圆二色谱Circular Dichroism (CD)47

1、圆二色谱圆二色谱Circular Dichroism (CD) Application 圆二色光谱仪通过测量生物大分子的圆二色光 谱从而得到生物大分子的二级结构。 可应用于：蛋白质折叠蛋白质构象研究, DNA/RNA反应, 酶动力学, 光学活性物质纯度测 量, 药物定量分析。天然有机化学与立体有机化 学, 物理化学, 生物化学与宏观大分子, 金属络合 物, 聚合物化学等相关的科学研究。 构 象 确定蛋白质构象最准确的方法是x-射线晶体衍 射，但对结构复杂、柔性的生物大分子蛋白质 来说，得到所需的晶体结构较为困难。二维、 多维核磁共振技术能测出溶液状态下较小蛋白 质的构象，可是对分子量较大的蛋白

2、质的计算 处理非常复杂。 圆二色光谱：研究稀溶液中蛋白质构象，快速、 简单、较准确 CD is very useful for looking at membrane proteins Membrane proteins are difficult to study. Crystallography difficult - need to use detergents Often even when structure obtained: Q- is it the same as lipid? CD ideal can do spectra of protein in lipid vesicl

3、es. We will look at Staphylococcal a-hemolysin as an example 主要内容主要内容 CD原理 蛋白质CD谱 CD实验要点 CD原理 圆二色性(circular dichroism, CD) 当平面偏振光通过具有旋光活性的介质时， 由于介质中同一种旋光活性分子存在手性不同 的两种构型，故它们对平面偏振光所分解成的 右旋和左旋圆偏振光吸收不同，从而产生圆二 色性 圆二色性的表示 椭圆度椭圆度 ，摩尔，摩尔椭圆度椭圆度 =2.303(AL AR)/4 = 3298(L - R)3300 (L - R) 在蛋白质研究中， 常用平均残基摩尔椭圆度

4、圆二色仪原理 蛋白质的CD谱 The peptide bond is inherently asymmetric NaCl; Anything optical active, e.g. Glutamate Summary CD is a useful method for looking at secondary structures of proteins and peptides. CD is based on measuring a very small difference between two large signals must be done carefully the Abs

5、 must be reasonable max between 0.6 and 1.2. Quarts cells path lengths between 0.0001 cm and 10 cm. 1cm and 0.1 cm common have to be careful with buffers TRIS bad - high UV abs Measure cell base line with solvent Then sample with same cell inserted same way around Turbidity kills - filter solutions

6、Everything has to be clean For accurate 2ndry structure estimation must know concentration of sample 核酸的CD信息 B-Z? Or Z-B？ 建议浓度：在吸光值0.2-0.8时浓度的5-10倍 圆二色谱在糖类化合物结构分析中的应用 碳水化合物的结构决定CD的强度和形状，而从CD获 得的构象信息的多少取决于样品结构的复杂程度； 对那些具有较好重复系列的糖类化合物而言，CD能提 供更为可靠的空间结构信息。 对那些结构比较复杂的糖类来说，即使不能直接测定 糖类化合物的绝对构象，利用一些经验规则，例如

7、， CD可以用来推断糖类是否具有gt构象，而且CD可以作 为探针来测定糖类化合物构象的变化，如胶体-溶液或 无序-有序的转变过程。 THANKS! iQeNbK8G5D1A-x*t$qZnVkSgPdMaI7F4C0z)v&s!pXmUjRfOcL9H6E2B+y(u%r#oWlThQeNbJ8G5D1A-w*t$qYnVkSgPdLaI7F3C0z)v&s#pXmUiRfOcK9H5E2B+x(u%rZoWkThQeMbJ8G4D1A-w*t!qYnVjSgPdLaI6F3C0y)v&s#pXlUiRfNcK9H5E2A+x(u$rZoWkThPeMbJ7G4D1z-w&t!qYmVjSgO

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