Ganesan Ashok, Moore Barry D, Kelly Sharon M, Price Nicholas C, Rolinski Olaf J, Birch David J S, Dunkin Ian R, Halling Peter J
WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, United Kingdom.
Chemphyschem. 2009 Jul 13;10(9-10):1492-9. doi: 10.1002/cphc.200800759.
We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm(-1) to 1632 cm(-1), indicating a shift from alpha-helical structure to beta-sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to beta-sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.
我们报道了基于圆二色性（CD）、漫反射红外傅里叶变换（DRIFT）和色氨酸（Trp）荧光的生物物理技术的发展，用于原位研究固定在固体颗粒上的酶的结构。使用固定在硅胶上的枯草杆菌蛋白酶卡尔伯格（SC）和固定在Lewatit VP.OC 1600（诺维信435）上的南极假丝酵母脂肪酶B证明了它们的适用性。从远紫外CD光谱和酰胺I振动带可以明显看出，SC在溶液中和固定状态下显示出几乎相同的二级结构。近紫外CD强度增加和Trp荧光降低表明在二氧化硅表面三级结构更刚性。固定化的SC失活后，这些技术揭示：a）近紫外CD信号几乎完全丧失，表明三级结构丧失；b）酰胺I振动带从1658 cm(-1) 移至1632 cm(-1)，表明从α-螺旋结构转变为β-折叠；c）远紫外CD中出现大幅蓝移和二色性降低，支持向β-折叠结构的转变；d）Trp荧光强度大幅增加，这反映了随着三级结构丧失分子内淬灭减少；e）荧光寿命分布发生重大变化，证实Trp环境发生了实质性变化。DRIFT测量表明压制KBr圆盘可能会扰乱蛋白质结构。对于固定在有机聚合物上的酶，可以获得不受载体材料干扰的近紫外CD光谱。然而，远紫外CD、DRIFT和荧光测量显示来自有机载体的强信号。总之，本文所述的光谱方法提供了迄今无法获得的结构信息，其适用性受到载体材料干扰而非颗粒性质的限制。
