the smaller part of domain II. DSC study of albumin in ethanol solutions has revealed stronger binding of ethanol to defatted than to nondefatted albumin. The interaction of ethanol with fatty acid binding sites located in subdomain IIA has been confirmed. Ethanol has been observed to be a stabilizer of the folded state of albumin at a lower concentration contrary to the high denaturant concentration where its binding to the unfolded protein predominates. The obtained results indicate that the influence of radiofrequency radiation (from several to tens MHz) on albumin unfolding events could be detected using ultrasensitive microcalorimeter. That influence is not observed immediately, however, the differences between DSC profiles for irradiated and nonirradiated albumin solutions have appeared during their storage. The changes in irradiated samples outpace nonirradiated ones. Calorimetric and spectroscopic results have shown the conformational restructuring of albumin under UV irradiation. The differences in response to UV radiation between nondefatted and fatty acid free albumins have been found. Albumin devoid of endoge-nous fatty acids has been suggested to be more susceptible to aggregation caused by UV A—C as well as 254 nm UVC radiation. DSC curve deconvolution results allow to conclude that the C-terminal fragment of albumin macromolecule, containing domain III, is the most liable part to UV radiation. The studies presented by the author have revealed the fundamental role of the presence of fatty acids for the thermal stability, conformational rearrangement and binding properties of albumin macromolecule.