圆二色谱原理.pptx

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,圆二色谱原理,光学活性分子对,左、右圆偏光,得吸收率得差值,1、自然光,光具有波、粒二象性,就是横电磁波,。,只有横波才有偏振现象,:,光矢量得,振动方向与光得传播方向垂直,在垂直于光传播方向得平面内,有不同得,振动方向,。,电场矢量,E,与磁场矢量,H,互相垂直、位相相同。,v,0,H,E,由于,感光作用,都就是,E,引起得,因而,将,E,作为光矢量,。,振动面,:,E,与光传播方向组成得平面。,E,传,播方向,振动面,从统计规律上说,自然光得光振动,:,在垂直于光速得平面上遍布所有方向,沿各方向振动得光矢量呈对称分布,相应光矢量得振幅(光强度)相等。,(1)高亮度得点光源;(2),日光色,。色温接近6000,K;(3),在可见光区连续光谱,;(4)高显色性,显色指数95;(5)在整个寿命期内维持光色特性;(6)高电弧稳定性;(7),热重启能力,;(8),启动后即能达到接近最大光输出,;(9)电弧光斑小、易聚光。,超高压短弧,氙灯,:,在高压纯净氙气中放电发光。,自然光,平面偏振光,圆偏振光,样品,椭圆偏振光,双折射现象,:,一束光射入,各向异性晶体,后有两束折射光。,尼科耳棱镜。,2、光得偏振,在生物样品中,肌肉纤维、骨骼与牙齿等具有各向异性,淀粉粒、染色体与纺锤体等具有双折射性,因此被用于组织细胞得化学研究。,名称,折射定律,折射率,传播方向,寻常光,(,o,光),遵守,对于晶体一切方向都具有相同得折射率,在入射面内,非常光,(,e,光),不遵守,折射率随方向而变化,不一定在入射面内,o,光与,e,光:频率相同、振动方向相互垂直、,平面偏振光,自然光入射到某些晶体(电气石、硫酸金鸡钠碱晶体等)时,晶片吸收振动面与晶轴垂直得光,而只允许振动面平行于晶轴得光通过。,大家学习辛苦了，还是要坚持,继续保持安静,自然光,平面偏振光,起偏器,检偏器,晶轴相互平行时，透射光强度最大；,晶轴相互垂直时，透射光强度为零；,晶轴夹角为,时，透射光强度与,cos,2,成正比。,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,.,也称线(完全)偏振光,简称偏振光。,它得振动面称为其偏振面。,振动方向保持不变,振幅发生周期性变化,E,之端点在空间得轨迹为一平面正弦曲线,投影到垂直于光传播方向得平面上为一直线段,3、平面偏振光(,Plane polarized light,),4、布儒斯特角,当入射光射到两种介质界面上时,反射光与折射光得偏振情况与入射光不同。,5、四分之一波片,使,o,光与,e,光得光程相差1/4波长得晶片。此时,相位差为,/2,透射得合成光就是长轴与晶轴重叠得,正椭圆偏振光,。,朝光源瞧,电场矢量方向按,顺时针,方向旋转得,称为,右圆偏振光,;,电场矢量方向按,逆时针,方向旋转得,称为,左圆偏振光,。,若入射平面偏振光得电矢量与晶体晶轴得夹角,45,则,Eo,=,Ee,合成光就是,圆偏振光,(,Circular polarized light),。,6、光得叠加,一束,自然光,可以瞧作就是两束相互垂直而没有相位关系得,平面偏振光,得加与。,旋转方向相反得,左、右圆偏光,透过一光学,活性物质后,透过得左、右圆偏光,振幅,相位,矢量与,相等,相同,平面偏振光,相等,不同,平面偏振光,方向改变,不等,相同,椭圆偏振光,不等,不同,椭圆偏振光,方向改变,振幅相等、振动方向相互垂直、相位相差1/4波长得,两平面偏振光合成圆偏振光,右旋,7、旋光色散(,Optical rotatory,dispersion,ORD),旋光性,:,光学活性分子对左、右圆偏光得,折射率,不同,透射光虽然仍然为平面偏振光,但其偏振面旋转了一定得角度。,旋光物质又称为光学活性物质,。,旋光度,:偏振面旋转得角度,旋光色散,:不同波长得偏振光得旋转角度不同。,旋光仪就是测量旋光性与波长得函数关系,从而得到一个旋光色散谱。,圆双折射,:某个物质对于左、右圆偏光得折射率分别为,n,L,与,n,R,若,n,L,=,n,R,则为光学各向同性物质;,若,n,L,n,R,则为光学各向异性物质;左、右圆偏光得相位改变,若,n,L,n,R,透射光得偏振面右旋。,若,n,L,n,R,透射光得偏振面左旋。,旋光现象就是圆双折射得一种特殊形式。,旋光物质使左、右圆偏振光得速度不同,即其色散大小得折射率不同,旋光现象得产生就是由于光学各向异性物质得折射率,n,L,n,R,得结果。,旋光,双折射,化合物在正常情况下,处于低能得基态,电子占据所有得成键轨道(,、n,轨道)。如果电子吸收了外界得能量,它就从基态跃迁到激发态能级。,如果所有得跃迁仅在基态得最低振动能级与第一激发态之间,吸收光谱将就是很狭窄而不连续得谱线。,由于分子得,价电子跃迁总伴随着振动与转动能级得跃迁,所以,紫外分光光度计测定物质得吸收光谱,虽然有一最大吸收峰值,但都就是具有一定波长宽度并相对平滑得曲线。,8、,光得吸收与圆二色性,(,circular dichroism,CD),光得吸收,若溶液中得溶质对某一波长得入射光有吸收,则透射光得光强度小于入射光。,圆二色性得存在将通过该物质传播得,左、右圆偏光,变成,椭圆偏振光,。并且只在发生吸收得波长处才能观察到。,理论计算得圆二色性与该椭圆偏振光得摩尔椭圆率得关系:,=100,/(,c,l)=3300(,L,-,R,),:,介质对圆偏振光得摩尔消光系数,c,:样品摩尔浓度,l,:样品厚度(,cm,),单位:/(,mol cm),或,cm,2,/dmol。,圆二色性得表示,吸收(率)差,=,L,-,R,A=A,L,A,R,椭圆度,摩尔,椭圆度,=2、303(,A,L,A,R,)/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,园二色双折射,9、,ORD,与,CD,光谱,同时产生,包括同样得分子结构信息:光学活性物质分子中得不对称生色团。,CD,反映光与分子间能量得交换,旋光性,则就是与分子中电子得运动有关。,可以由,Kronig-Krammers,转换方程相互转换。,CD,谱得极值处与吸收峰一致(科顿效应,cotton effect),因而分子中不同生色团对于谱得贡献比在,ORD,中更容易分辨。,ORD,就是渐近线,两端不回归,ORD,CD,吸收带附近,旋光度由负到正,正,Cotton,效应,正,CD,带,吸收带附近,旋光度由正到负,负,Cotton,效应,负,CD,带,CD,比,ORD,容易辨别重叠峰,CD,比,ORD,弱带易检测,CD,比,ORD,更容易拟合,CD,比,ORD,提供较多意义明确得信息,表明有三个生色团,其中两个有光学活性,CD,能够提供得信息:,primary structure,secondary structure,tertiary structure,Alpha-helix,beta-sheet,肽链骨架得构象变化,蛋白质得紫外吸收光谱主要有两个峰:,190-250,nm,:,肽键,280-290,nm,:,酪氨酸,tyr,色氨酸,trp,10、,J-810,圆二色光谱仪,S1-S2:,第一级单色器;,S2-S3:,第二级单色器;,M:,球面反光镜;,P:,晶体石英棱镜;,L:,透镜;,F:,滤光器;,由,CDM,交互形成得左、右旋圆偏光通过光学活性物质,透射光强度随时间得变化,当调制电压过其峰值(正与负)时,R,L,时,透过得,右旋圆偏光得光强对应于最大值,而左旋最小(实线);,R,L,时,透过得,左旋圆偏光得光强对应于最大值,而右旋最小(虚线)。,PMT,得输出信号由正比于,I,A,得直流分量与正比于,S,得交流分量所组成。,直流成分,I,A,=（,I,L,+,I,R,）/2,交流成分,S,相当于圆二色性,透射光强的变化频率与外加调制电压的频率相同。,圆二色光谱仪工作原理,用相同强度,相同频率得左、右旋圆偏光交替照射样品测得,透过样品后得强度,I,R,与,I,L,由于,I,R,与,I,L,十分接近,令,I,A,=(,I,L,+,I,R,)/2,S=,I,R,I,L,只放大相应于,S,得,PMT,输出电压,E,s(,交流信号),而不放大相应,I,A,得输出电压,E,A,。(,适当选取放大倍数,G,值,使,E,S,G,与,E,A,可比)。,Peltier Cell Holder,CD/Total Fluorescence,FMO,Stopped Flow,-20200,The peptide bond is inherently asymmetric and is always optically active,蛋白质得光学活性,1、氨基酸得圆二色性,可见光区,:各种氨基酸都没有光吸收。,紫外光区,:只有芳香族,色氨酸、酪氨酸与苯丙氨酸,有吸收。,氨基酸分子内得,紫外生色基团,:吲哚基、酚羟基、苯基、二硫键、咪唑基与羧基等。,2、肽得圆二色谱,活性肽构象与其生理功能得关系,肽得构象也就是蛋白质构象得一种模型。,肽得圆二色性为研究肽在溶液中得构象提供了重要信息。,Far-UV(250-170nm),Secondary Structure:,-helix,-sheet,-turn,random,Tertiary Structure Class:all-,+,/,all-I,all-II,Near-UV(320-250nm),:,aromatic side chain,Near-UV Near-infrared(300-1000nm),:,colored proteins,苯丙氨酸(,phenylalanine,Phe),:255、261与268,nm,附近;,酪氨酸(,tyrosine,Tyr),:277,nm,左右;,色氨酸(,tryptophan,Trp),:279、284与291,nm,附近;,二硫键得变化反映在整个近紫外区。,芳香氨基酸残基及二硫键,处于不对称微环境时,在,近紫外区,表现出,CD,信号,Far UV CD spectra of poly-L-Lys,1、100%,-,螺旋,2、100%,-,折叠,3、100%无规卷曲,-,band(nm),+,band(nm),-helix,222,208,192,-sheet,216,195,-turn,220-230(,weak),180-190(strong),205,polypro II helix,190,210-230,weak,Random coil,200,212,Main CD features of protein 2,ndary,structures,3、估算蛋白质,二级结构,含量,仅适合,含量较高得蛋白质！,将测得得,CD,曲线与标准曲线拟合。,x,a,+x,b,+x,c,=1、0,q,t,=x,a,q,a,+x,b,q,b,+x,c,q,c,改变,x,a,、,x,b,与,x,c,使,q,t,与样本曲线最佳拟合(最小二乘法,least squares minimization,),未知结构得蛋白得,CD,信号,myoglobin,q,t,=x,a,q,a,+x,b,q,b,+x,c,q,c,fits best with,x,a,=80%,x,b,=0%,x,c,=20%,agrees well with structure,78%helix,22%coil,For further details:,GCN4-p1,0,o,C:100%helix,75,o,C:0%helix,what about 50,o,C?,q,t,=x,o,q,o,+x,75,q,75,fits best with,x,o,=50%,x,75,=50%,Shows that at 50,o,C,1/2,of peptide,a,-helix dimer,1/2 of peptide,random coil monomer,Lau,Taneja and Hodges(1984)J、Biol、Chem、,259,:13253-13261,螺旋二聚体分解为单螺旋,三氟乙醇,(,trifluoroethanol,TFE,),诱导肽链形成螺旋,TFE,对卷曲螺旋,(coiled-coil),得影响,chymotrypsin,(all,b,),lysozyme,(,a,+,b,),triosephosphate isomerase(,a,/,b,),myoglobin,(all,a,),CD signal of a protein depends on its 2,ndary,structure,pH,变化引起得,肌球素变性前后,CD,谱得动力学变化,稀释前后得荧光与,CD,谱,从伸展到折叠,螺旋含量得变化得动力学检测,Kinetic Trace at 222nm(secondary structure region),Unfolding state(random),Refolding state(-Helix),Kinetic traces at 289nm(Aromatic side chain region),细胞色素,c,伸展与折叠状态得,CD,与荧光光谱,反映了芳环侧链(色氨酸残基)得变化,盐酸胍,中得细胞色素,c,(,伸展状态,),与,PBS,混合后得,CD,与荧光光谱,6,、磁圆二色谱(,Magnetic Circular Dichroism,MCD),磁性材料对左、右圆偏光得吸收不同而产生得二色性现象。,利用法拉第效应,在外加磁场得作用下,许多原来没有光学活性得物质也具有了光学活性,原来可测得,CD,谱得,在磁场中,CD,信号可增大几个数量级。,7000,Gauss,实验要点,Sample concentration,A good suggestion is to run in advance an absorption,UV-VIS spectra,、,CD spectroscopy calls for same requirements as UV-VIS:best S/N is obtained with absorbance level in the range,0、6 to 1、2,、Its usually difficult to get proper data when absorbance(,of sample+solvent,)is over 2 O、D、,样品杯:高度均匀得熔融石英,不会带来附加得圆二色性。,光程:0、01,cm-1cm,为了降低溶剂影响,一般提高样品浓度,用短光程。,cell pathlength,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 lot of ozone,harmful for the mirrors,below 195nm oxygen will absorb radiation,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 dilution or the use of shorter path cell are required、,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 single 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 patible to the natural bandwidth(NBW)of the bands 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 occasionally required、,2 nm in the far UV region,1 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,标准操作时谱带宽度选为1,nm;,高分辨率测量用较窄得狭缝宽度,此时,PMT,电压较高,信噪比差;,当样品得光密度很高、,CD,很小时,就要保持测试,CD,峰多需要得足够浓度,并用较宽得狭缝宽度。此时,由于样品,OD,值过高,可能存在有荧光或杂散光引起得某些假象。,Number of data point,data pitch,i、e、number of data points per nm,will not directly influence 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 the S/N will improve with the square root of the number of accumulations、,Averaging is very effective since it pensates short term random noise,but itll not pensate long term drifts(mainly of thermal origin)、So if long accumulations are used we remend a suitable long warm-up of the system 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、,Buffer Systems for CD Analyses,Acceptable:1、Potassium Phosphate with KF,K,2,SO,4,or(NH,4,),2,SO,4,as the salt、2、Hepes,2mM、3、Ammonium acetate,10mM、,Avoid:Tris;NaCl;Anything optical active,e、g、Glutamate,Typical Conditions for protein CD,Protein Concentration:0、2 mg/ml,Cell Path Length:1 mm,Volume,:,350,m,l,Need very little sample,:,0、1 mg,Buffer Concentration:5 mM or as low as possible while maintaining protein stability,一般将其始波长定在吸收带开始前50-100,nm,吸收带过后,CD,为0即可终止扫描。,对于未知样品,可先扫描较宽得波长范围以确定之。,举例,25bp RNA,片段(,15 79,),Thermal melting curves,for ssDNA(0.8 uM)in DEAB(3.3 mM)aqueous solution(a),DEAB(3.3 mM)/SDS(6.7 mM)mixed system(b),SDS aqueous solution(c)and water(d).The solid lines were obtained by fitting the data to a sigmoidal function.,CD spectra for ssDNA(2.7 uM),in 3.3 mM DEAB aqueous solution(a),in water(b),in 6.7mM SDS aqueous solution(c)and in DEAB(3.3 mM)/SDS(6.7 mM)mixed system(d)at 150C,