OXYGEN QUENCHING OF CHLOROPHYLL FLUORESCENCE IN CHLOROPLASTS

Abstract— Three phases of chlorophyll a fluorescence quenching by O₂ are observed in green plants. The effects of various inhibitors on photosynthetic partial processes in chloroplasts were investigated in attempts to (1) localize the O₂-quenching sites and (2) assess possible physiological significance of O₂-quenching. Our results localize the most sensitive (and presumably functionally important) phase to a site between plastoquinone and the photosystem I acceptor, chlorophyll (P₇₀₀), possibly plastocyanin. It is suggested that PC may transfer electrons to oxygen in addition to P₇₀₀.     

TEMPERATURE AND VISCOSITY DEPENDENCE OF FLUORESCENCE QUENCHING BY OXYGEN IN MODEL SYSTEMS

Abstract— The bimolecular rate constant, k _q, for the quenching of the fluorescence of pyrenebutyric acid and naphthaleneacetic acid by molecular oxygen has been studied as a function of temperature and viscosity, η. Fluorescence lifetime measurements were used to monitor the degree of quenching. Glycerol-water and sucrose-water mixtures were used to increase the bulk viscosity. Plots of log k _q vs log η show deviations from Stokes-Einstein behavior, but the data are consistent with the patterns that have been observed for other diffusion limited reactions. This work provides background information, regarding the viscosity dependence of oxygen quenching reactions, which is essential for the correct interpretation of oxygen quenching/viscosity dependence studies with more complex systems (i.e. quenching of tryptophan residues in proteins).     

RAPID EQUILIBRATION OF OXYGEN IN FLUORESCENCE QUENCHING REACTIONS

A simple means is described for the rapid equilibration of oxygen in fluorescence quenching reactions. This involves bubbling the gas through experimental solutions. With this procedure, data for Stern-Volmer plots can be obtained in 1–2 h, without bubbling it takes approximately ten times as long to collect such data.     

QUENCHING OF THE FLUORESCENCE OF 8-METHOXYPSORALEN BY PROTONS

Abstract— The quenching of 8-methoxypsoralen (8-MOP) fluorescence by protons was observed to occur at the diffusion controlled rates in aqueous solutions at room temperature. Enhanced basicity of 8-MOP in the excited state compared to the ground state is expected on theoretical grounds. The fluorescence yield. which we determined as 6.3 × 10^--⁴ at pH 1 is surprisingly low and indicative of extremely fast radiationless decay pathways. The fluorescence lifetime of 8-MOP in neutral aqueous solution is on the order of 1–2 ns.     

THE EFFECT OF ALIPHATIC ALCOHOLS ON FLUORESCENCE QUENCHING AND FLUORESCENCE POLARIZATION OF LUMINESCENCE PROBES IN HEXADECYLTRIMETHYLAMMONIUM BROMIDE MICELLES

Abstract— The fluorescence quenching of the indole chromophore by NO⁻₂ and the fluorescence depolarization of several luminescence probes in aqueous solutions containing hexadecyltrimethylammonium bromide (HDTBr) were measured as a function of added C₂–C₄ aliphatic alcohol concentration. The fluorescence decay profiles of pyrene in the micellar solutions were also measured to estimate the aggregation number of the micelles. The addition of n -butyl alcohol significantly reduces the fluorescence quenching rate and the aggregation number and increases the extent of fluorescence depolarization in HDTBr micellar systems. The addition of ethyl alcohol shows a similar but smaller effect.     

QUENCHING OF PYRENE FLUORESCENCE BY TRYPTOPHAN IN MICELLAR SOLUTIONS

Deactivation of excited pyrene incorporated to cationic (cetyltrimethylammonium chloride), anionic (sodium dodecyl sulfate) and neutral (Triton X-100) micelles by tryptophan has been investigated over a wide pH range. Data obtained allow an estimation of the tryptophan association to the micelles, of the tryptophan apparent p K in the micellar solutions, and of the dynamics of tryptophan incorporation to the micellar pseudophase. In particular, the data obtained at pH 7 allow an estimation of the effect of the micellar charge upon the binding capacity of the tryptophan zwitterion.     

QUENCHING OF CHLOROPHYLL FLUORESCENCE BY NITROBENZENE

Abstract—Nitrobenzene quenching of chlorophyll fluorescence in ethanol has been investigated. Steady state relative quantum yields have been measured and fluorescence decay rates were determined using both nanosecond photon counting and picosecond pulses from a mode-locked Nd³⁺ glass laser.
The fluorescence decay is described by
1( t )= I ₀ exp (- t/τ−At^1/2 )
the form predicted for decay governed by the kinetics of the continuum model of diffusion controlled reactions. From the parameters of the fluorescence decay, the encounter distance is 5–7 A° the mutual diffusion coefficient is 0.62 × 10^--⁵ cm2s^-1± 12%.
Some of the fluorescence quenching is also attributed to static quenching by a nitrobenzene-chlorophyll, ground-state complex. The equilibrium constant for formation of this ground-state complex was determined to be 4.1 M ^-1. The combined dynamic and static quenching model allows calculation of quantum yields of fluorescence in good agreement with the experimentally determined quantum yields.     

FLUORESCENCE QUENCHING OF INDOLE BY DIMETHYLFORMAMIDE

Abstract— The effect of dimethylformamide on the lifetime (T) and quantum yield ( F ) of indole has been determined. Negative Stern-Volmer deviations prevail for T and F , but F ⁰/ F T⁰/T at all DMF concentrations. The deviation between F ⁰/ F and T⁰/T is ascribed to changes in F ⁰ and T⁰ induced by the change in solvent composition at the higher quencher concentrations.     

烷基苄基铵对芘的荧光猝灭行为研究

广义的荧光猝灭系指所有能使荧光强度降低的物理和化学过程,但通常涉及的主要有动态猝灭和静态猝灭,二者均遵从Stern-Volmer方程,但由于猝灭作用本质不同,它们在荧光寿命变化、温度效应及吸收光谱等方面表现出差异可资区别.本文通过稳态荧光强度变化和荧光衰减速率的比较研究了具有不同烷基链长的十四烷基苄基二甲基氯化铵(Zeph)和苄基三甲基溴化铵(TMBA)对芘的荧光猝灭,并基于电导实验结果以及猝灭剂全反式构象从理论上计算了Zeph和TMBA的猝灭速率常数,讨论了荧光猝灭的性质和长链分子的构型。     

FLUORESCENCE QUENCHING OF PYRENE DERIVATIVES BY NITROXIDES IN MICROHETEROGENEOUS SYSTEMS

Abstract Fluorescence quenching of pyrene derivatives by 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO), 4-oxo-2,2,6,6-tetramethylpiperidinyl-1-oxy and 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy has been measured in homogeneous solvents and microheterogeneous systems: cetyltrimethylammonium chloride micelles, large unilamellar vesicles of dioctadecyldimethylammonium chloride and dipalmitoylphosphatidylcholine (DPPC) and rat liver microsomes. The extent of intraaggregate quenching is mostly determined by the quencher incorporation to the micro-phases. In particular, it is observed that quenching by TEMPO in vesicles is considerably faster when the bilayer is in the liquid crystalline state. This significant increase in quenching rate with the melting of the bilayer is not observed for the other TEMPO derivatives, indicating that the effect of the lipid organization upon the solubility is related to the hydrophobicity of the solute. The data obtained in rat liver microsomes at 37°C show a pattern very similar to that observed in DPPC vesicles in the liquid crystalline state.     

胭脂红酸的荧光发射寿命及荧光猝灭

以蒸馏水中的糖原悬浮液为参照物，测定了胭脂红酸的荧光发射寿命，τ＝０．０９３±０．０１０ｎｓ．研究了ＯＨ－和Ｉ－对胭脂红酸荧光的猝灭，后者遵守Ｓｔｅｒｎ－Ｖｏｌｍｅｒ方程，其猝灭常数ＫＱ＝１．２７±０．０９Ｌ·ｍｏｌ￣（－１），前者则不是遵守此方程的简单荧光猝灭，这可能是由于胭脂红酸中酚基的存在降低了最低激发态能量的原因。     

DEPTH PROFILES FOR PERMEATION OF WATER and OXYGEN IN VESICLES and TUBULE PHASES BY FLUORESCENCE QUENCHING STUDIES*

Static and dynamic fluorescence data from three structurally related probe molecules [(1-pyrenyl)acetic acid, 4-(l-pyrenyl)butanoic acid, and 12-(l-pyrenyl)dodecanoic acid] were employed to determine the influence of phase structure on oxygen and water permeability in small unilamellar vesicles and microtubules of l,2-bis(tricosa-10,12-diynoyl)-in-glycero-3-phosphocholine (DC₂₃PC). Since the pyrenyl units of the three reporter molecules probe environments at different depths in DC₂₃PC bilayers, the aggregated information provides a depth profile of both small-molecule permeability and order. The results indicate that the probe sites are somewhat more available to water molecules immediately below the chain melting transition temperature than above it: singlet lifetimes of the pyrenyl groups undergo a clear increase as the temperature is raised in the region of the transition. This observation is consistent with melted chains being able to wrap themselves more efficiently around pyrenyl units (and thereby protect them better from water molecules) than stiff chains. The gross picture that emerges is one in which pyrenyl groups experience water and oxygen quenching via opposing concentration gradients. The concentration of oxygen is greatest near the bilayer midplane and decreases toward the aqueous interface; the concentration of water increases from the midplane to the interface.     

QUENCHING OF SINGLET OXYGEN BY SENSITIZER IN DYE-SENSITIZED PHOTOOXYGENATION*

Abstract— A general method for the determination of the extent of the singlet oxygen (¹O₂) quenching by sensitizer in the dye-sensitized photooxygenation of olefins is used in the case of rose bengal (RB) in methanol and oil-soluble chlorophyll (M) in benzene with 2-methyl-2-pentene and tetramethylethylene as acceptors. Unlike RB, M which contains only a low percentage of pure chlorophyll (Chi), quenches ¹O₂. It is shown that this very cheap mixture can be used for kinetic studies and that the chemical quenching of ¹O₂ by M is very weak with respect to the physical quenching. The upper limit for the rate constants of physical and chemical quenching of ¹O₂ by Chi is estimated.     

DISTANCE-DEPENDENT FLUORESCENCE QUENCHING OF TRYPTOPHAN BY ACRYLAMIDE

Abstract We used GHz frequency-domain fluorometry to investigate the time-dependent intensity decays of N -acetyl -L-trytophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol at 20°C. The intensity decays of NATA became increasingly heterogeneous in the presence of acrylamide. The NATA intensity decays were not consistent with the Collins-Kimball radiation boundary condition (RBC) model for quenching. The steady-state Stern-Volmer plots show significant upward curvature. At low temperature in vitrified propylene glycol (-60%), where translational diffusion cannot occur during the lifetime of the excited state, quenching of NATA by acrylamide was observed. The Smoluchowski and RBC quenching models do not predict any quenching in the absence of translational diffusion. Hence, these frequency-domain and steady-state data indicate a through-space quenching interaction between NATA and acrylamide. The rate for quenching of NAT A by acrylamide appears to depend exponentially on the fluorophore-quencher separation distance. Comparison of the time-resolved and steady-state data provides a sensitive method to determine the distance dependence of the fluorophore-quencher interaction. The distance-dependent rate of quenching also explains the upward curvature of the Stern-Volmer plot, which is often observed for quenching by acrylamide. These results suggest that the distance-dependent quenching rates need to be considered in the interpretation of quenching data of proteins by acrylamide.     

QUENCHING OF METHYLCYCLOHEXANE FLUORESCENCE BY METHANOL*

We measured the fluorescence emission spectrum and intensity decays of methylcyclohexane (MCH) when excited by simultaneous absorption of two photons at 298 nm. The steady-state intensities and lifetimes were both decreased by methanol, which was found to be an efficient quencher of MCH fluorescence. Methanol quenching of MCH is clearly dynamic, but the exact mechanism of quenching is unclear. Dynamic quenching of MCH was also observed by water and n-propanol. These results suggest that alkane fluorescence from biopolymers, if observable, will only occur from regions of the macromolecules that are not exposed to water.     

酶催化-荧光猝灭法测定药物中的姜黄素

酪氨酸在辣根过氧化物酶催化下被H2O2氧化为强荧光物质S,姜黄素对其荧光产生猝灭作用,据此建立了测定姜黄素的新方法。姜黄素浓度c在0．10～16．0μg/mL范围内与F0／F（F和F0分别为姜黄素存在和不存在时产物S的荧光强度）呈线性关系,线性回归方程c=5．4552F0／F-5．4860,线性相关系数r=0．9996,检出限为0．02μg/mL,测定结果的相对标准偏差为0．79％（n=10）,加标回收率为94．7％～102％。研究了pH值、各物质的量、反应时间、干扰离子等对测定的影响。该法可用于药物中姜黄素含量的测定。     

QUENCHING OF SINGLET OXYGEN BY HUMAN PLASMA

Direct measurements of the decay of singlet oxygen phosphorescence at 1270 nm were made in human plasma diluted with various amounts of deuterium oxide. The Stern-Volmer plot of the singlet oxygen lifetimes was linear up to 15% plasma concentration (vol/vol). Extrapolation of these measurements to 100% plasma content gave a singlet oxygen lifetime of 1.04 +/- 0.03 microseconds in human plasma. Biological molecules accounted for 77% of the total singlet oxygen quenching while water accounted for 23% of the quenching. The contributions of various types of biological molecules to the total singlet oxygen quenching were calculated from their plasma concentrations and their quenching constants. Plasma proteins quenched most of the singlet oxygen. Uric acid also quenched a significant amount of singlet oxygen (12%). Tocopherols, carotenoids, ascorbic acid and bilirubin made only small contributions to the total singlet oxygen quenching (less than or equal to 4%).     

氨基乙酸邻苯甲酸-溴化十六烷基吡啶荧光猝灭体系检测痕量铜

研究了荧光猝灭法测定痕量铜的方法。在溴化十六烷基吡啶存在下,氨基乙酸邻苯甲酸可以和铜形成稳定的配合物,此配合物可以使配体荧光定量猝灭。反应的适宜酸度为pH5.0～6.0,最大发射波长λem为427nm,表观摩尔吸光系数ε=5.56×104L·mol-1·cm-1,Cu(Ⅱ)含量在0.2～12.8μg/10ml范围内符合比耳定律,相关系数为0.9999。应用于铝合金样品中痕量铜的测定,结果满意。     

FLUORESCENCE QUENCHING OF INDOLES BY N,N-DIMETHYLFORMAMIDE

Abstract. The quenching of the excited state of indoles by small polar molecules is believed to occur via the formation of excited state complexes. The rate of excited state complex formation of indoles with dimethylformamide is shown to be a function of the group appended to the indole ring at the 3- or 5-position. Substituents which can strengthen the charge transfer character of the exciplex are observed to enhance the rate of fluorescence quenching. In contrast to other amides, dimethylformamide appears to be a moderately good quencher of indole fluorescence, and may be a useful probe for proteins.