Synergetic effects of pressure and chemical denaturant on protein unfolding: stability of a serine-type carboxyl protease, kumamolisin.
Synergetic effects of pressure and chemical denaturant on protein unfolding: stability of a serine-type carboxyl protease, kumamolisin.
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DOI：
10.1016/j.bbapap.2005.12.010
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发表时间：
2006-03
期刊：
影响因子：
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通讯作者：
Y. Fujimoto;H. Ikeuchi;T. Tada;H. Oyama;K. Oda;S. Kunugi
中科院分区：
文献类型：
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作者：
Y. Fujimoto;H. Ikeuchi;T. Tada;H. Oyama;K. Oda;S. Kunugi
Kumamolisin是一种丝氨酸羧基蛋白酶，非常稳定，几乎不受化学变性剂（尿素）、压力（<500 MPa）或温度（<65 °C）的单一扰动而变性。为了研究这三种变性剂的协同作用，在各种条件下测量DSC、CD、固有荧光和四阶导数UV吸光度。通过对溶于8 M尿素溶液中的曲氨青霉素施加压力，在类似的含尿素条件下，观察到荧光发射光谱质量中心的显著红移，并且观察到四阶导数UV吸光度中两个主峰的相应蓝移。基于一个简单的双态模型分析了变性曲线，得到了热力学参数（ΔV、ΔG和m值），并讨论了变性剂的联合作用，特别是对大空腔和邻近Trp残基的影响.
Kumamolisin, a serine carboxyl proteinase, is very stable and hardly denatured by single perturbation of a chemical denaturant (urea), pressure (<500 MPa) or temperature (<65 °C). In order to investigate the cooperative effects of these three denaturing agents, DSC, CD, intrinsic fluorescence, and fourth derivative UV absorbance were measured under various conditions. By application of pressure to kumamolisin in 8 M urea solution, substantial red-shift in the center of fluorescence emission spectral mass was observed, and the corresponding blue-shift was observed for two major peaks in fourth derivative UV absorbance, under the similar urea-containing conditions. The denaturation curves were analyzed on the basis of a simple two-state model in order to obtain thermodynamic parameters (ΔV, ΔG, and m values), and the combined effects of denaturing agents are discussed, with the special interest in the large cavity and neighboring Trp residue in kumamolisin.