Borzova Vera A, Markossian Kira A, Chebotareva Natalia A, Kleymenov Sergey Yu, Poliansky Nikolay B, Muranov Konstantin O, Stein-Margolina Vita A, Shubin Vladimir V, Markov Denis I, Kurganov Boris I
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.
PLoS One. 2016 Apr 21;11(4):e0153495. doi: 10.1371/journal.pone.0153495. eCollection 2016.
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.
利用动态光散射、不对称流场-流分级分离和分析超速离心法研究了牛血清白蛋白（BSA）的热聚集。研究在0.1M磷酸盐缓冲液（pH7.0）中，BSA浓度为1mg/ml，固定温度（60℃、65℃、70℃和80℃）下进行。通过差示扫描量热法研究了蛋白质的热变性。实验数据分析表明，在65℃时，蛋白质展开阶段和蛋白质聚集的各个阶段在时间上明显分开。这种情况使我们能够提出以下BSA热聚集机制。蛋白质展开导致形成两种具有不同聚集倾向的非天然蛋白质形式。其中一种形式（高反应性展开形式，Uhr）的特征是聚集速率高。Uhr的聚集导致形成流体动力学半径（Rh,1）为10.3nm的初级聚集体。第二种形式（低反应性展开形式，Ulr）通过附着于由Uhr形式产生的初级聚集体而参与聚集过程，并具有自我聚集形成稳定小尺寸聚集体（Ast）的能力。当Ulr完全耗尽时，形成流体动力学半径（Rh,2）为12.8nm的二级聚集体。在60℃时，展开和聚集速率相当；在70℃时，初级和二级聚集体的形成速率相当；在80℃时，由于形成大尺寸聚集体，聚集初始阶段的记录变得复杂。
