Investigating the Hydrogen-Bonding Model of Urea Denaturation
Investigating the Hydrogen-Bonding Model of Urea Denaturation
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DOI：
10.1021/ja9016057
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发表时间：
2009-07-08
影响因子：
15
通讯作者：
Cremer, Paul S.
中科院分区：
文献类型：
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作者：
Sagle, Laura B.;Zhang, Yanjie;Cremer, Paul S.
用温敏聚合物聚（N-异丙基丙烯酰胺）（PNIPAM）测试了基于尿素的蛋白质变性的直接结合机制。聚合物的热力学测量。通过傅里叶变换红外（FTIR）光谱、斯托克斯半径测量和甲基化脲补充疏水性塌陷。实验结果表明，PNIPAM的低临界溶解温度（LCST）随着尿素的加入而降低。因此，尿素实际上促进了大分子的疏水性塌陷。此外，这些热力学测量与酰胺I带数据强烈相关，这表明LCST的降低与脲与聚合物的酰胺部分的直接氢键结合有关。此外，氢键被认为是高度合作，这是一致的交联（二价结合）机制。使用凝胶过滤色谱法，通过低于聚合物的LCST的斯托克斯半径测量来确认交联。最后，与甲基脲，二甲基脲，和四甲基脲的相变测量表明，这些取代的化合物导致PNIPAM的LCST随着甲基含量的增加而上升。通过FTIR，没有发现任何这些甲基化脲与聚合物酰胺部分直接结合的证据。这些结果与尿素诱导蛋白质变性的直接氢键机制不一致。
The direct binding mechanism for urea-based denaturation of proteins was tested with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM). Thermodynamic measurements of the polymer's. hydrophobic collapse were complemented by Fourier transform infrared (FTIR) spectroscopy, Stokes radius measurements, and methylated urea. experiments. It was found that the lower critical solution temperature (LCST) of PNIPAM decreased as urea was added to the solution. Therefore, urea actually facilitated the hydrophobic collapse of the macromolecule. Moreover, these thermodynamic measurements were strongly correlated with amide I band data which indicated that the decrease in the LCST was coupled to the direct hydrogen bonding of urea to the amide moieties of the polymer. In addition, the hydrogen bonding was found to be highly cooperative, which is consistent with a cross-linking (bivalent binding) mechanism. Cross-linking was confirmed by Stokes radius measurements below the polymer's LCST using gel filtration chromatography. Finally, phase transition measurements with methylurea, dimethylurea, and tetramethylurea indicated that these substituted compounds caused the LCST of PNIPAM to rise with increasing methyl group content. No evidence could be found for the direct binding of any of these methylated ureas to the polymer amide moieties by FTIR. These results are inconsistent with a direct hydrogen-bonding mechanism for the urea-induced denaturation of proteins.