Zheng Jiayin, Desbat Bernard, Rastogi Vipin K, Shah Saumil S, Defrank Joseph J, Leblanc Roger M
Department of Chemistry, University of Miami, Coral Gables, Florida 33124-0431, USA.
Biomacromolecules. 2006 Oct;7(10):2806-10. doi: 10.1021/bm060352j.
The secondary structure of organophosphorus hydrolase (OPH) at the air-water interface was studied using polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The shape and position of the amide I and amide II bands were used to estimate the surface conformation and orientation of OPH. The PM-IRRAS results indicated that the enzyme did not unfold for the range of surface pressure used (0-30 mN/m). At low surface pressures, the signal of amide I was very weak and the intensity was almost the same as amide II. Upon further compression, the PM-IRRAS signal and the ratio of the intensity of amide I and amide II both increase, implying an increased interfacial concentration of the enzyme. From the amide I/amide II ratio and the band position, it was deduced that the enzyme adopts a conformation which gives a higher occupied surface at low surface pressure and rotates to a more vertical orientation at high surface pressures. The compression and decompression of the OPH monolayer indicated that the fingerprint of the secondary structure at the air-water interface was reversible. PM-IRRAS was also used to investigate the pH effect of the subphase on the secondary structure of OPH. The secondary structure of OPH at the air-water interface was well defined when the pH of the subphase was near its isoelectric point (IP, pH 7.6). However, it adopted a different orientation when the subphase pH values were higher or lower than the IP with formation of random coil structure. The hydrolysis of organophosphorus compound paraoxon by OPH was also studied at the air-water interface by PM-IRRAS. The pH effect and the interaction with paraoxon both seem to orientate the enzyme more in the plane of the interface and to produce random coil structure.
采用偏振调制红外反射吸收光谱法（PM - IRRAS）研究了有机磷水解酶（OPH）在气 - 水界面的二级结构。利用酰胺I带和酰胺II带的形状及位置来估计OPH的表面构象和取向。PM - IRRAS结果表明，在所使用的表面压力范围（0 - 30 mN/m）内，该酶并未展开。在低表面压力下，酰胺I的信号非常弱，强度几乎与酰胺II相同。进一步压缩时，PM - IRRAS信号以及酰胺I与酰胺II强度之比均增加，这意味着酶在界面处的浓度增加。从酰胺I/酰胺II的比值和谱带位置推断，该酶在低表面压力下采取一种构象，使其在表面上占据更高的面积，而在高表面压力下旋转至更垂直的取向。OPH单分子层的压缩和解压缩表明，气 - 水界面二级结构的指纹图谱是可逆的。PM - IRRAS还用于研究亚相的pH值对OPH二级结构的影响。当亚相的pH值接近其等电点（IP，pH 7.6）时，OPH在气 - 水界面的二级结构明确。然而，当亚相pH值高于或低于等电点时，它会采取不同的取向并形成无规卷曲结构。还通过PM - IRRAS在气 - 水界面研究了OPH对有机磷化合物对氧磷的水解作用。pH值的影响以及与对氧磷的相互作用似乎都使酶在界面平面内的取向更多，并产生无规卷曲结构。
