Wu Sha, Ding Yanwei, Zhang Guangzhao
Hefei National Laboratory for Physical Sciences at the Microscale, Department of Chemical Physics, University of Science and Technology of China , Hefei, 230026, China.
Faculty of Materials Science and Engineering, South China University of Technology , Guangzhou, P. R. China 510640.
J Phys Chem B. 2015 Dec 31;119(52):15789-95. doi: 10.1021/acs.jpcb.5b08190. Epub 2015 Dec 16.
Folding and aggregation of proteins profoundly influence their functions. We have investigated the effects of thermal history, concentration and pH on the denaturation and refolding of lysozyme by using ultrasensitive differential scanning calorimetry (US-DSC) and sedimentation velocity (SV) via analytical ultracentrifugation (AUC). The former is sensitive to small energy change whereas the latter can differentiate the oligomers such as dimer and trimer from individual protein molecules. Our studies reveal that the degree of denaturation irreversibility increases as heating times increases. The denaturation temperature (Td) and enthalpy change (ΔH) are influenced by heating rate since the denaturation is not in equilibrium during the heating. We can obtain Td and ΔH in equilibrium by extrapolation of heating rate to zero. In a dilute solution, no aggregation but unfolding happens in the denaturation. However, when the concentration is above a critical value (∼15.0 mg/mL), lysozyme molecules readily form trimers or other oligomers. Lysozyme molecules unfold into stretched chains at pH > 6.0, which would further forms large aggregates. The formation of aggregates makes the refolding of lysozyme impossible.
蛋白质的折叠和聚集对其功能有深远影响。我们通过超灵敏差示扫描量热法（US-DSC）和分析超速离心沉降速度法（SV）研究了热历史、浓度和pH对溶菌酶变性和复性的影响。前者对小的能量变化敏感，而后者可以区分诸如二聚体和三聚体等寡聚体与单个蛋白质分子。我们的研究表明，随着加热时间的增加，变性不可逆程度增加。变性温度（Td）和焓变（ΔH）受加热速率影响，因为加热过程中变性并非处于平衡状态。通过将加热速率外推至零，我们可以得到平衡状态下的Td和ΔH。在稀溶液中，变性时不发生聚集而是发生解折叠。然而，当浓度高于临界值（约15.0 mg/mL）时，溶菌酶分子容易形成三聚体或其他寡聚体。在pH > 6.0时，溶菌酶分子解折叠成伸展链，这会进一步形成大的聚集体。聚集体的形成使得溶菌酶无法复性。
