Department of Chemistry, University of Perugia, Via Elce di Sotto 8, I-06123 Perugia, Italy.
J Phys Chem B. 2012 Sep 6;116(35):10779-85. doi: 10.1021/jp305939h. Epub 2012 Aug 27.
The dielectric behavior of native and heat-denatured lysozyme in ethanol-water solutions was examined in the frequency range from 1 MHz to 2 GHz, using frequency-domain dielectric relaxation spectroscopy. Because of the conformational changes on unfolding, dielectric methods provide information on the denaturation process of the protein and, at protein concentration high enough, on the subsequent aggregation and gelation. Moreover, the time evolution of the protein aggregation and gelation was monitored measuring, by means of dynamic light scattering methods, the diffusion coefficient of micro-sized polystyrene particles, deliberately added to the protein solution, which act as a probe of the viscosity of the microenvironment close to the particle surface. All together, our measurements indicate that heat-induced denaturation favors, at high concentrations, a protein aggregation process which evolves up to the full gelation of the system. These findings have a direct support from IR measurements of the absorbance of the amide I band that, because of the unfolding, indicate that proteins entangle each other, producing a network structure which evolves, in long time limit, in the gel.
使用频域介电松弛谱法研究了天然和热变性溶菌酶在乙醇-水溶液中的介电行为，频率范围从 1 MHz 到 2 GHz。由于展开时的构象变化，介电方法提供了有关蛋白质变性过程的信息，并且在蛋白质浓度足够高时，还提供了随后的聚集和胶凝过程的信息。此外，通过动态光散射方法测量微尺寸聚苯乙烯颗粒的扩散系数，监测蛋白质聚集和胶凝的时间演变，这些颗粒被故意添加到蛋白质溶液中，作为接近颗粒表面的微环境粘度的探针。总之，我们的测量结果表明，在高浓度下，热诱导变性有利于蛋白质聚集过程，该过程一直持续到系统完全胶凝。这些发现得到了酰胺 I 带吸光度的红外测量的直接支持，由于展开，吸光度表明蛋白质相互缠绕，产生一种网络结构，在长时间内演变成凝胶。
