Yang C-H, Wu P-C, Huang Y-B, Tsai Y-H
Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical University, No. 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, R.O.C.
J Biomol Struct Dyn. 2004 Aug;22(1):101-10. doi: 10.1080/07391102.2004.10506985.
Based on Fourier transform infrared (FTIR) microspectroscopy, the conformation of rhEGF under the influence of pH, heat treatment, chaotropic salts, concentration of salt and protein structure perturbants was studied. The FTIR spectrum of rhEGF showed that major secondary structures from amide I bands composed of 40.6% beta-sheets, 25.0% reverse turns, 16.5% random coils, 13.0% loops and 4.9% side-chain structures. At extreme pH conditions (pH < 4 and pH > 8), there were changes in intensity of the bands attributed to loop (1658 cm(-1)) and random coil structures, and these bands shifted to lower wavenumbers, indicating changes in protein conformation. Thermal denaturation of rhEGF occurred at 40-76 degrees C and the formation of intermolecular beta-aggregates was revealed by the FTIR spectra. Thermal-irreversible property of rhEGF after second-heating treatment suggested that rhEGF has a poor thermal stability. While investigating the stability of rhEGF in the presence of chaotropic salts, anions induced protein unfolding of rhEGF more significantly than cations. The optimal stabilizing effect was found at the 2 M NaCl added to rhEGF, and expressed the structure of rhEGF more stable on the many components. The bands of loop structure (1654 cm(-1)), beta-sheet (1638 cm(-1)) and intermolecular antiparallel beta-aggregation formation (1694, 1619 and 1612 cm(-1)) seem to be "marked" to be more sensitive in determining environmental changes of rhEGF for FTIR microspectroscopy.
基于傅里叶变换红外（FTIR）显微光谱技术，研究了pH值、热处理、离液盐、盐浓度和蛋白质结构扰动剂对重组人表皮生长因子（rhEGF）构象的影响。rhEGF的FTIR光谱表明，酰胺I带的主要二级结构由40.6%的β-折叠、25.0%的反向转角、16.5%的无规卷曲、13.0%的环和4.9%的侧链结构组成。在极端pH条件下（pH < 4和pH > 8），归属于环（1658 cm⁻¹）和无规卷曲结构的谱带强度发生变化，且这些谱带向低波数移动，表明蛋白质构象发生了变化。rhEGF的热变性发生在40 - 76℃，FTIR光谱揭示了分子间β-聚集体的形成。二次加热处理后rhEGF的热不可逆特性表明其热稳定性较差。在研究rhEGF在离液盐存在下的稳定性时，阴离子比阳离子更显著地诱导rhEGF的蛋白质解折叠。在向rhEGF中添加2 M NaCl时发现了最佳的稳定效果，并且在多种成分上rhEGF的结构表现得更稳定。环结构（1654 cm⁻¹）、β-折叠（1638 cm⁻¹）和分子间反平行β-聚集体形成（1694、1619和1612 cm⁻¹）的谱带似乎是在FTIR显微光谱法测定rhEGF环境变化时更敏感的“标记”。
