Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin.
Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, Uhr) is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of primary aggregates with the hydrodynamic radius (Rh,1) of 10.3 nm. The second form (low reactive unfolded form, Ulr) participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast). At complete exhaustion of Ulr, secondary aggregates with the hydrodynamic radius (Rh,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.
Department of Structural Biochemistry of Proteins, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
Vera A
VA
Borzova
Department of Structural Biochemistry of Proteins, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
Kira A
KA
Markossian
Department of Structural Biochemistry of Proteins, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
Natalia A
NA
Chebotareva
Department of Structural Biochemistry of Proteins, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.|Department of Biophysics, Kol'tsov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia.
Sergey Yu
SY
Kleymenov
Department of Chemical and Biological Processes Kinetics, Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia.
Nikolay B
NB
Poliansky
Department of Chemical and Biological Processes Kinetics, Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia.
Konstantin O
KO
Muranov
Department of Structural Biochemistry of Proteins, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
Vita A
VA
Stein-Margolina
Department of Biochemistry of Chloroplast, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
Vladimir V
VV
Shubin
Department of Structural Biochemistry of Proteins, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
Denis I
DI
Markov
Department of Structural Biochemistry of Proteins, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
Boris I
BI
Kurganov
D019540:Area Under Curve; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D006358:Hot Temperature; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D011489:Protein Denaturation; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods; D014461:Ultracentrifugation
D012710:Serum Albumin, Bovine