色谱的基本原理介绍_43页.ppt

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,*,色谱的基本原理介绍,Circular,Dichroism,CD,圆,二色,光,谱,1.,什么是圆,二色谱,?,旋光色散谱,2.,圆二色谱在生物,医学,研究中的应用,产生,特征,structure of membrane protein,cm,-1,Secondary structure,1624,4,sheet,1631,3,sheet,1637,3,sheet,1645,4,Random coil,1653,4,helix,1663,4,turn,1671,3,turn,1675,5,sheet,16832,turn,1689,2,turn,1694,2,turn,Crystal Structure of Mitochondrial Respiratory,Membrane Protein Complex II,.,Cell,2005,121,这是二十五年以来，我国科学家完全,立足于“本土”，第一次在该杂志上发,表完整的、系统性的、原创性研究成果,hybrid orbital,COOH,CH,3,H,NH,2,COOH,CH,3,NH,2,H,COOH,NH,2,CH,3,H,Chiral,L,alanine,COOH,CH,3,NH,2,H,D,alanine,X,Y,线,偏振光,X,Y,X,Y,频率相同,方向相反,振幅相同,相位相同,线,偏振光,参,照,面,频率相同,方向相反,振幅相同、,相位,不同,线,偏振光,2,1,参,照,面,y,y,a,x,x,R,L,=tg,-1,(|E,l,E,r,|)/(E,l,+,E,r,),椭圆偏振光,频率相同,方向相反,振幅,不同,、,相位,不同,参,照,面,合成光特性,合成效果,比较,频率,振幅,相位,是否,旋转,是否,椭圆,相同,相同,相同,否,否,相同,不,同,是,否,不,同,不,同,是,是,左、右,旋,圆偏振光,的,合成,椭圆偏振光,Optically Active Sample,Chiral,Right and Left hand circularly polarized,light,Preferential absorption of left hand polarized,?,振动方程,E,x,=,E,0 x,cos,(,t+,0,),E,x,=E,0 x,cos,(,t-L/v,)+,0,波动方程,E,x,=,E,ox,cos,t,-(2,L,/,)n,+,0,Or,CD(circular,dichroism,),与振幅,A,=,lgI,0,/I=(1/2,303)lnI,0,/I=,C,L,Beer-,Lanmbert,Law,=tg,-1,(|E,l,E,r,|)/(E,l,+,E,r,),=,2.303,A/4 (,弧度,),A,=A,l,-,Ar,=,C L,L,Optical active object,E,x,=,E,0 x,cos,t-(,2,L,/,)n,+,0,?,C:,摩尔浓度,L,:,光程,(,d,m),l,:,deg mol,-1,cm,-1,or,deg cm,2,dmol,-1,摩尔椭圆率,l,(the molar,ellipticity,),l,MRw,=,M,Rw,y,l,/100,y,l,(the specific,ellipticity,),平均残基椭圆率,l,MRw,(the mean residue,ellipticity,),l,=,M,w,y,l,/100l,=,/,C,L,旋光色散,与位相,旋光色散,ORD(optical,rotatory,dispersion),?,n,l,n,r,Chiral,Optical active object,=(,/,),n,D,n,=,n,l,-,n,r,a=,f(,l,),弧度,=(2,/,),n,L,a=,LC,圆双折射(,Circular,birefringence):,E,x,=,E,0 x,cos,t,-(,2,L,/,)n,+,0,平均残基旋光度,m,l,(the mean residue,rotation),m,l,=,a,l,M,Rw,/100,C,:,g/mL,L,:dm,M,w,:molecular weight,M,Rw,:the mean residue M,w,l,(the specific rotation),摩尔比旋光度,j,l,(the molar rotation),j,l,=,a,l,M,w,/100,y,l,=,l,/,C,w,L,Plane,(linearly),polarized,light,基本原理,(,principle,),Right and Left hand circularly polarized,light,Optically Active Sample,Preferential absorption of left hand polarized,Chiral,E,x,=,E,0 x,cos,t,-(,2,l,/,)n,+,0,Light,n,l,n,r,y,y,a,x,x,R,L,l,=,f(,l,),a,l,=,f(,l,),ORD,CD,Kronig-Kramers,transition,relationship between CD and ORD,and Cotton effect,Cotton effect,l,0,e,l,0,l,0,+,-,+,-,Positive cotton,effect,Negative cotton,effect,Instrument,www-,structure.llnl.gov,/,CD of amino acid and peptides,Amino acid,吲哚基，酚羟基，苯基，二硫键，咪唑基，羧基。,酪氨酸，色氨酸，苯丙氨酸，胱氨酸，脯氨酸,在近紫外区（,240-300nm),对,CD,谱有贡献,羧基在,200-210nm,显示正峰。,在波长大于,300nm,的区域，包括可见区域，对,CD,谱的贡献主要来自像血红素一类含有,金属离子,的生色团，这一波段,CD,谱常用于研究金属离子的氧化态、配位体以及链,-,链相互作用。,含有金属离子的生色团,Secondary,structure,of macromolecule,a,-helix,a,-helix:,19,0,nm(+),208nm,222nm(-),q,x E,-3,0,200,210,220,230,240,nm,b,-sheet:,195nm(+),antiparallel,:red shift,parallel:blue shift,215-,21,7nm,(-),b,-sheet,Unordered structure:,(-)below 200nm,(+)218nm weak band,unordered,b,-turn:,180-190nm(-),200-205nm(+),225nm(+)band,weak red shift,Far UV,（,190-240nm),Content of secondary structure,、,、,分别为,螺旋,、,折叠和,非周期结构在波长,处的椭圆率值,f,、,f,、,f,分别为,螺旋,、,折叠和,非周期结构在,蛋白质中,所占重量百分比。,图：三种,LRR,（,lysine-rich repeat),家族肽的,CD,谱,利用,CD,谱研究蛋白质的构象变化,TFE,Unfolding/folding,216nm,222nm,Conformational varies,190,200,210,220,230,240,250,260,-1.5,-1.0,-0.5,0.0,0.5,1.0,1.5,2.0,a5 in 5%SDS/PBS,a5 in 50%TFE/PBS,a5 in pH7,a5 in 50%TFE/H,2,O,a5 in H,2,O,X1E-4 deg.cm,2,/,dmol,-1,wavelength(nm),216nm,图,A,Bob1,（,1-65,）和,Oct/DNA,复合物的,CD,谱,蛋白质,-,蛋白质、蛋白质,-,核酸结合的研究,克山病区粮饲养的豚鼠心肌线粒体膜的研究,模型动物心肌线粒体膜的,CD,谱,（,a,：病区粮喂养组；,b,：病区粮补加蔬菜喂养组；,c,：正常对照组）,模型动物心肌线粒体细胞色素,C,氧化酶的,CD,谱,（,a,：病区粮喂养组，,15,周前死亡；,b,：病区粮喂养组，存活的；,c正常对照组）,烟草花叶病毒侵染膜蛋白,磷脂膜诱导的蜂毒素二级结构的变化,蜂毒素的,CD,谱,1,：蜂毒素插入单层膜的,CD,谱；,2,：蜂毒素吸附在单层膜上时的,CD,谱；,3,：在,Tris,-HCL,缓冲液中的蜂毒素,CD,谱,1,-,螺旋,84%,，,-,折叠,1%,无规卷曲,15%,2,-,螺旋,27,%,，,-,折叠,20,%,无规卷曲,5,3,%,Near UV spectra:,to investigate the,tertiary structure,Protein Concentration,:,0.5 mg/ml,Cell Path Length,:,0.5 mm,Stabilizers(Metal ions,etc.):,minimum,Buffer Concentration:,5,mM,or as low as possible,while maintaining protein stability,Typical,Initial Concentrations:,One may find that the protein concentration needs to be adjusted to produce the best data.Changing this has a profound effect on the data,so small increments or decrements are called for.If that does not produce reasonably good data,a change in buffer composition may be necessary.It would also be a good idea to check the sample for unforeseen aggregation via Dynamic Light Scattering(DNA repair enzymes are an especially good example of this behavior).If buffer poses a problem,cells with shorter path(0.1 mm)and a correspondingly increased protein concentration and longer scan time can help.,When CD is applied to proteins what is the main,chromophore,(absorbing group)that is looked at?What wavelength range does it absorb in?,Does the CD spectrum of a protein give most information about the,(i),primary structure,(ii),secondary structure or,(iii),tertiary structure?,Draw the CD spectrum of an,-helix.Remember to label the axes and mark important points with their values.,Why is nitrogen gas used in CD instruments?,Does CD give information about where the helices are in a protein?,Membrane proteins are difficult to study by CD.True or false?Why?,思 考 问 题,参考文献,生物物理学,赵南明，周海梦，高教出版社施普林格出版社2000,330,园二色性和旋光色散在分子生物学中的应用,鲁子贤，崔涛，施庆洛，科学出版社1987,Circular,Dichroism,and Linear,Dichroism,Alison Rodger and,Bengt,Nordn,Oxford University Press 1997,分析仪器手册,朱良漪，孙亦梁，陈耕燕，化学工业出版社,1997,p 247,Circular,Dichroism,and Optical Rotary Dispersion of Proteins and Polypeptides,A.J.Alder,N.J.Greenfield and,G.D.Fasman,Meth.,Enzymology,27,675(1973).,Computed Circular,Dichroism,Spectra for the Evaluation of Protein Conformation,N.Greenfield and,G.D.Fasman,Biochemistry,8(10),4108(1996),C.R.Cantor and,P.R.Schimmel,Biophysical Chemistry,Vol.2,Chapter 8(1980),Effect of,Trifluoroethanol,on Protein Secondary Structure,F.D.Soennichsen,J.E.VanEyk,R.S.Hodges and B.D.Sykes,Biochemistry,31(37),8791(1992),Protein Secondary Structure and Circular,Dichroism,:A Practical Guide,W.C.Johnson,PROTEINS,7,205-214(1990),