CircularDichroism.ppt

1、圓二色譜Circular Dichroism (CD),Application,圓二色光譜儀通過測(cè)量生物大分子的圓二色光譜從而得到生物大分子的二級(jí)結(jié)構(gòu)。 可應(yīng)用于：蛋白質(zhì)折疊蛋白質(zhì)構(gòu)象研究, DNA/RNA反應(yīng), 酶動(dòng)力學(xué), 光學(xué)活性物質(zhì)純度測(cè)量, 藥物定量分析。天然有機(jī)化學(xué)與立體有機(jī)化學(xué), 物理化學(xué), 生物化學(xué)與宏觀大分子, 金屬絡(luò)合物, 聚合物化學(xué)等相關(guān)的科學(xué)研究。,構(gòu) 象,確定蛋白質(zhì)構(gòu)象最準(zhǔn)確的方法是x-射線晶體衍射，但對(duì)結(jié)構(gòu)復(fù)雜、柔性的生物大分子蛋白質(zhì)來(lái)說，得到所需的晶體結(jié)構(gòu)較為困難。二維、多維核磁共振技術(shù)能測(cè)出溶液狀態(tài)下較小蛋白質(zhì)的構(gòu)象，可是對(duì)分子量較大的蛋白質(zhì)的計(jì)算處理非常復(fù)雜。

2、圓二色光譜：研究稀溶液中蛋白質(zhì)構(gòu)象，快速、簡(jiǎn)單、較準(zhǔn)確,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 vesicles. We will lo

3、ok at Staphylococcal a-hemolysin as an example,主要內(nèi)容,CD原理 蛋白質(zhì)CD譜 CD實(shí)驗(yàn)要點(diǎn),CD原理,圓二色性(circular dichroism, CD),當(dāng)平面偏振光通過具有旋光活性的介質(zhì)時(shí)，由于介質(zhì)中同一種旋光活性分子存在手性不同的兩種構(gòu)型，故它們對(duì)平面偏振光所分解成的右旋和左旋圓偏振光吸收不同，從而產(chǎn)生圓二色性,圓二色性的表示,橢圓度，摩爾橢圓度 =2.303(AL AR)/4 = 3298(L - R)3300 (L - R) 在蛋白質(zhì)研究中， 常用平均殘基摩爾橢圓度,圓二色儀原理,蛋白質(zhì)的CD譜,The peptide bond

4、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 must be reasonable max between

5、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 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？ 建議濃度：在吸光值0.2-0.8時(shí)濃度的5-10倍,圓二色譜在糖類化合物結(jié)構(gòu)分析中的應(yīng)用,碳水化合物的結(jié)構(gòu)決定CD的強(qiáng)度和形狀，而從CD獲得的構(gòu)象信息的多少取決于樣品結(jié)構(gòu)的復(fù)雜程度； 對(duì)那些具有較好重復(fù)系列的糖類化合物而言，CD能提供更為可靠的空間結(jié)構(gòu)信息。 對(duì)那些結(jié)構(gòu)比較復(fù)雜的糖類來(lái)說，即使不能直接測(cè)定糖類