New Insights in the Interpretation of Tryptophan Fluorescence

Origin of tryptophan fluorescence is still up to these days a quiz which is not completely solved. Fluorescence emission properties of tryptophan within proteins are in general considered as the result of fluorophore interaction within its environment. For example, a low fluorescence quantum yield is supposed to be the consequence of an important fluorophore–environment interaction. However, are we sure that the fluorophore has been excited upon light absorption? What if fluorophore excitation did not occur as the result of internal conformation specific to the fluorophore environment? Are we sure that all absorbed energy is used for the excitation process? Fluorescence lifetimes of Trp residues are considered to originate from rotamers or conformers resulting from the rotation of the indole ring within the peptide bonds. However, how can we explain the fact that in most of the proteins, the two lifetimes 0.5 and 3 ns, attributed to the conformers, are also observed for free tryptophan in solution? The present work, performed on free tryptophan and tyrosine in solution and on different proteins, shows that absorption and excitation spectra overlap but their intensities at the different excitation wavelengths are not necessarily equal. Also, we found that fluorescence emission intensities recorded at different excitation wavelengths depend on the intensities at these excitation wavelengths and not on the optical densities. Thus, excitation is not equal to absorption. In our interpretation of the data, we consider that absorbed photons are not necessary used only for the excitation, part of them are used to reorganize fluorophore molecules in a new state (excited structure) and another part is used for the excitation process. A new parameter that characterizes the ratio of the number of emitted photons over the real number of photons used to excite the fluorophore can be defined. We call this parameter, the emission to excitation ratio. Since our results were observed for fluorophores free in solution and present within proteins, structural reorganization does not depend on the protein backbone. Thus, fluorescence lifetimes (0.5 and 3 ns) observed for tryptophan molecules result from the new structures obtained in the excited state. Our theory allows opening a new way in the understanding of the origin of protein fluorescence and fluorescence of aromatic amino acids.     

Fluorescence of albumin solutions containing Pb2+ and Na+ ions

Solutions of bovine serum albumin with metal ions Pb²⁺ and Na⁺ were studied by fluorescence analysis in the visible and UV ranges in relation to such parameters of the medium as the concentration of macromolecules, the pH, and the ionic strength of the solution. The formation of nanoparticles, protein clusters, in aqueous solutions of albumin containing ions of such a heavy metal as lead was revealed by fluorescence polarization analysis. This investigation is of practical value for the solution of ecological and medical problems.     

Inclusion Complexation of 2-Amino-7-bromofluorene by β-Cyclodextrin: Spectral Characteristics and the Effect of pH

Spectral characteristics of 2-amino-7-bromofluorene (2ABF) have been studied in aqueous beta-cyclodextrin (beta-CDx) solution. Enhancement in the fluorescence intensity of the neutral from of 2ABF was observed due to the formation of 1:1 complex with beta-CDx. The formation of this complex was confirmed by time-resolved fluorescence spectroscopy. The ground state pKa value for the monocation-neutral equilibrium of 2ABF in beta-CDx shows no change with that in aqueous solution, but the excited state pKa value changes. Based on its photophysical and photoprototropic characteristics in beta-CDx, the structure of the 1:1 inclusion complex is proposed.     

Origin of Fluorescence Lifetimes in Human Serum Albumin. Studies on Native and Denatured Protein

Human serum albumin consists of a single polypeptide of 585 amino acid residues with 1 Trp residue. In the present work, we measured fluorescence lifetimes of the protein in both native and denatured states. The results indicate that Trp emission occurs with three lifetimes in both states. Lifetimes values and contribution to the global emission decay differ between the two states. Data are interpreted as the results of an emission occurring from three substructures of the tryptophan formed in the excited state. Two of these substructures are already present for the tryptophan free in solution. The third lifetime is the result of the interaction between the tryptophan residue and surrounding microenvironment. The populations of these substructures characterized by the pre-exponential parameters of the fluorescence lifetimes are dependent on the fluorophore microenvironment and on the global protein structure.     

Singlet Excited State Pyridinic Deprotonation of the N<Subscript>9</Subscript>-methylbetacarboline Cations in Aqueous Sodium Hydroxide Solutions

The singlet excited state pyridinic deprotonation of the 9-methyl-9H-pyrido[3,4-b]indole, MBC, cations has been studied in aqueous NaOH solutions by absorption, steady state and time resolved fluorescence measurements. This excited state reaction proceeds through a stepwise mechanism involving different ground and excited state hydrogen bonded MBC-(water)_n complexes. Thus, in aqueous NaOH solutions of MBC above pH 8, two ground state hydrogen bonded MBC-water adducts, namely PC and PTC, coexist in equilibrium. Upon excitation, the PC behaves as an independent fluorophore, whereas the PTC reacts with water molecules during its excited state lifetime to give the intermediate CL*. This exciplex is the precursor of the excited state cation, C*. In almost all the pH range, C* is practically the only existing species in the singlet excited state of MBC. In concentrated NaOH solutions beyond the pH range, C* deprotonates giving CL* and PTC* species.     

Singlet-triplet conversion of He(1s2s) by metal surfaces

The singlet-triplet conversion of the excited He(1s2s) metastable atom, approaching the metal surface with relatively low work function, is delt with on the basis of an extended Newns-Anderson model allowing the atom to have spin singlet and triplet states. After a canonical transformation, we show that a virtual process through the ionic states, of both the positive and the negative ions, makes the conversion possible. The numerical results show that in a certain range of parameters, the conversion possibly takes place in a region rather far from the surface.     

Interaction of the Iron(II) Cage Complexes With Proteins: Protein Fluorescence Quenching Study

Interaction of the iron(II) mono- and bis-clathrochelates with bovine serum albumin (BSA), β-lactoglobulin, lysozyme and insulin was studied by the steady-state and time-resolved fluorescent spectroscopies. These cage complexes do not make significant impact on fluorescent properties of β-lactoglobulin, lysozyme and insulin. At the same time, the monoclathrochelates strongly quench a fluorescence intensity of BSA and substantially decrease its excited state lifetime due to their binding to this protein. This occurs due to the excitation energy transfer from a tryptophan residue to a cage molecule or/and to the change of the tryptophan nearest environment caused by either clathrochelate binding or an alteration of the BSA conformation. The effect of the iron(II) bis-clathrochelate on BSA fluorescence is much weaker as compared to its monomacrobicyclic analogs as a result of an increase in its size.     

Fluorescent Studies of Salicylaldehyde and Other Related Carbonyl Compounds for the Selective and Sensitive Detection of Zinc(II) Ions in Aqueous Solution

Salicylaldehyde was found to have a high selectivity for zinc ions with simultaneous enhancement of fluorescence in aqueous buffer solution at optimum pH 8.5. The stoichiometry of the complex was determined to be 1:1 with a K(a) value of 3.4 × 10(4) M(-1) at 298 K. The fluorescence of the complex is not affected by common anions and Zn(2+) binds preferentially to salicylaldehyde in the presence of alkali, alkaline earth and heavy metal cations (Hg(2+), Cd(2+), Cr(3+) and Ni(2+)). This property is not observed with related phenolic compounds bearing a carbonyl group such as esters, amides, carboxylic acids and ketones.     

Specific features of the radiative relaxation of excited states of polymethine dyes of different ionicity in films of photoconductive and nonphotoconductive polymers

The spectral and luminescent properties of film composites based on photoconductive poly-N-epoxipropylcarbazole and nonphotoconductive polyvinylbutyral with admixtures of cationic and anionic polymethine dyes, as well as the effect of an external magnetic field on these properties, are studied. It is found that the magnetic field affects the intensity and kinetics of the delayed fluorescence and recombination luminescence of the cationic dye in photoconductive films. This is explained by specific features of photogeneration of charge pairs, namely, by the participation of the singlet and triplet excited states of dye molecules in this process, as well as by the singlet-triplet conversion in dye molecules and photogenerated charge pairs.     

The Effect of Heteroatom Position on the Spectral-Luminescent Properties of Monochloroanilines

The effect of position of a heteroatom in the phenyl radical of aniline on the spectral, geometric, and photophysical characteristics of free molecules of monochloroaniles is studied by electron microscopy, fluorescence, and quantum chemistry. The position of energy levels of singlet and triplet states of monochloroanilines are found. It is shown that substitution by chlorine in aniline results in an increase in the singlet-triplet conversion and emergence of a new path of the internal conversion in the triplet-state system.     

Sub-Structures Formed in the Excited State are Responsible for Tryptophan Residues Fluorescence in β-Lactoglobulin

Origin of tryptophan residues fluorescence in β-lactoglobulin is analyzed. Fluorescence lifetimes and spectra of β-lactoglobulin solution are measured at pH going from 2 to 12 and in 6 M guanidine. Tryptophan residues emit with three lifetimes at all conditions. Two lifetimes (0.4–0.5 ns and 2–4 ns) are in the same range of those measured for tryptophan free in solution. Lifetimes in the denatured states are lower than those measured in the native state. Pre-exponential values are modified with the protein structure. Data are identical to those already obtained for other proteins. Fluorescence lifetimes characterize internal states of the tryptophan residues (Tryptophan sub-structures) independently of the tryptophan environments, the third lifetime results from the interaction that is occurring between the Trp residues and its environment. Pre-exponential values characterize substructures populations. In conclusion, tryptophan mission occurs from substates generated in the excited state. This is in good agreement with the theory we described in recent works.     

Effect of heavy-metal ions on dynamic characteristics of collagen molecules in solutions

Effect of heavy (Pb²⁺, Cs⁺) and light (Na⁺) metal ions on the molecular-dynamic characteristics of type-I collagen in aqueous solution was studied using the method of dynamic light scattering. It was found that the dependence of the translational diffusion coefficient D _t from pH solutions has a nonlinear form with a pronounced extremum close to the isoelectric point of the protein (pI 6.0). For pure aqueous solution of protein there is a maximum of D _t in isoelectric point. For collagen solutions with the addition of heavy-metal salts the minimum of D _t was observed near the isoelectric point. This fenomenon is connected with the formation of protein nanoclusters in solution. With concentration of heavy metal ions increasing translational diffusion coefficient Dt decreases, which shows on increasing of aggregation effect. The addition of sodium ions in aqueous solution of collagen containing heavy metal ions sharp decreasing of the translational diffusion of molecules is observed. That can be connected with the rise of scattering particles masses.     

一种新型酰腙配体及其Sn配合物的荧光特性研究

用稳态荧光和时间分辨荧光研究了一种新型酰腙配体2-羟基甲醛-5-氯水杨酰腙(H3L)及Sn配合物(n-Bu2)Sn(HL)晶体、溶液和旋涂膜的光谱特性与分子结构的关系。实验结果表明,对H3L而言,与其稀溶液相比,晶体及旋涂膜的荧光强度依次增强,荧光峰位都有所红移,荧光寿命有所延长,其单分子跃迁能为240.2kJ.mol-1;对[(n-Bu2)Sn(HL)]而言,其晶体稳态荧光强度比在溶液中强且荧光峰位红移,旋涂膜产生了荧光猝灭,单分子跃迁能为230.4kJ.mol-1;与H3L相比较,(n-Bu2)Sn(HL)晶体的荧光强度要强接近4倍,荧光寿命变长。这些现象的物理机制是分子的共轭体系越大、分子的刚性越大其荧光强度越强,荧光寿命越长。     

载铅高硅质粉尘对大肠杆菌壁膜的损伤作用机制

大气颗粒物与微生物共存时的健康效应受到了越来越多的关注。以大气颗粒物中石英与重金属铅为研究对象,粉尘浓度为1.6 g·L-1,制备载带不同浓度铅高硅质粉尘,以人体常见菌-大肠杆菌为受试对象,探讨载铅高硅质粉尘对大肠杆菌细胞壁膜损伤的机理。采用噻唑蓝(MTT)测定微生物的细胞活力后发现,与对照组相比,大肠杆菌与载铅石英粉尘作用2 h后,细胞活力表现出单一铅离子组大于载铅石英粉尘组,并且呈现重金属剂量效应。PI的摄入量测试表明,高浓度载铅粉尘组中摄入量分别高出对照组36%与46%,单一重金属组摄入量也有较高的增长,而激光共聚焦显微镜观察图中,染毒组均出现不同程度的红色荧光,可以发现载铅石英粉尘作用后的细菌细胞壁膜通透性明显升高,利用探针标记,采取荧光分光光度法测定荧光强度显示胞内和溶液中活性氧逐渐增多,载铅粉尘组(Q+Pb-2,Q+Pb-3)胞内ROS分别较对照组高出2倍和2.5倍,参照前人研究,发现溶液中ROS变化主要与重金属离子在其表面结合态数量决定。综合分析,活性氧在诱使细胞膜损伤过程中起到决定性作用。红外表征中,细胞膜表面磷酸二脂基团、蛋白质甲基振动及酰胺带等基团与载铅石英粉尘作用后均发生明显峰位偏移,均与载铅粉尘发生较强相互作用,一定程度上影响细胞壁膜完整性。综上,重金属与粉尘共同作用使得细胞膜通透性发生变化,细胞壁膜的完整性改变,影响细胞活力,最终导致细菌死亡,活性氧及重金属等的作用导致细胞膜的损伤可能是载重金属高硅质粉尘的一种毒性作用机制。     

CdTe量子点与罗丹明B水溶液体系下的双光子激发荧光共振能量转移

采用时间分辨荧光光谱技术研究了在双光子激发下不同尺寸的量子点与罗丹明B 之间的荧光共振能量转移. 研究结果表明, 在800 nm的双光子激发条件下, 体系间能量转移效率随着供体吸收光谱与受体荧光光谱的光谱重叠程度增加而增加; 理论分析表明, 供体和受体间的Förster半径增加是导致其双光子能量转移效率增大的物理原因. 同时, 研究了罗丹明B浓度对荧光共振能量转移效率的影响. 研究结果表明, 量子点的荧光寿命随着罗丹明B浓度的增加而减小; 量子点与罗丹明B之间的荧光共振能量转移效率随着罗丹明B浓度的增加而增加; 当罗丹明B浓度为3.0×10^-5 mol·L^-1时, 双光子荧光共振能量转移效率为40.1%.     

Mechanism for quenching of the luminescence of 9,10-anthraquinone vapor by oxygen

The fluorescence of 9,10-anthraquinone, 1-aminoanthraquinone, 1,4-diaminoanthraquinone, and 1,5-diaminoanthraquinone is not quenched by oxygen because the singlet-triplet energy difference in these compounds is less than the energy needed for excitation of the triplet state of oxygen to the singlet state. Luminescence of 9,10-anthraquinone is quenched because it is mainly phosphorescence, for which the singlet-triplet difference is sufficient for quenching by a mechanism involving singlet oxygen formation. The weak fluorescence of 9,10-anthraquinone is not quenched. The resistance of the fluorescence of 9,10-anthraquinone vapor to quenching by oxygen and the quenching of its phosphorescence explain the different effects of oxygen on the luminescence of α-substituted and β-substituted anthraquinones known from the literature, and indicate that their singlet excited state cannot convert triplet oxygen to singlet oxygen. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 1, pp. 79–4, January–February, 2008.     

Red-Edge Excitation Study of Nonexponential Fluorescence Decay of Indole in Solution and in a Protein

Proteins are known to be heterogeneous systems with a hierarchy of internal motions. However, those properties are often ignored when the complex fluorescence decay of tryptophan residues is compared to model studies with indole derivatives in solution. Here two simple models are presented, which illustrate different aspects of protein organization: (1) Trp zwitterion in buffer exemplifies ground-state heterogeneity and (2) indole in water/glycerol mixture exemplifies excited-state reconfiguration of solvate. Both systems are known to produce nonexponential fluorescence decay, attributed to the existence of multiple species (rotamers) or to the effects of slow dipolar relaxation, for (1) and (2), respectively. In the latter case a substantial dependence of decay on the excitation wavelength is expected. Indeed such dependence is observed for indole in water/glycerol mixture but not for Trp zwitterion in buffer. Therefore, excitational dependence can be used as a criterion to distinguish effects of multiple conformations in the ground state from effects of excited state reactions on tryptophan decays in proteins. The example of the bee venom peptide melittin indicates that both phenomena are important for interpretation of heterogeneity of decay, and therefore, caution should be exercised when assigning individual decay components to conformational subspecies in proteins.     

Origin of Tryptophan Fluorescence Lifetimes. Part 2: Fluorescence Lifetimes Origin of Tryptophan in Proteins

Fluorescence intensity decays of L-tryptophan in proteins dissolved in pH 7 buffer, in ethanol and in 6 M guanidine pH 7.8 and in lyophilized proteins were measured. In all protein conditions, three lifetimes were obtained along the emission spectrum (310–410 nm). The two shortest lifetimes are in the same range of those obtained for L-Trp in water or in ethanol. Thus, these two lifetimes originate from specific two sub-structures existing in the excited state and are inherent to the tryptophan structure independently of the surrounding environment (amino acids residues, solvent, etc.) In proteins, the third lifetime originates from the interactions that are occurring between tryptophan residues and neighboring amino acids. Populations of these lifetimes are independent of the excitation wavelength and thus originate from pre-defined sub structures existing in the excited state and put into evidence after tryptophan excitation. Fluorescence decay studies of different tripeptides having a tryptophan residue in second position show that the best analysis is obtained with two fluorescence lifetimes. Consequently, this result seems to exclude the possibility that peptide bond induces the third fluorescence lifetimes. Indole dissolved in water and/or in ethanol emits with two fluorescence lifetimes that are completely different from those observed for L-Trp. Absence of the third lifetime in ethanol demonstrates that indole behaves differently when compared to tryptophan. Thus, it seems not adequate to attribute fluorescence lifetime or fluorescence properties of tryptophan to indole ring and to compare tryptophan fluorescence properties in proteins to molecules having close structures such as NATA which fluoresces with one lifetime.     

（7—甲氧基—4—亚甲基）香豆素基—单氮杂18—冠—6的合成及其？…

合成、鉴定了题示光敏离子载体（ＭＭＣ－ＭＡＣ（Ｏ５）。其中不同ｐＨ值水溶液中吸收和荧光光谱的变化,计算得该剂基态和激发态分子的酸性常数（ｐＫａ＝８．８４,ｐＫａ＾＊＝５．１１）。基于在不同溶剂中荧光光谱的变化,由Ｓｏｌｖａｃｈｒｏｍｉｃ法,借助Ａｌｃｈｅｍｙ２０００量化计算软件,估算得该分子的基态和激发态偶极矩,分别为３．１１Ｄ和８．１３Ｄ。同时发现,ＭＭＣ－ＭＡＣ（Ｏ５）水溶液除氧后其荧光强度奇