【正文】 ironment of binding sites. FLUORESCENCE APPLlED TO PROTElNS 1. Analytical detection ANALYTlCAL APPLlCATIONS Fluorescence intensity F is useful to biochemists in observing the presence of a macromolecule. For instance, biopolymers emerging from a high pressure liquid chromatograph (HPLC) might be monitored with a fluorescence detector. We see in Figure the results for the isolation of the protein melittin from honey bee venom on an HPLC. Melittin has a tryptophan residue that can be excited at 280 nm to fluoresce over a range of wavelengths that include 340 nm. Two proteins are eluted from the HPLC that have 340 nm fluorescence, one at min and one at min. The fraction at min has melittin activity (hemolysis of red blood cells). Monitoring the elution with normal UV absorbance at 214 nm (Figure ) reveals a number of other fractions that contain biological molecules, but these do not have to be considered because they do not fluoresce. By using fluorescence for analysis, identification of the proper fraction is simplified. Figure The fluorescence (a) and electronic absorption (b) for a melittin preparation emerging from a high pressure liquid chromatograph. The melittin elutes at min. FLUORESCENCE APPLlED TO PROTElNS 2. Changes in quantum yield Quantum yield is a useful quantity ? Quantum yield is a useful quantity ? Changes in the quantum yield are often diagnostic of changes in the molecular environment of a chromophore ? For example, the quantum yield of a fluorophore will often increase when the molecule is taken up from solution and bound to a macromolecule such as DNA or a protein. ? Measurement of the quantum yield provides a simple way to measure binding . 利用蛋白質(zhì)的天然熒光檢測(cè)蛋白質(zhì)的結(jié)構(gòu)變化 氨基?；冈谑榛蛩徜囎饔孟聝?nèi)源熒光發(fā)射譜隨作用時(shí)間的變化 ,隨作用時(shí)間的增加 ， 熒光強(qiáng)度逐漸減小 。 表明隨作用時(shí)間的增加 ， 該蛋白質(zhì)逐漸變性 ， 發(fā)生去折疊 ， 蛋白質(zhì)內(nèi)部的熒光生色團(tuán)逐漸暴露在極性水環(huán)境中 。 Changes in the fluorescence emission spectra of aminoacylase during denaturation in mM LDS. Repeated scan of the fluorescence emission spectra with an excitation wavelength of 295 nm after mixing the enzyme with LDS solution. The final concentration of the enzyme was ?M. Reaction times of the enzyme denaturaion for curves from top to bottom were 0, 1,3,5,8,10,15,20,25,30,40 and 50 min, respectively. FLUORESCENCE APPLlED TO PROTElNS 3. The effects of energy transfer 共振能量轉(zhuǎn)移 參考文獻(xiàn) 1. PAUL R. SELVIN, Fluorescence Resonance Energy Transfer, in ―METHODS IN ENZYMOLOGY.‖ VOL 246 ,p300334 2,3,4 見(jiàn)所提供的閱讀文獻(xiàn) Energy transfer Under favorable circumstances excitation energy can be transferred from one fluorophore to another: ? Transition dipole interaction between the two fluorophores ? An appreciable overlap of the fluorescence spectrum of the donor with the absorption spectrum of the acceptor ? The requirement of dipoledipole interaction between the fluorophores leads to a strong dependence of energy transfer on the distance between the participating groups 如果兩種不同的熒光生色團(tuán)離得較近，且其中一種生色團(tuán)的熒光發(fā)射譜與另一種生色團(tuán)的激發(fā)譜有相當(dāng)程度的重疊。則當(dāng)?shù)谝环N熒光團(tuán)被激發(fā)時(shí)，另一種熒光團(tuán)卻因第一種生色團(tuán)激發(fā)能的轉(zhuǎn)移而被激發(fā)，這種現(xiàn)象稱為共振能量轉(zhuǎn)移。 共振能量轉(zhuǎn)移 Dependence of efficiency of energy transfer on the sixth power of the separation r Figure The efficiency of energy transfer changes rapidly with the distance between donor and acceptor. Efficiency of energy transfer The efficiency of transfer is given by efficiency = Here R0 is the distance for efficiency of transfer, and is characteristic of the donoracceptor pair and the medium between them. 60 )/(11Rr? R0稱為臨界距離 ， 定義為能量轉(zhuǎn)移效率為 50%時(shí)兩個(gè)生色團(tuán)之間的距離 ， 對(duì)于每個(gè)供體一受體時(shí) ， R0是常數(shù) 。 R0可以根據(jù)受體的吸收譜和供體的發(fā)射譜 、 介質(zhì)的折射系數(shù) 、 供體和受體躍遷電偶極矩的朝向因子 、 供體在沒(méi)有受體存在時(shí)的量子產(chǎn)率等參數(shù)估算出來(lái) 。 根據(jù)轉(zhuǎn)移效率和 R0， 即可求出兩個(gè)生色團(tuán)之間的距離 。這種測(cè)定生色團(tuán)距離的方法 ， 常被人稱為光譜尺 。 R0 FOR SOME DONORACCEPTORS (nm) ? R0 is ordinarily found to be of the order of 2 nm ? Fluorescence transfer serves as a useful yardstick for the distances between groups in macromolecules such as proteins phe phe phe tyr tyr tyr tyr trp trp trp Tyr he m e trp he me Tyr DNS trp DNS trp ANS DNS DNS DNS he me Tyr NA DH trp NADH Flu Rho ? Energy transfer is a mon phenomenon in proteins ? It can be used for investigating their structure ? Tyrosine and tryptophan groups in proteins often satisfy both requirements for transfer ? Specific amino acids can be labeled with a fluorophore such as dimethyl aminonapthalene5sulfonate (DNS) ? Energy transfer used to measure the distance between the dye and other aromatic groups in the protein AMP (cAMP) fluorosensor ? cAMPdependent protein kinase consists of two regulatory and two catalytic subunits. cAMP causes the dissociation of these four subunits and the activation of the enzyme. ? The dye fluoroscein was attached to the catalytic subunit and the dye rhodamine attached to the regulatory subunit. The inactive tetramer exhibits energy transfer of the excited fluoroscein to the nearby rhodamine. Addition of cAMP dissociates the tetramer. ? The dissociation eliminates energy transfer, which in turn increases the fluorescence of fluoroscein at 520 nm and decreases the fluorescence of rhodamine at 580 nm. ? The fluorescence at 520 nm relative to 580 nm is a sensitive measure of cAMP concentration. Figure Dissociation of the four subunits in cAMPdependent protein kinase increases the fluorescence of fluoroscein at 520 nm at the expense of the fluorescence of rhodamine at 580 nm in the cAMP fluorosensor. No added cAMP (?? ), ?M cAMP (....), ?M cAMP (? ? ?), and 53 ?M cAMP (? ? ? ? ?). Visualizing cAMP with fluorescence Fluorescent probes have wide application. As a particularly beautiful example, DeBerna