《圆二色谱原理与应用ppt课件.ppt》由会员分享，可在线阅读，更多相关《圆二色谱原理与应用ppt课件.ppt（52页珍藏版）》请在三一办公上搜索。
1、圆二色谱Circular Dichroism (CD) 原理与应用,光的性质：波粒二象性诞生,人类对光的研究起源很早，但对光本质的认识经历了一个较漫长的过程。光究竟是波还是粒子？光的波动说与微粒说之争从十七世纪初开始， 其间牛顿、惠更斯、托马斯杨、菲涅耳、爱因斯坦、波尔等多位著名的科学家努力揭开了遮盖在“光的本质”外面那层扑朔迷离的面纱，至二十世纪初以光的波粒 二象性告终，前后共三百多年的时间。,粒子性：光是某种粒子即光量子，具有粒子的性质 如 反射，散射等现象。,波动性：即光具有波动性，有衍射、干涉等性质,墨子和他的学生做了世界上最早的“小孔成像”实验，并对实验结果作出了光沿直线传播的科学解
2、释，并用此原理解释了物体和投影的关系。,爱因斯坦提出了光量子论，解释了光电现象，揭示了微观客体的波粒二重性,光源发射光量子,从演示中可以看出：光源发射的单个光量子的运动轨迹是一个轨迹为余弦 函数的简谐振动 轨迹方程为 xAcos（t）,而真实光源向外发射的是连续的光量子 如图所示:图中不同颜色的小球代表不同的光量子 所有光量子在一个平面上我们称之为平面偏振光 它的轨迹方程为xAcos（t） 公式中 A为振幅与光强度的平方成正比 为初始相位 t为时间,我们平时看到的光为自然光具有: 1.方向随机性 自然光源向外发射方向性是随机的光量子 2. 不连续性 会产生无数初始相位不同的光量子,糖葫芦,z
3、传播方向 360度旋转的轨迹,这两个性质决定自然光具有如图所示的性质,既自然光可以看作圆柱形向前传播的光,两束光的合成:,物理力学力的矢量合成:,F1,F3,F2,物体受到的两个力F1,F2等同于物体受到这两个力的矢量合成F3,光的合成也遵循矢量合成:,其合成为电场矢量的合成与光强度的平方成正比,x,y,t,t1,t0 t1 t2 t3 t4,t0,t2,t3,t4,合成平面偏振光,图相互垂直的片面偏振光,2-1=0,取 t0,t1,t2,t3,t4时间的 截面,2-1=pi/2 AX=AY pi=3.141592,圆偏振光,x=AXcos(Wt+1)y=AYcos(Wt+2),x,y,如果A
4、X=AY合成轨迹为圆,如果AX= AY合成轨迹为椭圆,垂直传播方向,下面我们研究以下两束圆偏振光的合成:,两束旋转方向相反的圆偏振光如果振幅相同（振幅与光强度的平方成正比）矢量合成为平面偏振光。如果振幅不等则根据公式合成为下图右图所示的椭圆偏振光！,圆二色性,当自然光入射到电气石晶片的时,晶片强烈地吸收振动平面与晶轴垂直的光波,而只允许振动平面平行与晶轴的光波通过,因此通过晶片的光就变为具有一定平面的偏振光.,各向异性的物质对不同方向的光吸收不同如图所示：,平面不对称的分子具有各向异性 如氨基酸核酸、手性分子 也对偏振光有调节活性,当两束圆偏振光照射到含有这类分子的溶液时:,sample so
5、lution,溶液对左旋和右旋的圆偏振光的吸收不同这会导致EL不等于ER根据我们前面对振幅不同的两束圆偏正光的叠加现象可以判断透过样品溶液的光变为一束椭圆偏振光,当溶液中的样品的结构不同,或成分不同我们可以得到不同的椭圆反过来我们可以通过检测两束旋转方向相反的圆偏振光透过样品所产生的椭圆的不同来判断样品所含的成分,和结构信息。,泰勒一阶展开式,in proper units (CD spectroscopists use decimol),Per residue,2),为了方便比较我们用来描述椭圆的信息,度,圆二机器测量值,光谱学家一般采用摩尔椭圆率 和 Deltaepslon大家形成统 一的
6、单位易于比较,tan(i0)n2/n1 时i0 被称为布鲁斯特角 此时的反射光为平面偏振光，利用该原理可制造起偏器。,双折射：,o-ray,e-ray,当一束光经过各向异性的晶体或其他状态的介质时产生相互垂直的两束偏振光 o, e,当光离开晶体时的2-1=2*pi*l/(n0-ne)*入 四分之一波片 如果l=入/4 /(n0-ne) 则2-1=pi/2 此时如果入射光与光轴夹角为45度 时o,e的振幅相同则o,e合成的为圆偏振光.,45deg,45deg,光轴,光轴,偏振光的产生:,圆二色谱仪器刨面图M为镜子,p为起偏器 ls代表等和水晶,CD特点,测量的非常小CD测量的为2.32*(AL-
7、AR) 弧度以样品有圆二信号一定要有紫外吸收但有紫外吸收不一定由远而信号,CD谱在蛋白质研究中的应用,The peptide bond is inherently asymmetric & is always optically active,蛋白质分子的固有不对称性决定了蛋白质的光学活性蛋白质的结构的不同表现出其对圆二色性特征的差异所以我们可以通过圆二信号来测量蛋白质的结构信息,图为一些蛋白的(或肽)的标准园二谱图,我们以肌红蛋白为例,193nm,208nm,223nm,我们从图中看到了193,208,223特征峰与上一图中全螺旋的蛋白的特征峰基本相同我们可以从这个信息断定我们样品的结构主要
8、为螺旋结构,当我们得到一个图谱可以通过下表来和前面提到的标准图谱来判断我们样品的结构信息,对于螺旋性较高的蛋白可以通过下式初步估算螺旋的含量: 但是准确性较差不推荐, chymotrypsin (all b) lysozyme (a + b) triosephosphate isomerase(a/b) myoglobin (all a),但一般蛋白的结构信息是较复杂的并非标准的螺旋或折叠之类的结构 对于这些蛋白结构的分析我们要选择合适的软件和算法进行分析,CD谱的分析算法很多但是最主要的都是基于最小二乘法:,v1v2v3v4v5v6v7v8v9v10v11v13v14v15v16.,180n
9、m,260nm,Helix,a +,v1v2v3v4v5v6v7v8v9v10v11v13v14v15v16.,sheet,b +,v1v2v3v4v5v6v7v8v9v10v11v13v14v15v16.,c=,测量的样品谱,v1v2v3v4v5v6v7v8v9v10v11v13v14v15v16.,Random coil,a/(a+b+c)=螺旋含量,b/(a+b+c)=折叠含量,c/(a+b+c)=无规卷曲含量,通过最最小二乘法求解出最优a,b,c的值,Recommended Methods,For determination of globular protein conformati
10、on in solution: SELCON, CDNN and K2D.For determination of polypeptide conformation: LINCOMB with a suitable polypeptide set of references.For determining the effects of mutation s, ligands and perturbants on protein structure: LINCOMB.For evaluating the number of folding states giving rise to a set
11、of spectra: The CCA algorithm and SVD.,The Protein Circular Dichroism DataBank (PCDDB)http:/pcddb.cryst.bbk.ac.uk /,On Line Circular Dichroism Analysis http:/public-1.cryst.bbk.ac.uk/cdweb/html/,Cca分析 http:/www2.chem.elte.hu/cgi-bin/ccaform061122-5lyxPGDxoLsM6-00,园二二级结构分析相关文献http:/lamar.colostate.ed
12、u/sreeram/PDF/index.html,下载软件分析: http:/www2.umdnj.edu/cdrwjweb/#Instructions,Cdpro分析:定期更新:SELCON3, CDSSTR,and CONTIN CLUSTERhttp:/lamar.colostate.edu/sreeram/CDPro/,从CD谱分析准确性,对全、 /和变性蛋白质的准确度90-100%对 + 的准确度为85%对全的准确度为75%,Applications of CD in structural biology,Determination of secondary structure of
13、 proteins that cannot be crystallised Investigation of the effect of e.g. drug binding on protein secondary structure Dynamic processes, e.g. protein folding Studies of the effects of environment on protein structure Secondary structure and super-secondary structure of membrane proteins Study of lig
14、and-induced conformational changes Carbohydrate conformation Investigations of protein-protein and protein-nucleic acid interactions Fold recognition,CD实验要点,仪器操作及参数:,Nitrogen flushing,Flushing the optics with dry nitrogen is a must:Xe lamp has a quartz envelope, so if operated in air itll develop a
15、lot of ozone, harmful for the mirrorsbelow 195nm oxygen will absorb radiation,氮气流量15-20 for 180nm 20 for 180nm,温度:圆二信号同紫外和荧光一样对温度十分敏感 实验平行体系温度要保持一致 房间空调,同时仪器控温,HT plot,The HT plot is very important, since readings above 600-650V mean that not enough light is reaching the detector so a sample dilutio
16、n or the use of shorter path cell are required.Furthermore the HT plot is in realty a single beam spectra of our sample, since there is a direct relation between HT and sample absorbance. By data manipulation HT conversion into absorbance and buffer baseline subtraction is possible. Alternatively si
17、ngle beam absorbance scale can be used already in CH2 during data collection, loosing however a bit the alerting functions of this channel.,Bandwidth (SBW) selection,Setting of slits should be as large as possible (to decrease noise level), but compatible to the natural bandwidth (NBW) of the bands
18、to be scanned.As a rule SBW should be kept at least 1/10 of the NBW, otherwise the band will be distorted.If NBW is not known a series of fast survey spectra at different SBW will help proper selection. Trade in of accuracy versus sensitivity (i.e. the use of larger than theoretical SBW) is occasion
19、ally required.2 nm in the far UV region1 nm in the aromatic region (where fine structures may be present), optimal band-pass (as large as possible, but not loosing information) can be determined after a trial,Number of data point,data pitch, i.e. number of data points per nm, will not directly influ
20、ence the noise level. However if post run further data processing will be applied to reduce the noise, its advisable to collect as many data points as possible to increase the efficiency of the post run filtering algorithm,Accumulation,another way to improve S/N is to average more spectra. Here too
21、the S/N will improve with the square root of the number of accumulations.Averaging is very effective since it compensates short term random noise, but itll not compensate long term drifts (mainly of thermal origin). So if long accumulations are used we recommend a suitable long warm-up of the system
22、 and/or the use of a sample alternator (to collect sequentially sample and blank and average their subtracted values).For long overnight accumulations its essential that room temperature is well kept stable.,扫描速度和响应时间:,扫描速度和响应时间对不同样品体系可能有所不同要得到较好可重复的光谱数据必须找到合适的扫描速度和响应时间.一般蛋白质扫描速度 :20-100nm/min响应时间:
23、4-8秒,样品装备,2. For stubborn dirt or proteins,a) You may use concentrated HNO3, aqua regia or other acid wash.b) Do NOT use alkali, hydrofluoric acid, or phosphoric acid on the quartz cell.Avoid scratching the cell.c) Clean with distilled H2O then soak with 2-3% sodium lauryl sulfate for a few minutes.
24、 Rinse thoroughly (10 times) with water, then EtOH 3 times, and acetone 3 times.3. After cleaning, avoid touching the sample cell. Fingerprints can cause artifacts in the data.,1. Routine cleaning.a) Wash with water or solvent.b) Wash with volatile solvent (e.g., acetone), then stream clean dry air
25、into the cell. The house air may contain oils so it is recommended to avoid it or to use glass filter in a pipette.,二、石英杯的清洗:,Circular Dichroism Calibration Procedure,1cm cell. 0.06% (w/v) At 291 nm, the result should be 190.4 1%, or 188.5 to 192.3 mdeg. This doesnot included error in making the cam
26、phor standard.,1-mm cell. 0.06% 18.7 mdeg at 290.5 nm and 238.9 mdeg at 192.5nm,Ammonium camphor sulfonate: solid ammonium d-camphor-10-sulfonate, FW 249.33From KATAYAMA CHEMICAL INDUSTRY CO LTD,溶剂的选择:样品往往需要溶解在不同的体系中,溶剂的选择一定要尽可能的避免 溶剂在我们要考察的波长范围内有吸收值,下面的表格列出了常用溶剂的圆二吸收峰位 在选择溶液前一定要要考察好溶剂的性质,Avoid: Tri
27、s; NaCl; Anything optical active, e.g. Glutamate,尽量避免Tris 氯离子,当溶液需要特定的缓冲体系时尽可能的稀释到较低的离子浓度一般为5mM/L,溶剂的吸收!,样品的浓度: 根据选择的园二比色杯的光程长度的不同浓度也不相同 1cm cell样品浓度 0.01-0.04mg/ml 但是 190左右的峰很不明显 0.5cmcell样品浓度 0.2-0.5 mg/ml 190左右的峰明显 慎重改变样品的浓度因为浓度的改变可能会导致样品的性质的改变如聚 集状态亚基的结合等,其它: 样品要有较高的纯度95% 所用试剂尽量用光谱纯 要确定蛋白的分子量和浓度 防止气泡干扰 超声波除气泡 防止粒子的散射 使用0.02um滤膜对过滤样品,Protein Concentration: 0.2 mg/mlCell Path Length: 5 mmVolume 200 ml Stabilizers (Metal ions, etc.): minimumBuffer Concentration : 5 mM or as low as possible while maintaining protein stability scan speed 100nm/min responce 4sec band width 2,
