Steady-state Fluorescence Emission Spectra Research Articles

Comparative studies on inter- and intramolecular electron transfer processes within 4-methoxybenzo[b]thiophene (4MBT) and p-chloroacetophenone (PCA) reacting systems by using steady-state and laser flash photolysis techniques

Methods of synthesis and characterization of the bichromophores (4MBA and 4MBAS) comprising 4-methoxybenzo[b]thiophene (4MBT) as donor and p-chloroacetophenone (PCA) as acceptor linked together by an olefinic (unsaturated in the former bichromophore and saturated in the case of the latter one) bridge have been presented in this paper. The photophysics of these bichromophores has been extensively studied both by steady-state and laser flash photolysis techniques. An attempt was made to compare the monomolecular photophysical properties of the bichromophores with the photophysics of the bimolecular systems where its constituent reacting partners (same donor and acceptor molecules as within the bichromophores) being unlinked will react by diffusion-assisted process. Both from the steady-state electronic absorption and fluorescence emission spectra it shows that the bichromophores exhibit charge transfer (CT) complex both in the ground and excited states. This observation demonstrates that mutual orientation between the donor and the acceptor moieties largely facilitates the formation of such complex. In intermolecular interactions it appears that a weak excited singlet (S 1) CT complex of contact nature is formed, whereas in intramolecular systems (bichromophores 4MBA and 4MBAS) relatively stronger excited CT complexes of two types: cis (folded) and trans (extended) isomers for 4MBA and two conformers (gauche and staggered) for 4MBAS are formed, as evidenced from steady-state and time-resolved measurements. Transient absorption measurements clearly demonstrate the occurrences of photoinduced intermolecular ET reactions, whereas in intramolecular system direct excitation of ground state CT by using the third harmonic (355 nm) of a nanosecond Nd:YAG laser system produce ion-pair (D +–A −) state both in the singlet and triplet states. Transient absorption spectral measurements by laser flash photolysis technique reveal that energy wasting charge recombination process within the bichromophore is largely precluded on changing the nature of the spacer from unsaturated to more flexible saturated one. The necessity of incorporating two spacers (cyclophane type) instead of one to enhance the charge separation rate and to minimize the energy wasting recombination process is hinted.

Spectroscopic investigations of poly(propyleneimine)dendrimers using the solvatochromic probe phenol blue and comparisons to poly(amidoamine) dendrimers.

The physical and chemical properties of PPI dendrimers' interior were investigated using the fluorescent, solvatochromic probe phenol blue. In aqueous solutions of each generation studied, two discrete dye populations were clearly observed. PPI dendrimers were shown to form a tight, nonpolar association with the vast majority of available dye, within the dendrimer interior, near the core. In the steady-state fluorescence emission spectra, a microenvironment of decreasing polarity in increasingly larger-generation PPI dendrimers (up to G3) was seen for the associated probe. Each of the remaining larger-generation dendrimers provided a microenvironment of essentially equal polarity. Fluorescence anisotropy values for phenol blue in the PPI dendrimers demonstrated the dye's sensitivity to the changing molecular volumes of the dendrimer generations. Model compounds that mimicked PPI's surface groups and branching moieties were used to better define the associated dye's location. The mimics further confirmed that phenol blue was associated inside the dendrimer, where it did not interact with the dendrimer surface groups. The comparison of amine-terminated PPI and PAMAM dendrimers clearly demonstrated the effects of their structural differences and the ability of phenol blue to have sensed those differences, including the initiator core length, branching unit length, and branching unit chemical composition.

Spectroscopic Investigations of Polyamido Amine Starburst Dendrimers Using the Solvatochromic Probe Phenol Blue

The physical and chemical properties of PAMAM-AT dendrimers' interior were investigated using the fluorescent, solvatochromic probe phenol blue. In aqueous solutions of each generation studied except G0, two discrete dye populations were clearly observed. PAMAM-AT dendrimers were shown to form a tight, nonpolar association with the majority of available dye within the dendrimer interior, probably near its core. In the absorption and steady-state fluorescence emission spectra, a microenvironment of decreasing polarity in increasingly larger-generation PAMAM-AT dendrimers (up to G3) is seen for the associated probe. The remaining larger-generation dendrimers (G4−8) all provide a microenvironment of essentially equal polarity. Fluorescence anisotropy values for phenol blue in the PAMAM-AT dendrimers demonstrate the dye's sensitivity to the changing molecular volumes of the dendrimer generations. Model compounds that mimic PAMAM-AT's surface groups and branching moieties were used to better define the associa...

Oxidative response and membrane modification of diabetic platelets challenged with PAF

Alterations in the functional activities of platelets (PLT) in type I diabetes have been widely observed. These changes play a key role in the development of cardiovascular complications in diabetes. Various functional activities of PLT are the result of the interaction of numerous stimuli with PLT plasma membrane. This study was designed to evaluate the oxidative response and membrane modifications of diabetic PLT stimulated by platelet activating factor (PAF). The oxidative response was assessed by employing luminol- and lucigenin-amplified chemiluminescence. Luminol-amplified chemiluminescence is sensitive to the release of hydrogen peroxide whereas lucigenin-amplified chemiluminescence is sensitive to the production of superoxide anion. Membrane fluidity and polarity were studied using fluorescence spectroscopy. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). The diabetic group consisted of 20 type I diabetic children with good metabolic control. Our results show a significant decrease in the luminol- and lucigenin-amplified chemiluminescence of PAF stimulated PLT in the diabetic group with respect to controls. These data indicate a decrement in the release of reactive oxygen species by diabetic PLT. We observed a significant increase in steady-state fluorescence anisotropy of diabetic PLT membrane that reflects a decrease in membrane fluidity. Laurdan showed a blue shift of the fluorescence emission and excitation spectra in diabetic PLT with respect to the control group, indicating a decrease in membrane polarity. The addition of PAF to PLT induced a red shift of Laurdan spectra in both groups, indicating an increase in membrane polarity. Our study demonstrates an altered oxidative response to PAF stimulation of diabetic PLT, probably due to altered generation or handling of reactive oxygen species, and alterations in the physico-chemical properties of the plasma membrane which could influence various functional activities of PLT.

Plasma membrane polarity of polymorphonuclear leucocytes from children with primary ciliary dyskinesia.

Polymorphonuclear leucocytes (PMN) from subjects with primary ciliary dyskinesia (PCD) can have abnormal locomotory systems. The locomotory activity of PMN is the result of biochemical events mediated by the plasma membrane. In this study we investigated plasma membrane polarity of PMN from children with PCD. Membrane polarity was studied in 11 children with PCD and in healthy controls by measuring the steady-state fluorescence excitation and emission spectra of 2-dimethylamino[6-lauroyl]naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surroundings. Laurdan shows a marked steady-state emission red shift in polar solvents, with respect to nonpolar solvents. Moreover, the effect of the microtubule disassembling agent colchicine on PMN membrane polarity was evaluated. Our results show a red shift of the fluorescence excitation and emission spectra of Laurdan in PMN from the PCD group with respect to the control group. These data indicate an increase in membrane polarity of PMN from the PCD group. Treatment of PMN with colchicine induced a red shift in the Laurdan excitation and emission spectra with the same trend observed in PMN from the PCD group. PMN from children with PCD are characterized by an increased plasma membrane polarity. These changes could be the basis of the modifications in the locomotory activities of PMN. The observed alterations may be attributed to abnormalities in the cytoskeleton.

Plasma Membrane Fluidity and Polarity of Polymorphonuclear Leukocytes from Children with Type I Diabetes Mellitus

Polymorphonuclear leukocytes (PMN) from diabetic subjects have been found to be abnormal in various functional activities. These activities are mediated by the plasma membrane. This study was designed to evaluate plasma membrane fluidity and polarity in children with type I diabetes mellitus using fluorescence spectroscopy. PMN membrane fluidity and polarity were assessed in a group of 32 diabetic children. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy and fluorescence decay of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH), whereas membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). TMA-DPH and Laurdan are known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer. Our data show a significant increase in steady-state fluorescence anisotropy in diabetic PMN that reflects a decrease in membrane fluidity, and a decrease in TMA-DPH lifetime distribution indicating a decrease in membrane heterogeneity. Laurdan shows a blue shift of the fluorescence emission and a red shift of the excitation spectra in diabetic PMN with respect to the control group, indicating a decrease in membrane polarity. The results demonstrate a decrease in the phospholipid order at the membrane surface and a decrease in membrane polarity in diabetic PMN. These alterations in the physico-chemical properties of the plasma membrane could be the basis of the modifications in functional activities of PMN. The changes in the plasma membrane of PMN could be the result of metabolic and chemical modification associated with type I diabetes.

Isolation of pigment-binding early light-inducible proteins from pea.

The early light-inducible proteins (ELIPs) in chloroplasts possess a high sequence homology with the chlorophyll a/b-binding proteins but differ from those proteins by their substoichiometric and transient appearance. In the present study ELIPs of pea were isolated by a two-step purification strategy: perfusion chromatography in combination with preparative isoelectric focussing. Two heterogeneous populations of ELIPs were obtained after chromatographic separation of solubilized thylakoid membranes using a weak anion exchange column. One of these populations contained ELIPs in a free form providing the first isolation of these proteins. To prove whether the isolated and pure forms of ELIP bind pigments, spectroscopic and chromatographic analysis were performed. Absorption spectra and TLC revealed the presence of chlorophyll a and lutein. Measurements of steady-state fluorescence emission spectra at 77 K exhibited a major peak at 674 nm typical for chlorophyll a bound to the protein matrix. The action spectrum of the fluorescence emission measured at 674 nm showed several peaks originating mainly from chlorophyll a. It is proposed that ELIPs are transient chlorophyll-binding proteins not involved in light-harvesting but functioning as scavengers for chlorophyll molecules during turnover of pigment-binding proteins.

Identification of a Catalytic Aspartyl Residue ofd-Ribulose 5-Phosphate 3-Epimerase by Site-directed Mutagenesis

Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-type kcat of 7.1 x 10(3) s-1 is lowered to 3.3 x 10(-4) s-1 in D186A, 0.13 s-1 in D186N, and 1.1 s-1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the beta-carboxyl of Asp186 facilitates catalysis by >10(7)-fold. Relative to the wild-type enzyme, the Km for D-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp186 serving as an electrophile to facilitate alpha-proton abstraction. This study is the first to identify a catalytic group of the epimerase.

Fluorescence Study on the Conformational Change of an Amino Group-Containing Polymer Chain Grafted onto a Polyethylene Microfiltration Membrane

The fluorescence probe technique was used to investigate the characteristics of an amino group-containing polymer chain grafted onto a polyethylene microfiltration (MF) membrane. The amino group-containing polymer chain labeled with a dansyl group that served as a fluorescence probe was grafted onto a polyethylene MF membrane by radiation-induced graft polymerization. The conformational changes of the grafted polymer chain (graft chain) in various solvents were monitored by considering that the steady-state fluorescence emission spectrum of the dansyl group was affected by the polarity of the solvent, the polyethylene, and the graft chain itself. The shift of the emission peak wavelength of graft chains with different lengths demonstrated that the graft chain containing amino groups stretched in water and methanol and shrank in dimethylformamide, acetone, and benzene. These results corresponded to changes in solvent permeability through the membrane pore to which the amino group-containing polymer chains were grafted.

Fluorescence Lifetime Measurements and Spectral Analysis of Adamantyldiazirine

We report the first confirmed fluorescence lifetime measurement for a diazirine. We have obtained time-correlated single-photon counting fluorescence decays for adamantyldiazirine in a variety of solvents, over a wide range of temperatures (77−320 K) and across the diazirine absorption band (330−371 nm). The fluorescence lifetime of the primary decay component is on the order of 240 ps at ambient temperature and increases at lower temperatures. Arrhenius treatment of the fluorescence lifetime data indicates that the rate-limiting barrier for activated processes in the diazirine excited state is between 2.7 and 2.9 kcal/mol. Adamantyldiazirine's fluorescence lifetime appears to be unaffected by deuteration of the solvent, solvent polarity, or excitation energy. We also report and discuss the steady-state absorption and fluorescence emission spectra of adamantyldiazirine in a variety of solvents, as well as the infrared spectrum (KBr). We interpret the spectra with the help of ab initio (RHF/6-31G* and CIS/6-31G*), density functional (B3LYP/6-31G*), and semiempirical (PM3) calculations. The fluorescence quantum yield of adamantyldiazirine at ambient temperature was calculated to be ∼0.0012. Analysis of our data in the light of previous research leads us to conclude that little or no intermolecular chemistry is attributable to photoexcited adamantyldiazirine in solution at ambient temperature. Rather, fluorescence competes with one or more intramolecular photochemical processes.

Characterization of a Solvatochromic Fluorophore: 1-(2,2-Dicyanovinyo)-2,5-Dimethoxybenzene (DCN-2,5-DMOB)

Absorption spectra and steady-state fluorescence emission spectra for l-(2,2-dicyanovinyl)-2,5-dimethoxybenzene in five solvents were determined. Although the absorption spectra demonstrate little solvatochromism, emission peaks show a red shift of roughly 90 nm between cyclohexane and methanol or acetonitrile, which appears to indicate charge transfer associated with a relaxed, as opposed to a vertical, excited state. Semiempirical gas phase AM1 calculations on this compound and the related unsubstituted 2,2-dicyanovinyl benzene indicate a dihedral twist of roughly 35° between the phenyl and the dicyanovinyl planes for both molecules in their ground states, as well as substantial polarity associated with the ground states of these compounds.

Bacteriochlorophyll organization and energy transfer kinetics in chlorosomes from Chloroflexus aurantiacus depend on the light regime during growth

We have used measurements of fluorescence and circular dichroism (CD) to compare chlorosome-membrane preparations derived from the green filamentous bacterium Chloroflexus aurantiacus grown in continuous culture at two different light-intensities. The cells grown under low light (6 μmol m(-2) s(-1)) had a higher ratio of bacteriochlorophyll (BChl) c to BChl a than cells grown at a tenfold higher light intensity; the high-light-grown cells had much more carotenoid per bacteriochlorophyll.The anisotropy of the QY band of BChl c was calculated from steady-state fluorescence excitation and emission spectra with polarized light. The results showed that the BChl c in the chlorosomes derived from cells grown under high light has a higher structural order than BChl c in chlorosomes from low-light-grown cells. In the central part of the BChl c fluorescence emission band, the average angles between the transition dipole moments for BChl c molecules and the symmetry axis of the chlorosome rod element were estimated as 25° and 17° in chlorosomes obtained from the low- and high-light-grown cells, respectively.This difference in BChl organization was confirmed by the decay associated spectra of the two samples obtained using picosecond single-photon-counting experiments and global analysis of the fluorescence decays. The shortest decay component obtained, which probably represents energy-transfer from the chlorosome bacteriochlorophylls to the BChl a in the baseplate, was 15 ps in the chlorosomes from high-light-grown cell but only 7 ps in the preparation from low-light grown cells. The CD spectra of the two preparations were very different: chlorosomes from low-light-grown cells had a type II spectrum, while those from high-light-grown cells was of type I (Griebenow et al. (1991) Biochim Biophys Acta 1058: 194-202). The different shapes of the CD spectra confirm the existence of a qualitatively different organization of the BChl c in the two types of chlorosome.

Photophysics of High Concentration Systems at a Solution / Solid Interface: An Evanescentwave Study

ABSTRACTFor many species the study of photophysical behaviour by fluorescence spectroscopy has been confined to low concentration as a result of the influence of the inner filter effect. In this paper we report how the total internal reflection technique of evanescent wavefluorescence spectroscopy (EWIFS) allows both the collection of distortion free steady-state fluorescence emission spectra and quantitative analysis of such species, in ‘bulk’ solution environment, to significantly higher concentration than can be achieved with a conventional ( right-angle ) geometry. A study of two species, the laser dye rhodamine 101 and anovel meso-substituted zinc prophyrin is presented.

Sequential Electron Transfer Leading to Long-Lived Charge Separated State in a Porphyrin–Oxochlorin–Pyromellitdimide Triad

Abstract The synthesis and excited-state dynamics are described for fixed-distance porphyrin–oxochlorin–pyromellitdimide triads (P–C–Im) and related reference compounds. In zinc-oxochlorin–pyromellitdimide (ZnP–Im), the 1(ZnC)* is quenched by the attached Im through intramolecular charge separation (CS) in benzene, THF, and DMF, while the 1(H2C)* in the corresponding free base is not significantly quenched by the Im even in polar DMF. In the steady-state fluorescence emission spectra, only the emission from the 1(C)* is commonly observed, indicating an efficient intramolecular singlet–singlet excitation energy transfer from P to C. Of these, the fluorescence intensities of the 1(H2C)* in ZnP–H2C and ZnP–H2C–Im are significantly reduced in polar DMF solution and this is attributed to the intramolecular CS that gives (ZnP)+–(H2C)−–Im and (ZnP)+–(H2C)−, respectively. The (ZnP)+–(H2C)−–Im ion pair is clearly shown, by picosecond absorption spectroscopy, to be converted into a secondary, longer-lived charge separated state (ZnP)+–H2C–(Im)− via charge-shift reaction in competition with wasteful charge recombination to the ground state. The (ZnP)+–H2C–(Im)− state is formed in 0.09 quantum yield from ZnP–1(H2C)*–Im and has a lifetime of 0.24 μs in DMF.

Steady-state fluorescence emission studies on polyazacyclophane macrocyclic receptors and on their adducts with hexacyanocobaltate(III)

The steady-state fluorescence emission spectra of the azacyclophanes 2,5,8,11-tetraaza[12] paracyclophane (L1), 2,6,9,13-tetraaza[14]paracyclophane (L2), 14,15,17,18-tetramethyl-2,5,8,11-tetraaza[12]paracyclophane (L3) and 16,17,19,20-tetramethyl-2,6,9,13-tetraaza[14]paracyclophane (L4) as a function of pH have been measured. The fully protonated species of each cyclophane gives the highest fluorescence-emission quantum yield. The shapes of the titration curves have been explained by the existence of an electron-transfer quenching effect from a non-protonated amine to the benzene chromophore. This effect is greater for macrocycles in which the first deprotonated amine group is closer to the benzene. The association constants for the interaction of the four fully protonated macrocycles with K3[Co(CN)6] have been measured either by potentiometry or from fluorescence-emission measurements, and increase in the order L3≈ L4 < L1≈ L2. The photoaquation quantum yields of K3[Co(CN)6] have been measured in the presence of the macrocycles L1 and L2, and indicate that three of the CN nitrogens of the complex are involved in adduct formation with the fully protonated macrocycles, as supported by molecular modelling.

Yeast TATA binding protein interaction with DNA: fluorescence determination of oligomeric state, equilibrium binding, on-rate, and dissociation kinetics.

A combination of steady-state, stopped-flow, and time-resolved fluorescence of intrinsic tryptophan and extrinsically labeled fluorescent DNA is utilized to examine the interaction of yeast TATA binding protein (TBP) with DNA. TBP is composed of two structural domains, the carboxy domain (residues 61-240), which is responsible for DNA binding and initiation of basal level transcription, and an amino terminal domain (residues 1-60), whose function is currently unknown. The steady-state fluorescence emission spectrum of the single tryptophan in the amino terminal domain of TBP undergoes a huge (30-40 nm) red-shift upon interaction with stoichiometric amounts of TATA box containing DNA. From time-resolved tryptophan fluorescence anisotropy studies, we demonstrate that, in the absence of DNA, the protein exists as a multimer in solution and it contains (at least) two primary conformations, one with the amino terminus associated tightly with the protein(s) in a hydrophobic environment and one with the amino terminus decoupled away from the rest of the protein and solvent-exposed. Upon binding DNA, the protein dissociates into a monomeric complex, upon which only the solvent-exposed amino terminus conformation remains. Kinetic and equilibrium binding studies were performed on TATA box containing DNA which was extrinsically labeled with a fluorescent probe Rhodamine-X at the 5'-end. This "fluorescent" DNA allowed for the collection of quantitative spectroscopic binding, kinetic on-rate, and kinetic off-rate data at physiological concentrations. Global analysis of equilibrium binding studies performed from 500 pM to 50 nM DNA reveals a single dissociation constant (Kd) of approximately 5 nM. Global analysis of stopped-flow anisotropy on-rate experiments, with millisecond timing resolution and TBP concentrations ranging from 20 to 600 nM (20 nM DNA), can be perfectly described by a single second-order rate constant of 1.66 x 10(5) M(-1) s(-1). These measurements represent the very first stopped-flow anisotropy study of a protein/DNA interaction. Stopped-flow anisotropy off-rate experiments reveal a single exponential k(off) of 4.3 x 10(-2) min-1 (1/k(off) = 23 min) From the ratio of on-rate to off-rate, a predicted Kd of 4.3 nM is obtained, revealing that the kinetic and equilibrium studies are internally consistent. Deletion of the amino terminal domain of TBP decreases the k(on) of TBP approximately 45-fold and eliminates classic second-order behavior.

Disparate contributions of Tyr 10 and Tyr 109 to fluorescence intensity of rabbit skeletal muscle troponin C identified using a genetically engineered mutant

Intrinsic tyrosines, as monitored by fluorescence spectroscopy, are sensitive reporters of local, Ca 2+-induced conformational changes in troponin C (TnC). Rabbit skeletal TnC contains two tyrosines (Y10 in the N-helix, and Y109 in site 3 in the C-terminal domain) in distinct microenvironments: their individual contributions to total fluorescence intensity are elucidated here utilizing bacterially synthesized rabbit skeletal TnC (sTnC4) and a genetically engineered variant, termed 109YF, lacking one of the tyrosines (Y109 replaced with F109). The steady-state fluorescence emission spectra following excitation at 280 nm were recorded in EGTA (Ca 2+-free) and Ca 2+-saturated (pCa4) solutions. For the wild-type sTnC4, pCa4 causes a significant (46%) increase in the peak fluorescence intensity over the value in EGTA. For the mutant 109YF, the EGTA fluorescence is only marginally affected (74% of the wild-type F EGTA), but interestingly the Ca 2+ effect is completely suppressed (Δ F = F pCa4 - F EGTA = 2% of the wild-type value). These results indicate that the two tyrosines make disparate contributions to the fluorescence spectrum of wild-type sTnC, both in the presence and absence of Ca 2+; whereas Y10 in the N-helix is dominant in Ca 2+-free solution, Y109 is the sole contributor to the Ca 2+ effect. Furthermore, to explain the biphasic fluorescence response of Y109 obtained during Ca 2+ titrations, the findings yield the most unequivocal evidence that Ca 2+-induced conformational changes in the trigger sites operating the contractile switch modify properties of the C-terminal sites in TnC pari passu.

Probing the Interaction of PAF with Human Platelet Membrane Using the Fluorescent Probe Laurdan

Changes in membrane polarity of human platelets during the interaction with PAF were investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-lauroyl)naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surroundings. Laurdan shows a marked steady-state emission red-shift in polar solvents, with respect to non-polar solvents. Our results demonstrate that platelet activation factor (PAF) (10(-7) M) induces a red-shift of the fluorescence emission spectra of Laurdan. These changes were not observed in the presence of the PAF antagonist, L-659,989. These data suggest that the interaction of PAF with its specific receptor and the signalling pathways involved in platelet activation are accompanied by an increase in polarity at the hydrophobic-hydrophilic interface of human platelet membranes.

Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants

Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389-396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683-6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741-750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence emission from mechanical pulp sheets

Corrected steady-state fluorescence emission spectra of various mechanical pulps and celluloses were measured using an excitation wavelength of 320 nm. Regardless of its source, cellulose displayed a relatively high characteristic emission. The emission spectra of mechanical pulps were red-shifted with respect to that of cellulose, and peak shapes were influenced by the pulping process, by hydrogen peroxide bleaching and by monochromatic UV irradiation of the sheets. Changes in emission spectra owing to bleaching or UV irradiation occurred principally in the 370–440 nm and 500–550 nm regions, and were similar to changes in reflectance spectra reported in the literature. This suggests that, to a first approximation, emission spectra of mechanical pulps may be interpreted as a decrease in emission of cellulose caused by light absorption by lignin; changes due to bleaching and irradiation may be attributed to changes in lignin absorption.