여러 가지 Perturbing media 에서의 알부민의 비가역적 열변성 = Study of Thermal Denaturation of Bovine Serum Albumins in Various perturbing Media by High Sensitivity Differential Scanning Calorimetry

다국어 초록 (Multilingual Abstract)

The thermal denaturation mechanisms of bovine serum albumin (BSA) and its reduced form of cysteinylBSA in PBS buffer (pH 7.0, ionic strength=0.1 or 0.2) were investigated on the basis of kinetic analysis in which in-eversible two-state transition (N→D) was assumed. Several perturbants such as urea, N-methylurea, N,N`-dimethylurea, tetrnmethylurea, acetamide, thiourea, guanidine-HCl, sorbitol and glycerol were employed to perturb the system and to see their effect on the thermal denaturation of BSA and disulfide-broken cysteinyl-BSA. The thermal transition parnmeters, T_m (denaturation peak temperature), ΔH_(cal) (endothermic heat) and C_p (heat capacity) of the two proteins were measured on a Setaram micro-DSC (differential scanning calorimeter) in the presence or absence of those perturbants. The activation energies (E_a) of thermal denaturation of BSA and cysteinyl-BSA were estimated from the analysis of DSC-scan profiles. The estimated activation energies (E_a) ranged from 216 to ∼380 KJ/mol depending on the type and the concentration of the perturbant employed. The activation energy of thermal denaturation of unreduced BSA is higher than that of cysteinyl-BSA, which means that the 17 disulfide bonds contribute to the stabilization of BSA. It was also found that urea, methyl substituted ureas and high-concentration guanidine-HCl destabilized BSA, but the polyhydroxyl perturbants of sorbitol and glycerol stabilized the protein, which was determined from both T_m and E_a data. However, increasing acetamide concentration caused a gradual decrease of T_m and a grndual increase of E_a of BSA Thus, this indicates that the two determinants (T_m and E_a) for thermal stability of proteins are independent of each other. Apparent deviation from the two-state mechanism of BSA thermal denaturation in acetamide-containing PBS buffer was estimated by calculating the van`t Hoff enthalpies (ΔH_(VH)) and comparing them with the calorimetric enthalpies (ΔH_(cal)). An increase of acetamide concentration generally decreased the deviation from the two-state mechanism.