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.     

DEACTIVATION OF PYRENE BY INDOLE IN MICELLAR SOLUTIONS

Abstract— The deactivation rate of excited pyrene by indole strongly depends on the polarity of the media. In micellar systems (Triton X-100, cetyltrimcthylammonium chloride (CTAC) and sodium dodecylsulfate (SDS) the deactivation efficiency is enhanced due to the high local concentration of indole in the micellar pseudophase. Quantitative interpretation of the data in CTAC and SDS micelles requires to take into account indole exchange between the micelles and the aqueous phase. In SDS micelles, where due to their smaller size the exchange process is more relevant, the exit and entrance rates are (3.0 ± 0.6) x 10⁶ and (1.2 ± 0.3) x 10¹⁰ M ⁻¹s⁻¹ respectively. Intramicellar bimolecular quenching constants are (1.1 ± 0.2) x 10⁸ M⁻¹ s⁻¹ (1.4 ± 0.2) x 10⁸ M ⁻¹ s⁻¹ and (1.5 ± 0.2) x 10⁸ M ⁻¹ s⁻¹ in Triton X-100, SDS and CTAC respectively. These rates are similar to those measured in ethanol rich ethanol-water homogeneous solutions. This is in agreement with the average polarity sensed by both pyrene and indole in the micellar pseudophases.     

STUDIES OF SODIUM TAUROCHOLATE MICELLES USING FLUORESCENT PROBE MOLECULES

Abstract Micelles formed by sodium taurocholate (NaTC) and mixed micelles formed by NaTC with detergents (SDS, reduced Triton X-100 and CTAC) were studied with fluorescent probes. Pyrene was used as an indicator of the polarity of the micellar binding site by comparison of the fluorescence spectra and vibronic band intensity ratios of pyrene in the different systems. Perylene was used as a fluorescence polarization probe to study the rigidity of the NaTC and mixed micelles. The fluorescence lifetime of perylene in the different systems was also measured. Results of the studies were compared with measurements of the probes in cyclohexane, ethanol and aqueous beta-cyclodextrin. Perylene was found to be more rigidly bound in the NaTC micelles than in the detergent micelles. Insertion of small amounts of reduced Triton X-100 into the NaTC micelles appears to increase the rigidity. The binding sites of NaTC and CTAC have similar polarities, and are more polar than those of SDS and reduced Triton X-100. Insertion of any of the detergents into the NaTC micelle decreases the polarity of the binding site, possibly by reducing the penetration of water into the micelle.     

FLUORESCENCE CHARACTERISTICS OF INDOLES IN NON-POLAR SOLVENTS: LIFETIMES, QUANTUM YIELDS AND POLARIZATION SPECTRA

Abstract— Fluorescence lifetimes, quantum yields and polarization spectra were measured for indole, 3-methylindole and 2,3-dimethylindole in non-polar solvents. The results indicate simultaneous emission from thermally equilibrated ¹L_a and ¹L_b levels, with ¹L_a→¹ A dominating the 2,3-dimethylindole emission, and ¹L_b→¹ A dominating the indole emission. These results are consistent with previous assignments of the 0-0 transitions in absorption for these compounds. Radiative rates are: ¹L_a→¹ A , 2·0 × 10⁸ S^-1 and ¹L_b→¹ A . 0·62 → 10⁸ S^-1. In addition, the temperature dependence of the excitation and emission spectra are presented, which show that aggregation occurs with these indoles in hydrocarbons below approximately - 110°C. Possible applications to tryptophyl emission in the hydrophobic interiors of proteins are briefly discussed.     

COMPETITION BETWEEN THE SINGLET OXYGEN AND ELECTRON TRANSFER MECHANISMS IN THE PORPHYRIN-SENSITIZED PHOTOOXIDATION OF l-TRYPTOPHAN AND TRYPTAMINE IN AQUEOUS MICELLAR DISPERSIONS

Abstract— Kinetic studies of the hematoporphyrin–sensitized photooxidation of l -tryptophan and tryptamine at pH 10 in either homogeneous aqueous solutions or in aqueous dispersions of Triton X–100 and cetyltrimethylammonium bromide micelles indicate that the indole substrates are attacked via a mixed type I (electron transfer from triplet dye)/type II (¹O₂-involving) mechanism. Both reactive intermediates, generated by micelle-solubilized hematoporphyrin, can diffuse to attack substrate molecules located in either the bulk aqueous phase or a different micelle. In particular, incorporation of the substrate into a micelle has only minor effects on its reactivity toward¹O₂, although the ¹O₂—indole interaction appears to be more efficient in cationic micelles owing to a favourable orientation of the target with respect to the attacking species. On the other hand, the electron transfer from triplet porphyrin to a micellized substrate is virtually non-operative when the latter is located in an anionic micelle, whereas in neutral or cationic micelles, the efficiency of the process is again controlled by the substrate orientation. Studies of tryptamine photooxidation sensitized by meso-tetra-(4-sulfonato-phenyl) porphine in the presence of sodium dodecylsulphate micelles lend further support to the abovementioned conclusions.     

RESONANCE RAMAN SPECTRA OF THE Pr-FORM OF PHYTOCHROME

Abstract— Resonance Raman spectra of the P_r-form of oat phytochrome have been obtained at 77 K. Interference from phytochrome fluorescence is avoided by employing far-red 752 nm excitation. Vibrational assignments are suggested for the tetrapyrrole chromophore in phytochrome by comparison with previously published model compound spectra and by examining the characteristic shifts induced by deuteration of the pyrrole nitrogens. The lack of carbonyl intensity, the frequencies of the 1626 and 1644 cm^-1 C=C stretching modes, and the presence of an intense mode at 1326 cm^-1 are all consistent with a protonated structure for the tetrapyrrole chromophore in P_r. This suggests that the -50 nm red-shift of the protein-bound chromophore absorption compared to the chromophore in vitro is caused by protonation of the pyrrole nitrogen.     

RESOLUTION OF OVERLAPPING BANDS IN THE NEAR-UV ABSORPTION SPECTRUM OF INDOLE DERIVATIVES

Abstract— The second derivative spectra of tryptophan in water and in ethylene glycol at 22°C have been integrated in order to obtain the corresponding primitive functions. The integration was carried out by making use of Tchebychev polynomials. The results show that the integrated primitive functions do not correspond to the original absorption spectra of tryptophan in various solvents, but they reflect only the contributions of the ¹L_b bands of the indolic chromophore. The identification of the electronic component, which generates the second derivative spectrum, was based on the solvent insensitivity of the derivative peaks. The comparison between the absorption spectra reported in this paper and those calculated for the ¹L_b←¹A electronic transition of indole confirmed the assumption that the derivation process eliminates the broad, although more intense, contributions coming from the ¹L_a←¹A electronic transition.     

SPECTROSCOPIC STUDIES OF COUMARIN IN MICELLES

Steady-state and time-resolved emission spectroscopic techniques have been employed to characterize the coumarin species and identify which species is solubilized in the hydrocarbon core micelles of triton X-100 (neutral), hexadecyltrimethyl ammonium bromide (cationic) and dodecyl lithium sulfate (anionic) solutions under physiological conditions at 77 K. The emission and absorption spectra for the following species of coumarin—monomer, hydrogenbonded complex, molecular aggregation and strong hydrophobic aggregates—were recorded in methylcyclohexane (MCH), ethanol, buffers and aqueous solutions. The fluorescence and phosphorescence emissions of monomer in MCH at 77 K are assigned as resulting from ¹(*)₁ and ³(*)₁ states, respectively, originated from the ethylenic bond and carbonyl of the pyrone ring. Molecular orbital calculations using the Hydrogenic Atoms in Molecule, version 3, method were carried out to help interpretation of the spectroscopic results. The photophysical properties from each species are used to probe which species penetrates in the hydrophobic region of micelles. It was found that a fifth species of coumarin assigned as the "action species" is solubilized into the interior of micelles. These observations could lend some insight into the mechanism of transporting coumarins across the membrane.     

SOLVENT EFFECTS ON THE ELECTRONIC ABSORPTION AND FLUORESCENCE SPECTRA OF INDOLE-4-CARBOXYLIC ACID: PROTOTROPIC EQUILIBRIA IN AQUEOUS SOLUTIONS

Abstract— The absorption and fluorescence spectra of indole-4-carboxylic acid in various solvents have indicated that the -COOH group is more planar with respect to the indole ring in the first excited singlet state (S₁) than in the ground (S₀) state. Relatively large Stokes' shifts indicate that polarisability and dipole moment of the molecule are increased predominantly upon excitation. Prototropic reactions in the S₀ and S₁ states are the same. The -COO^- and -COOH+₂ groups are not coplanar in the S₀, but coplanar in the S₁ state. pH-dependent fluorescence spectra have revealed that both protonation and deprotonation of the -COOH group increase the basicity of the molecule upon excitation.     

Ionic quenching of naphthalene fluorescence in sodium dodecyl sulfate micelles

Micellar effects on luminescense of organic compounds or probes are well established, and here we show that quenching is highly favored in aqueous sodium dodecyl sulfate (SDS) micelles, which concentrate a naphthalene probe and cations of lanthanides, transition metals, and noble metals. Interactions have been studied by steady state and time-resolved fluorescence in examining the fluorescence suppression of naphthalene by metal ions in anionic SDS micelles. The quenching is collisional and correlated with the unit charge and the reduction potential of the metal ion. The rate constants, calculated in terms of local metal ion concentrations, are close to the diffusion control limit in the interior of SDS micelles, where the microscopic viscosity decreases the transfer rate, following the Stokes-Einstein relation.     

TYPE I AND TYPE II MECHANISMS IN THE PHOTOOXIDATION OF L-TRYPTOPHAN AND TRYPTAMINE SENSITIZED BY HEMATOPORPHYRIN IN THE PRESENCE AND ABSENCE OF SODIUM DODECYL SULPHATE MICELLES

Abstract— Oxygenated aqueous solutions (pH 10) of L-tryptophan or tryptamine containing hematopor-phyrin and/or specific quenchers of the possible reactive intermediates were irradiated with visible light in the presence and in the absence of dispersions of sodium dodecyl sulphate micelles. The rate of loss of the indole derivatives was followed over several min. In the absence of dispersed micelles, indole derivatives appeared to be photooxidized largely by a type I mechanism involving electron transfer from triplet hematoporphyrin to the indole moiety. A type II (^lO₂-involving) mechanism was of minor importance. The presence of sodium dodecyl sulphate micelles, where the porphyrin was solubilized in a monomeric state, exerted only a slight influence on the competition between the two reaction pathways described above in the case of tryptophan, which was essentially all present in the aqueous phase. On the other hand, tryptamine, when dissolved within micelles, underwent photooxidative attack exclusively by ¹O₂ which was generated within a micelle, diffused through the aqueous phase and penetrated into another (tryptamine-containing) micelle; the reduced efficiency of the latter photoprocess was probably a consequence of the low probability of ¹O₂ penetration into charged micelles.     

PHOTOELECTRIC EFFECTS FROM CHLOROPHYLL a IN BILAYER MEMBRANES

Abstract— The photophysical and photochemical properties of thiophene derivatives have been studied by fluorescence and by 353 nm laser flash spectroscopy. α-Terthienyl and its derivatives show a moderate fluorescence quantum yield (less than 0.1) in cyclohexane, ethanol, or TritonX–100 micelles. An additional thiophene ring increases this value to 0.2 in ethanol or micelles. The transient triplet state of the six thiophenes is characterized by strong absorptions (ε⋍ 50000 M ^-1 cm^-1) in the visible region. These triplet states are very long lived. They react with oxygen, producing singlet oxygen very efficiently because of their high quantum yield of triplet formation (0.1 to 0.3). They do not react with excellent hydrogen or electron donors such as indole, N-acetyl tryptophanamide or cysteine. The hydrophobic thiophenes investigated are, therefore, Type II photodynamic agents almost exclusively.     

DEUTERIUM-ISOTOPE EFFECT ON THE FLUORESCENCE YIELDS AND LIFETIMES OF INDOLE DERIVATIVES–INCLUDING TRYPTOPHAN AND TRYPTAMINE

Abstract— The fluorescence yields and lifetimes of indole, five of its alkyl detivatives, tryptophan, and tryptamine have been determined in degassed, heavy and light water at room temperature. All of the compounds have radiative lifetimes nearly identical to the parent compound indole, and a comparison of these results with recently reported data on tryptophyl derivatives disclosed a striking uniformity in radiative lifetimes between indole and many amino acids and peptides which contain the indole group as the fluorescence unit. The fluorescence rate k _f in H₂O, was found to be 4.5 × 10⁷ sec^-1. The nonradiative decay rates were found to vary between 5.1 and 46 × 10⁷ sec^-1 and from a study of the deuterium-solvent isotope effect and the deuterium-substituent effect a mechanism for nonradiative deactivation is proposed which includes an isotopically dependent proton transfer and a pathway involving energy loss via the ring carbon hydrogen vibrations. Tryptophan at pH 7 was found to have a unique nonradiative decay scheme not evidenced at a pH 1 or pH 10.     

ABSORPTION SPECTRA AND ACID-BASE DISSOCIATION OF THE 4-ALKYL DERIVATIVES OF 7-HYDROXYCOUMARIN IN SELF-ASSEMBLED SURFACTANT SOLUTION: COMMENTS ON THEIR USE AS ELECTROSTATIC SURFACE POTENTIAL PROBES

Abstract— The absorption spectra of the un-ionized and ionized forms of 4-heptadecyl-7-hydroxycoumarin (HHC) in aqueous self-assembled surfactant solution have been investigated. From a comparison with the absorption spectra of 7-hydroxycoumarin, 7-hydroxy-4-methylcoumarin (MHC) and HHC in neat organic solvents and organic solvent/water mixtures it is shown that the 7-hydroxycoumarin chromophore of HHC in self-assembled surfactant solution resides, on average, in an interfacial microenvironment which has a lower effective dielectric constant than that of the bulk aqueous solution. The absorption spectrum of the ionized form of HHC in aggregates of self-assembled surfactant molecules with cationic quaternary ammonium headgroups is found to be consistent with there being specific molecular interaction between the anionic chromophore and the quaternary ammonium headgroup. pH titrations performed with MHC in pure water and in four molar aqueous solutions of sodium chloride and tetra-methylammonium chloride indicate that the acid-base dissociation of HHC in charged micelles and vesicles should not be substantially influenced by any interfacial salt-effects, and that the acid-base dissociation of HHC in cationic micelles and vesicles with quaternary ammonium headgroups should not be markedly affected by the specific molecular interaction that exists. Estimates of the electrostatic surface potentials of a number of self-assembled surfactant aggregates are made by utilising the acid-base dissociation of HHC and assuming that the nonionic micelles of n -dodecyl octaoxyethylene glycol monoether (C₁₂E₈) can serve as a reference state of zero surface potential. The validity of this assumption in relation to both micelles and vesicles is discussed in detail.     

An Insertion or Deletion in the Extramembrane Loop Connecting Helices E and F of Archaerhodopsin-1 Affects in vitro Refolding and Slows the Photocycle

Abstract— Upon addition of retinal, archaeopsin-1 expressed in Escherichia coli (_ecaO-1002) regenerated the chromophore in dimyristoyl phosphatidylcholine (DMPC), 3-[(3-cholamidopropyl) dimethylammonio]-l-propanesulfonate (CHAPS) and sodium dodecyl sulfate(SDS) mixed micelles as efficiently as the same opsin prepared from halobacteria. Introduction of an insertion or a deletion of five amino acids into the surface loop connecting helices E and F changed the secondary and tertiary structures of _ecaO-1002 in SDS, and diminished regeneration of the chromophore. The effect of the insertion and deletion on the in vitro refolding was specific to archaeopsin because the same insertion introduced at the corresponding position of bacterioopsin (bO) did not affect chromophore regeneration. The photocycle of the regenerated _ecaR-1002 decreased in DMPC/CHAPS/SDS mixed micelles compared with that of aR-1 in the claret membrane, which was consistent with the reported behavior of bO. Unexpectedly, the insertion and deletion in loop EF perturbed the photocycle of the regenerated _ecaR-1002. The accumulation of long-lived N- and O-like intermediates suggested that the insertion and deletion slowed down the proton uptake steps at the cytoplasmic surface.     

Microemulsions studies: Correlation between viscosity,electrical conductivity and electrochemical and fluorescent probe measurements

Microemulsion systems involving brine and dodecane, and stabilized by sodium dodecylsulfate and both pentanol and heptanol have been investigated. Results of various experiments including conductivity and viscosity measurements, electrochemical diffusion coefficients and fluorescent probe studies have been gathered and compared in order to gain additional understanding of the microemulsion structure. The diffusion coefficients of hydrophilic hydroquinone and hydrophobic ferrocene obtained from the Levich equation at the rotating disc electrode, vary as the self-diffusion coefficients of water and dodecane, respectively; the results are consistent with those obtained by other workers from tracer or NMR self-diffusion studies. The fluorescence analysis of the polarity sensed by pyrene and the microviscosity felt by dipyrenylpropane suggests that the progressive addition of pentanol and dodecane to SDS micelles leads to solubilizate the probes more in the droplet interior where they experience a more hydrophobic environment. The systematic study of the two microemulsion systems provides insight into the microscopic properties of the oil domains in which the fluorescent probes are assumed to be located. In the system stabilized by n-heptanol as cosurfactant, the microviscosity sensed by P(CH₂)₃P is shown to be much lower than the bulk viscosity of the microemulsion. All the results evidence the well-known structural transitions: water continuous, bicontinuous and oil continuous in the single monophasic area of the brine/ SDS/n-pentanol/dodecane system; premicellar aggregates and water swollen micelles in the W/O area of the brine/SDS/n-heptanol/dodecane system.     

MICROTUBULE ASSEMBLY INHIBITION BY PORPHYRINS and RELATED COMPOUNDS

Abstract— Fourth-derivative spectrophotometry has been applied to the analysis of the ¹L_a O-O band of the indole chromophore. Using several tryptophan models, it is shown that when this band is the most red-shifted, it produces a minimum at about 300 nm in the fourth-derivative spectrum. In proteins this red shift and the 300-nm minimum results from a charge-perturbation effect acting through space on the indole group of tryptophan residues. It is concluded that fourth-derivative spectrophotometry can be used for monitoring the effect of charges on tryptophan residues     

FLUORESCENCE PROBE FOR MICROENVIRONMENTS: A NEW PROBE FOR MICELLE SOLVENT PARAMETERS AND PREMICELLAR AGGREGATES

Abstract— A previous study on the electronic spectroscopy of p -N,N-dialkylaminobenzylidenemalononitrile, 1, has been extended to a larger variety of organic solvents and to micelles of ionic and nonionic surfactants. By comparing the fluorescence emission (λ_F and φ_∫) of 1 in micelles and in homogeneous organic solvents, the effective polarity and the microviscosity of the micellar environments of potassium dodecanoate, sodium dodecyl sulfate, cetyltrimethylammonium bromide and Triton X-100 micelles have been determined to be 40, 40, 36 and 28, respectively and 23, 31, 34 and 28 cP, respectively. These results indicate that the fluorescence probe is located in the micelle–water interface of a micelle and this region of a micelle is polar and viscous. 1 has also been studied in different surfactants with varying surfactant concentrations. The φ_∫ of 1, a microviscosity gauge for micellar aggregates, remains unchanged at the critical micelle concentrations of various surfactants, but decreases at much lower surfactant concentrations. This is attributable to the formation of premicellar aggregates of surfactant molecules below their critical micelle concentrations.     

Photoionization of Indole, N-Methylindole and Tryptophan In Aqueous Solution upon Excitation at 193 nm

Abstract— The 193 nm photoionization of aqueous indole, A'-meth-ylindole and tryptophan–as a function of pH and under several saturating gas conditions–has been studied by laser photolysis using optical and conductometric detection methods. Monophotonic ionization leads to production of the cation radicals and hydrated electrons, the quantum yield of electron ejection is 0.3–0.4. The cation radicals have pK_a values of 4.5, <5 and 4.5 for indole, N -methylindole and tryptophan, respectively. Above these pH values, the cation radical deprotonate rapidly, having lifetimes of 1.0, ≅6 and 1.1 μs, respectively. Under N₂O, neutral indolyl radical production is accompanied by formation of an OH adduct radical (<1 μs). The conductivity results in Ar- and N₂O-saturated solution support the deprotonation mechanism and indicate that in the acidic pH range, the cation radical decays by release of protons with kinetics on the millisecond time scale.     

Interaction between casein and sodium dodecyl sulfate

The interaction of the anionic surfactant sodium dodecyl sulfate (SDS) with 2.0 mg/ml casein was first investigated using isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence spectra. ITC results show that individual SDS molecules first bind to casein micelles by the hydrophobic interaction. The micelle-like SDS aggregate is formed on the casein chains when SDS concentration reaches the critical aggregation concentration (c1), which is far below the critical micellar concentration (cmc) of SDS in the absence of casein. With the further increase of SDS concentration to the saturate binding concentration c2, SDS molecules no longer bind to the casein chains, and free SDS micelles coexist with casein micelles bound with SDS aggregates in the system. DLS results show that the addition of SDS leads to an increase in the hydrodynamic radius of casein micelles with bound surfactant at SDS concentration higher than 4 mM, and also an increase in the casein monomer molecule (or submicelles) at SDS concentration higher than 10 mM. Fluorometric results suggest the addition of SDS leads to some changes in the binding process of hydrophobic probes to casein micelles.