紫外-荧光在蛋白质分析中的应用.pptVIP

紫外与荧光光谱在蛋白质研究中的应用 曹傲能 主要内容 UV/vis基础、实验要点、应用 Fluorescence基础、实验要点、常规荧光、淬灭、荧光共振能量传递(FRET) 谱学方法 基本原理1-Lambert Law The fraction of light absorbed by a transparent medium is independent of the incident intensity, and each successive layer of the medium absorbs an equal fraction of the light passing through it. Log10(I0/I)=kl 基本原理2-Beer’s Law The amount of light absorbed is proportional to the number of molecules of the chromophore through which the light passes. k=?c Deviations from the Beer-Lambert law 强吸收 高浓度 噪声与误差 等吸收点(Isosbestic point) 等吸收点常作为体系中只有两种成分(状态)的指示 可作为参比波长 若有等吸收点,则不需要为为每个谱作基线 单光路 双光路 双波长 二极管阵列检测器 二极管阵列光谱仪动力学实验 波长选择 常规吸收光谱实验要点 紫外/可见光谱在蛋白质研究中的应用 浓度测定 构象变化研究 相互作用研究 蛋白质主链的紫外吸收 肽键在250 nm的远紫外区有较大吸收 蛋白质浓度测定 ?190nm? 10000 l/mol.cm ?190nm比?280nm大~100倍 但溶剂吸收也较大 溶剂的吸收 蛋白质中氨基酸的紫外吸收光谱 确定蛋白质的消光系数 可以根据蛋白质序列预测蛋白质的消光系数 ProtParam:/tools/protparam.html Tyr的吸收谱与pH有关 pH titration can be used to determine- whether Tyr is internal or external- polarity of environment 环境极性的影响 Blue shift of spectra in polar solvent Weaker absorption of Tyr in polar solvent 蛋白质构象变化研究 蛋白质折叠/变性研究 荧光光谱 量子产率 ?F = 发射的光子数/吸收的光子数 荧光强度 IF = I0 (1-10-?cl) ?F  若?cl很小，则近似： IF = I0(?cl)?F Inner filter effect Inner filter effect 所以，实验中，A?,EX0.1 Raman and Rayleigh Scattering Two potential sources of background radiation are Raman and Rayleigh scattering. Both of these phenomena arise due to vibrational changes induced in molecules by incident radiation. Both can also be described as scattered light. The Raman lines are of different frequency from the incident light and usually of longer wavelength. Rayleigh radiation is scattered light of the same frequency as the incident light. Rayleigh Scattering 强度与r6/ ?4成正比 不能通过减空白来消除 选择合适的激发波长 从比激发波长大(10nm)处开始收谱 减少带宽 Raman scattering 因水中O-H收缩(3300cm-1):1/ ?RA = 1/ ?EX – 0.00033 Environmental Sensitivity Fluorophores can be affected by a large number of environmental factors including such