Principles of Fluorescence Spectroscopy XMUGXQ PFS0501 Principles of Fluorescence Spectroscopy Chemistry Department XMU 20040300

XMUGXQ PFS0501 Chapter Five Quenching of Fluorescence 20040300

Quenching of fluorescence XMUGXQ PFS0501 Quenching of fluorescence 5.1 Introduction 5.2 Stern-Volmer equation 5.3 Modified Stern-Volmer equation 5.4 factors influencing quenching 5.5 quenching mechanisms 5.6 Application 20040300

5.1 introduction Fluorescence Quenching XMUGXQ PFS0501 5.1 introduction Fluorescence Quenching Any processes decreasing the fluorescence intensity Excited-state reactions Molecular rearrangements Ground-state complex formation Collision Quencher Any species causing the decrease in the fluorescence General quencher Specific quencher 20040300

Dynamic quenching and static quenching XMUGXQ PFS0501 Dynamic quenching and static quenching Dynamic quenching Collision quenching relaxation (10-12 s) S0 S1 hvA hvF  knr Q kq[Q] 20040300

Dynamic quenching Diffusion control Effected by viscosity of solvent XMUGXQ PFS0501 Dynamic quenching Diffusion control Effected by viscosity of solvent In general, without permanent change in the fluorophore No changes in the absorption spectrum Decreasing the lifetime Intensify with temperature increasing Change into 20040300

Static quenching MQ* → MQ +  MQ* → MQ + hvF2 relaxation (10-12 s) S0 XMUGXQ PFS0501 MQ* → MQ +  MQ* → MQ + hvF2 relaxation (10-12 s) S0 S1 hvA2 hvF2  knr MQ relaxation (10-12 s) S0 S1 hvA1 hvF1  knr M 20040300

Static quenching Forming a new species XMUGXQ PFS0501 Static quenching Forming a new species Changing the absorption spectrum How about excitation spectrum? Change? Or not change? Depend on MQ emitting or not No change in the lifetime How about the effect of temperature? Depend on the thermodynamic properties of M and MQ 20040300

Quenchers oxygen Causing ISC halogens Aromatic and aliphatic amines XMUGXQ PFS0501 Quenchers oxygen Causing ISC halogens Aromatic and aliphatic amines Forming excited charge-transfer complexes Carboxyl groups Nitroxides Nitromethane and nitro compounds Heavy atoms more…… 20040300

5.2 Stern-Volmer eqution F0 and F XMUGXQ PFS0501 5.2 Stern-Volmer eqution F0 and F Fluorescence intensities in the absence and presence of quencher, respectively [Q] Concentration of quencher KSV Stern-Volmer quenching constant, given by kq0 KD dynamic quenching KS static quenching 20040300

Dynamic quenching For steady-state measurement XMUGXQ PFS0501 Dynamic quenching For steady-state measurement In the presence of quencher In the absence of quencher relaxation (10-12 s) S0 S1 hvA hvF  knr Q kq[Q] 20040300

For quantitative measurement XMUGXQ PFS0501 Dynamic quenching For quantitative measurement Stern-Volmer equation Because Stern-Volmer equation 20040300

Dynamic quenching 0 Lifetime in the absence of quencher kq XMUGXQ PFS0501 Dynamic quenching 0 Lifetime in the absence of quencher kq Bimolecular quenching constant Typically, 11010 mol-1 L s-1 kq = f(Q)k0 Quenching efficiency f(Q) The diffusion-controlled bimolecular rate constant k0 R Molecular radius (RF+RQ) collision radius D Diffusion coefficients N Avogadro’s number 20040300

Example Oxygen quenches The fluorescence of tryptophan XMUGXQ PFS0501 Example Oxygen quenches The fluorescence of tryptophan 25°C, O2, Dq = 2.5 10-5 cm2/s; tryptophan, DF = 0.66 10-5 cm2/s The collision radius R = ( RF + Rq ) = 5 Å k0 =1.2  1010 mol-1 L s-1 Thus Measured KD = 32.5 mol-1 L How to measure? Given  = 2.7 ns, Thus kq =1.2  1010 mol-1 L s-1 f(Q) = kq/k0 = 1 20040300

The effect of lifetime on quenching XMUGXQ PFS0501 The effect of lifetime on quenching Using oxygen as the quencher According to Stern-Volmer equation KD =kq0 Typically, kq = 11010 mol-1 L s-1 Typical fluorescence lifetime 0 = 10-8 s Thus, KD = 102 mol-1 L When Typical phosphorescence lifetime 0 = 10-3 s Thus, KD = 107 mol-1 L When What do these calculations suggest? 20040300

Static quenching May or may not fluoresce According to F = Kc Thus XMUGXQ PFS0501 Static quenching May or may not fluoresce According to F = Kc Thus 20040300

Stern-Volmer constant XMUGXQ PFS0501 Stern-Volmer constant KD = kq0 Related with lifetime, controlled by diffusion Increasing with temperature increasing T↑ 1.0 [Q] F0/F KS Formation constant Endothermal reaction Exothermal reaction T↑ 1.0 [Q] F0/F T↑ 1.0 [Q] F0/F 20040300

Combined dynamic and static quenching XMUGXQ PFS0501 Combined dynamic and static quenching Violation of Stern-Volmer equation 1.0 [Q] F0/F Suggest the combination of dynamic and static quenching 20040300

Correction to Stern-Volmer equation XMUGXQ PFS0501 Correction to Stern-Volmer equation the fraction of fluorescence, due to static quenching F0 F FQ the fraction of fluorescence, due to dynamic quenching hv F* FQ F* Q* 20040300

Correction to Stern-Volmer equation XMUGXQ PFS0501 Correction to Stern-Volmer equation F* FQ F F0 hv Q* fs fS.fD 20040300

Correction to Stern-Volmer equation XMUGXQ PFS0501 Correction to Stern-Volmer equation Kapp apparent Stern-Volmer constant 20040300

Correction to Stern-Volmer equation XMUGXQ PFS0501 Correction to Stern-Volmer equation [Q] KDKS KD+KS 20040300

Modified Stern-Volmer equation in interpreting “sphere of action” XMUGXQ PFS0501 Modified Stern-Volmer equation in interpreting “sphere of action” Where  is the volume of the sphere. The radius of the sphere is slightly larger than the sum of the radii of the fluorophore and the quencher. There exists a high probability that quenching will occur before these molecules diffuse apart. 20040300

Example 1 0/ x F0/F Oxygen quenching of tryptophan XMUGXQ PFS0501 20040300

XMUGXQ PFS0501 Example 2 F0/F F0/FQ ■ 0 /  Acrylamide(丙烯酰胺）quenching of N-acetyl-L-tryptophan-amide(N-乙酰-L-色氨酸酰胺） 20040300

XMUGXQ PFS0501 Example 3 From JRL. P.245 Acrylamide quenching of dihydroequilenin (DHE，二氢马萘雌甾酮) in buffer containing 10% sucrose（蔗糖） at 11°C 20040300

XMUGXQ PFS0501 Example 4 10-methylacridinium chloride quenching of guanosine-5’-monophosphate （鸟嘌呤核苷-5‘- 单磷酸） 20040300

XMUGXQ PFS0501 Example 4 20040300

5.4 factors influencing quenching XMUGXQ PFS0501 5.4 factors influencing quenching Steric effect Example 1 20040300

The ethidium bromide-DNA complex XMUGXQ PFS0501 The ethidium bromide-DNA complex Oxygen quenching of ethdium bromide fluorescence Why smaller than 11010? 20040300

XMUGXQ PFS0501 Example 2 20040300

XMUGXQ PFS0501 Charge effect I O2 F 20040300

Example 1 Copolymer 1 1% tryptophan + 99% glutamic acid XMUGXQ PFS0501 Example 1 Copolymer 1 1% tryptophan + 99% glutamic acid Copolymer 2 3% tryptophan + 97% lysine At neutral pH glutamic acid nagatively charged Lysine positive charged What happens to the fluorescence of tryptophan in the presence of oxygen and iodide, respectively? 20040300

XMUGXQ PFS0501 Example 1 20040300

XMUGXQ PFS0501 Example 2 氯化十二烷基三甲铵 十二烷基硫酸钠 20040300

Micro-environment Fluorescence quenching of trypsinogen 胰蛋白酶原荧光猝灭 XMUGXQ PFS0501 Micro-environment Fluorescence quenching of trypsinogen 胰蛋白酶原荧光猝灭 20040300

Different in micro-environment XMUGXQ PFS0501 Different in micro-environment Downward-curving Stern-Volmer plot Partial quenching 1.0 [Q] F0/F Fin Fex I In the absence of quencher F0 = Fin,0 + Fex,0 20040300

In the presence of quencher XMUGXQ PFS0501 Modified Stern-Volmer equation in interpreting the difference in micro-environment In the presence of quencher Total fluorescence 20040300

Deriving modified equation XMUGXQ PFS0501 Deriving modified equation 20040300

Deriving modified equation XMUGXQ PFS0501 Deriving modified equation Let Then Modified Stern-Volmer equation 20040300

Deriving modified equation XMUGXQ PFS0501 Deriving modified equation 1 /(KDfex) 1 / fex 1/[Q] 20040300

Iodide quenching of tryptophan fluorescence in lysozyme(溶菌酶） XMUGXQ PFS0501 Example Native protein Iodide quenching of tryptophan fluorescence in lysozyme(溶菌酶） Denatured protein Native protein 1/fex = 1.5, fex = 0.66 Denatured protein 1/fex = 1.0, fex = 1.0 20040300

XMUGXQ PFS0501 example 20040300

Localization of membrane-bound fluorophores XMUGXQ PFS0501 Localization of membrane-bound fluorophores 20040300

Localization of membrane-bound fluorophores XMUGXQ PFS0501 Localization of membrane-bound fluorophores 20040300

5.5 quenching mechanisms 5.5.1 Due to energy transfer XMUGXQ PFS0501 5.5 quenching mechanisms 5.5.1 Due to energy transfer Nonradiative energy transfer, due to dipole-dipole interaction of donor and acceptor D donor A acceptor Rate of energy transfer depends Overlap of spectra Relative orientation Distance between A and D 20040300

Principle of energy Rate of energy transfer D quantum yield of D XMUGXQ PFS0501 Principle of energy Rate of energy transfer D quantum yield of D D lifetime of D r distance between A and D n refrcative index N Avogadro’s number k orientation factor J overlap integral 20040300

The extinction coefficient of A at  XMUGXQ PFS0501 Overlap integral The corrected fluorescence intensity of D in -d, the total intensity normalized to unity The extinction coefficient of A at  The unit is (mol / L)-1 cm3 20040300

XMUGXQ PFS0501 Orientation factor randomize by rotational diffusion prior to energy transfer k = 2/3 20040300

Förster distance R0 The distance between A and D when XMUGXQ PFS0501 Förster distance R0 The distance between A and D when relaxation (10-12 s) S0 S1 hvA hvF  knr A(S0) A(S1) R0 > r, energy transfer decay dominate R0 < r, usual radiative and nonradiative decay dominate 20040300

Förster distance (in cm) Rate of energy transfer XMUGXQ PFS0501 Förster distance (in cm) Rate of energy transfer Efficieney of energy transfer 20040300

Example 100% transfer -萘 丹磺酰 D A 聚脯氨酸 n =1-12, r = 12- 46Å XMUGXQ PFS0501 Example 100% transfer -萘 丹磺酰 D A 聚脯氨酸 n =1-12, r = 12- 46Å Excited D, measure F of A no D F290= 2.3I0bcAA(A+ED) 0% transfer A’= A+ED 20040300

n =1, 100% transfer, the ratio of absorbance = the ratio of emission XMUGXQ PFS0501 Example n =1, 100% transfer, the ratio of absorbance = the ratio of emission 20040300

XMUGXQ PFS0501 example x = 5.9±0.3 20040300

Sensitized fluorescence and phosphorescence XMUGXQ PFS0501 Sensitized fluorescence and phosphorescence D(S1) D(S0) D(T1) A(T1) A(S1) A(S0) Sensitized fluorescence Sensitized phosphorescence For D, quenched For A, sensitized 20040300

Example Self-association of ATPase（腺苷三磷酸酶）molecules in lipid vesicles XMUGXQ PFS0501 Example Self-association of ATPase（腺苷三磷酸酶）molecules in lipid vesicles Mg2+-Ca2+-ATPase labeled individually with IAEDANS and IAF Acceptor Donor 20040300

Intra-molecule energy transfer XMUGXQ PFS0501 Intra-molecule energy transfer 20040300

Intra-molecule energy transfer XMUGXQ PFS0501 Intra-molecule energy transfer Trp HSA Tyr:Trp=18:1 R0 = 14Å Comparable to the diameter of most proteins 20040300

5.5.2 duo to photochemical reaction XMUGXQ PFS0501 5.5.2 duo to photochemical reaction 荧光猝灭所涉及的光化学反应类型 光化学反应荧光猝灭是指沿着激发态超平面发生的反应，导致激发态聚集数减少而引起的荧光猝灭。 单分子光化学反应 绝热光化学反应 产生另一种形态的发光分子，其发射波长不同于原来的发光体 TICT 非绝热光化学反应 产生另一种具有稳定基态的物质，不伴随光子的发射 光分解 20040300

激发态分子与其他分子发生反应，生成了稳定的或不稳定的新物质 XMUGXQ PFS0501 双分子光化学反应 激发态分子与其他分子发生反应，生成了稳定的或不稳定的新物质 光氧化还原 生成稳定的基态物质 激基二聚体 不具有稳定的基态 20040300

Excited state hypersurface R* XMUGXQ PFS0501 光化学反应的机制 Excited state hypersurface R* R P* P S0 S1 hvF hv’F TICT Dual fluorescence R* R P* P S0 S1 hvF hv’F Photoreaction 20040300

光化学反应的机制 P* R* S1 hvF P S0 P’ R XMUGXQ PFS0501 光化学反应的机制 R* R P* P S0 S1 hvF P’ Maurice R. Eftink, Fluorescence quenching: theory and application in “Topic in fluorescence spectroscopy” V2 principles, ed. By J.R.Lakowicz, p 53-120. Herbert C. Cheung, Resonance energy transfer, in “Topic in fluorescence spectroscopy” V2 principles, ed. By J.R.Lakowicz, p 128-171. 20040300

5.6 Application Q F Molecular beacons A D Q F Target DNA F Q XMUGXQ PFS0501 5.6 Application Q F Molecular beacons A D Q F Target DNA F Q ssDNA binding protein Denaturing reagent 20040300

XMUGXQ PFS0501 Molecular beacons Hairpin probe show no fluorescence, do not need separation Key type, almost no background fluorescence Specific probe Reference Xiaohong Fang, Tianwei Heffery, John Perlette, Weihong Tan (University of Florida, USA) Kemin Wang (Hunan University, P.R. CHINA), Anal. Chem. 2000, Dec.1 747A-753A 20040300