Aggregation of bovine insulin probed by DSC/PPC calorimetry and FTIR spectroscopy
Aggregation of bovine insulin probed by DSC/PPC calorimetry and FTIR spectroscopy
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DOI：
10.1021/bi034879h
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发表时间：
2003-09-30
期刊：
影响因子：
2.9
通讯作者：
Winter, R
中科院分区：
文献类型：
--
作者：
Dzwolak, W;Ravindra, R;Winter, R
采用压力微扰量热法（PPC）、差示扫描量热法（DSC）和时间分辨傅里叶变换红外光谱（FTIR）研究了牛胰岛素在pH 1.9时的聚集。聚集过程表现出两个不同的阶段。在第一阶段，一个中间的熔融球状构象状态是短暂形成的，反映了松散的三级接触和强大的H/D-交换。随后是天然二级结构的解折叠。胰岛素的解折叠是快速的、吸热的、部分可逆的，并且伴随着约0.2%的体积膨胀。第二阶段由实际的聚集：一个放热的不可逆过程揭示成核控制动力学的典型特征。与第二阶段相关的体积变化很小。DSC扫描的浓度依赖性不支持单体中间体模型。虽然胰岛素在环境压力下的聚集是快速和定量的，但如高压FTIR光谱所揭示的，低至300 bar的压力足以完全防止聚集。这可以解释为高压对胰岛素的热解折叠具有不利影响，因此防止了易于聚集的中间体的出现。比较了在水和重水中的聚集情况，发现同位素取代对聚集的两个阶段有不同的影响。在重水中，一个更明显的体积膨胀伴随着展开阶段，而只有第二相转移到更高的温度。
Pressure perturbation calorimetry (PPC), differential scanning calorimetry (DSC), and time-resolved Fourier transform infrared spectroscopy (FTIR) have been employed to investigate aggregation of bovine insulin at pH 1.9. The aggregation process exhibits two distinguished phases. In the first phase, an intermediate molten globule-like conformational state is transiently formed, reflected by loose tertiary contacts and a robust H/D-exchange. This is followed by unfolding of the native secondary structure. The unfolding of insulin is fast, endothermic, partly reversible, and accompanied by a volume expansion of approximately 0.2%. The second phase consists of actual aggregation: an exothermic irreversible process revealing typical features of nucleation-controlled kinetics. The volumetric changes associated with the second phase are small. The concentration-dependence of DSC scans does not support a monomer intermediate model. While insulin aggregation under ambient pressure is fast and quantitative, pressure as low as 300 bar is sufficient to prevent the aggregation completely, as high-pressure FTIR spectroscopy revealed. This is explained in terms of the high pressure having an adverse effect on the thermal unfolding of insulin, and therefore preventing occurrence of the aggregation-prone intermediate. A comparison of the aggregation in H2O and D2O shows that the isotopic substitution has diverse effects on both the phases of aggregation. In heavy water, a more pronounced volume expansion accompanies the unfolding stage, while only the second phase shifts to higher temperature.