Chalmers University of Technology, Department of Chemistry and Chemical Engineering, Physical Chemistry, SE-41296 Gothenburg, Sweden.
Phys Chem Chem Phys. 2018 Sep 19;20(36):23202-23213. doi: 10.1039/c8cp01063c.
Fluorescence spectroscopy of protein-bound molecular rotors Cy3 and Cy5 is used to monitor the effective viscosity inside the pores of two types of mesoporous silica (SBA-15 and MCF) with pore diameters between 8.9 and 33 nm. The ratio of the peak intensities is used to measure viscosity independently of solvent polarity, and the response of the lipase-bound dyes is calibrated using glycerol/water mixtures (no particles). The two dyes are either attached to the same protein or separate proteins in order to investigate potential effects of energy transfer (FRET) on the fluorescence properties, when using them as reporter dyes. The effective viscosity inside the pores at infinite protein dilution is one order of magnitude higher than in bulk water, and the effect of protein concentration on the measured viscosity indicates a stronger effect of protein-protein interactions in the pores than in similarly concentrated protein solutions without particles. In MCF-particles with octyl-groups covalently attached to the pore walls, a more efficient uptake of the lipase resulted in FRET between the protein-bound dyes even if the two dyes were attached to different proteins. In contrast to the unmodified particles the intensity-ratio method could therefore not be used to measure the viscosity, but the presence of FRET in itself indicates that octyl-protein interactions lead to a non-homogenous protein distribution in the pores. The dye labels also report a less polar pore environment as sensed by the proteins through a redshift in the dye emission. Both observations may help in understanding the higher efficiency of lipase immobilization in octyl-modified particles.
使用蛋白质结合分子转子 Cy3 和 Cy5 的荧光光谱法监测两种介孔二氧化硅（SBA-15 和 MCF）的有效粘度，其孔径在 8.9nm 至 33nm 之间。通过峰值强度比来独立于溶剂极性测量粘度，并且使用甘油/水混合物（无颗粒）对脂肪酶结合染料的响应进行校准。为了研究能量转移（FRET）对荧光性质的潜在影响，当将两种染料用作报告染料时，将它们附着在相同的蛋白质上或不同的蛋白质上。在无限稀释蛋白质的情况下，孔内的有效粘度比在体相水中高一个数量级，并且测量的粘度随蛋白质浓度的变化表明蛋白质-蛋白质相互作用在孔内的影响比在没有颗粒的类似浓度的蛋白质溶液中更强。在带有共价连接到孔壁上的辛基基团的 MCF 颗粒中，即使两种染料附着在不同的蛋白质上，脂肪酶的更有效吸收也导致了蛋白质结合染料之间的 FRET。与未修饰的颗粒相比，因此不能使用强度比方法来测量粘度，但是 FRET 的存在本身表明辛基-蛋白质相互作用导致孔内蛋白质分布不均匀。染料标签还通过染料发射的红移报告了蛋白质感觉到的极性较小的孔环境。这两种观察结果都有助于理解脂肪酶在辛基修饰的颗粒中更高的固定化效率。
