Murayama Koichi, Ozaki Yukihiro
Department of Chemistry, School of Science and Technology, Kwansei-Gakuin University, 2-1, Gakuen, Sanda, Hyogo 669-1337, Japan.
Biopolymers. 2002;67(6):394-405. doi: 10.1002/bip.10142.
The molten globule-like states of ovalbumin (OVA) in acid aqueous solutions are investigated by generalized two-dimensional (2D) Fourier transform near-IR (FT-NIR) correlation spectroscopy. This new method allows us to explore the changes in hydration and the secondary structure simultaneously. FT-NIR spectra are measured for OVA aqueous solutions with concentrations of 1, 2, 3, 4, and 5 wt % over a pH range of 2.4-5.4. Concentration-perturbed 2D correlation spectra are calculated for the spectra in the 4850-4200 and 7500-5350 cm(-1) regions at different pH values. The 2D NIR synchronous spectrum in the 4850-4200 cm(-1) region shows a significant change upon going from pH 5.4 to 3.6. An autopeak at 4265 cm(-1) that is due to a combination of a symmetric CH(2) stretching mode and a CH(2) bending mode of side chains seen at pH 5.0 disappears completely in the synchronous spectrum at pH 3.6. This suggests that some amino acid residues of OVA are subjected to microenvironmental changes with decreasing pH. More remarkable changes are observed in the synchronous spectra at pHs below 2.8. A band near 4600 cm(-1) arising from a combination of amide B and amide II modes (amide B/II) shifts downward with considerable broadening between pH 3.0 and 2.4, suggesting that the strength of the hydrogen bonds of amide groups of OVA changes significantly. The synchronous and asynchronous spectra in the 4850-4200 cm(-1) region show that the intensities of the bands attributable to amide groups and side chains of OVA and that of the band near 4800 cm(-1) arising from water change in phase with the increase in the concentration above pH 2.8, but they vary out of phase below pH 2.8. The 2D synchronous map in the 7500-5350 cm(-1) region also shows marked changes upon going from pH 2.8 to 2.6. A broad autopeak at around 6950 cm(-1) assigned to free water and bound water with weak hydrogen bonds becomes very weak in the synchronous spectrum at pH 2.6, while broad autopeaks around 6450 cm(-1) suddenly appear that are due to bound water with several hydrogen bonds and the first overtone of an NH stretching mode of the amide groups of OVA. Therefore, it is very likely that protein hydration and the hydrogen bonds of amide groups change simultaneously in a narrow pH region of 2.8-2.6. It is probably that below pH 2.6 the protein assumes a molten globule-like state in which the whole molecule is very flexible, and side chains (but not the backbone chain) fluctuate significantly.
通过广义二维（2D）傅里叶变换近红外（FT-NIR）相关光谱研究了酸性水溶液中卵清蛋白（OVA）的熔融球状状态。这种新方法使我们能够同时探索水合作用和二级结构的变化。在2.4 - 5.4的pH范围内，测量了浓度为1、2、3、4和5 wt%的OVA水溶液的FT-NIR光谱。计算了不同pH值下4850 - 4200和7500 - 5350 cm⁻¹区域光谱的浓度扰动二维相关光谱。4850 - 4200 cm⁻¹区域的二维近红外同步光谱在从pH 5.4变为3.6时显示出显著变化。在pH 5.0时出现的由于侧链对称CH₂伸缩模式和CH₂弯曲模式组合产生的4265 cm⁻¹处的自峰在pH 3.6的同步光谱中完全消失。这表明随着pH值降低，OVA的一些氨基酸残基受到微环境变化的影响。在pH低于2.8的同步光谱中观察到更显著的变化。由酰胺B和酰胺II模式（酰胺B/II）组合产生的4600 cm⁻¹附近的谱带在pH 3.0和2.4之间向下移动并显著变宽，表明OVA酰胺基团的氢键强度发生了显著变化。4850 - 4200 cm⁻¹区域的同步和异步光谱表明，在pH高于2.8时，随着浓度增加，OVA酰胺基团和侧链以及水产生的4800 cm⁻¹附近谱带的强度同相变化，但在pH低于2.8时它们异相变化。7500 - 5350 cm⁻¹区域的二维同步图谱在从pH 2.8变为2.6时也显示出明显变化。在pH 2.6的同步光谱中，分配给自由水和弱氢键结合水的约6950 cm⁻¹处的宽自峰变得非常弱，而在约6450 cm⁻¹处突然出现宽自峰，这是由于具有多个氢键的结合水和OVA酰胺基团NH伸缩模式的一级倍频产生的。因此，很可能在2.8 - 2.6的狭窄pH区域内蛋白质水合作用和酰胺基团的氢键同时发生变化。可能在pH低于2.6时蛋白质呈现熔融球状状态，其中整个分子非常灵活，侧链（而非主链）波动显著。
