Douglas Magde

Fluorescence Quantum Yields and Their Relation to Lifetimes of Rhodamine 6G and Fluorescein in Nine Solvents: Improved Absolute Standards for Quantum Yields¶

Abstract Absolute fluorescence quantum yields are reported for the rhodamine 6G cation and the fluorescein dianion dyes in nine solvents. This information is combined with previously reported fluorescence lifetimes to deduce radiative and nonradiative decay rates. Along the alcohol series from methanol to octanol, rhodamine 6G displays an increasing radiative rate, in parallel with the square of the refractive index increase, and a slightly decreasing nonradiative rate. Fluorescein is different: the apparent radiative rate actually decreases, suggesting that the emissive species is perturbed in some fashion. For both dyes, fluorescence yields are enhanced in D2O, rising to 0.98, in parallel with a corresponding increase in lifetimes. Protonated solvents invariably give shorter lifetimes and lower quantum yields, contrary to some previous speculation. From this work and an analysis of existing literature values, more precise values have been obtained for two previously proposed absolute quantum yield standards. The yield of fluorescein in 0.1 N NaOH(aq) is 0.925 ± 0.015, and for rhodamine 6G in ethanol, it is 0.950 ± 0.015. In both cases, the solutions are assumed to be in the limit of low concentration, excited close to their long-wave absorption band and at room temperature but may be either air-saturated or free of oxygen.

SOLVENT DEPENDENCE OF THE FLUORESCENCE LIFETIMES OF XANTHENE DYES

Fluorescence lifetimes of Ave representative xanthene dye species‐the rhodamine B zwitterion (RB=), the rhoda‐mine B cation (RB+), the rhodamine 6G cation (R6G+), the rhodamine 101 zwitterion (R101) and the fluorescein dianion (F2‐)‐were measured in H2O, D2O and in a series of alcohol solvents ranging from methanol to octanol. The lifetimes of both RB= and RB+ increased markedly as the solvent was varied from water to octanol. In contrast, the lifetimes of R6G+ and R101± decreased slightly over the alcohol series and that of F2‐ increased only slightly in the same series. For all the dyes studied the fluorescence lifetimes observed in D2O were slightly longer than those in H2O. Possible causes for the variations observed are discussed.

Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide

Changes in mean arterial pressure were monitored in rats following 50% isovolemic exchange transfusion with solutions of chemically modified hemoglobins. Blood pressure responses fall into three categories: 1) an immediate and sustained increase, 2) an immediate yet transient increase, or 3) no significant change either during or subsequent to exchange transfusion. The reactivities of these hemoglobins with nitric monoxide (⋅NO) were measured to test the hypothesis that different blood pressure responses to these solutions result from differences in ⋅NO scavenging reactions. All hemoglobins studied exhibited a value of 30 μm −1 s−1 for both⋅NO bimolecular association rate constants and the rate constants for ⋅NO-induced oxidation in vitro. Only the ⋅NO dissociation rate constants and, thus, the equilibrium dissociation constants varied. Values of equilibrium dissociation constants ranged from 2 to 14 pm and varied inversely with vasopressor response. Hemoglobin solutions that exhibited either transient or no significant increase in blood pressure showed tighter⋅NO binding affinities than hemoglobin solutions that exhibited sustained increases. These results suggest that blood pressure increases observed upon exchange transfusion with cell-free hemoglobin solutions can not be the result of ⋅NO scavenging reactions at the heme, but rather must be due to alternative physiologic mechanisms.

Molecular dynamics of chemical reactions in solution: Experimental picosecond transient spectra for I2 photodissociation

Picosecond transient electronic absorption spectroscopy is used to investigate the molecular dynamics of I2 reaction in solution following dissociative excitation into the A state. Our aim is to provide a sufficiently detailed set of experimental observations so that theoretical treatments of the molecular dynamics of a solution reaction can be rigorously tested. We report here measurements obtained in 13 solvents, pumping at 680 nm with four probe wavelengths of 840, 690, 630, and 595 nm. The time scale of the transient electronic spectral response varies considerably among the different solvents. For a given solvent, the response in general varies strongly with probe wavelength, being slower for higher probe photon energy. This indicates that the spectral response times are not due to recombination times, but are more likely due to the time for vibrational or perhaps electronic decay of already recombined molecules. This conclusion is reinforced by the presence of transient absorption in the near infrar...

Kinetics of CO Ligation with Nitric-oxide Synthase by Flash Photolysis and Stopped-flow Spectrophotometry

Interaction of CO with hemeproteins has physiological importance. This is especially true for nitric-oxide synthases (NOS), heme/flavoenzymes that produce ⋅NO and citrulline from l-arginine (Arg) and are inhibited by CO in vitro. The kinetics of CO ligation with both neuronal NOS and its heme domain module were determined in the presence and absence of tetrahydrobiopterin and Arg to allow comparison with other hemeproteins. Geminate recombination in the nanosecond time domain is followed by bimolecular association in the millisecond time domain. Complex association kinetics imply considerable heterogeneity but can be approximated with two forms, one fast (2–3 × 106 m −1 s−1) and another slow (2–4 × 104 m −1s−1). The relative proportions of the two forms vary with conditions. For the heme domain, fast forms dominate except in the presence of both tetrahydrobiopterin and Arg. In the holoenzyme, slow forms dominate except when both reagents are absent. Geminate recombination is substantial, ∼50%, only when fast forms predominate. Stopped-flow mixing found dissociation constants near 0.3 s−1. These data imply an equilibrium constant such that very little CO should bind at physiological conditions unless large CO concentrations are present locally.

Luminescent oligo(tetraphenyl)silole nanoparticles as chemical sensors for aqueous TNT

Colloidal oligo(tetraphenyl)silole nanoparticles in THF/H2O suspensions show increased luminescence and offer a method to detect TNT in an aqueous environment.

TIME‐RESOLVED FLUORESCENCE STUDIES OF SPINACH CHLOROPLASTS–EVIDENCE FOR THE HETEROGENEOUS BIPARTITE MODEL

Abstract— Fluorescence lifetimes of spinach chloroplasts were measured with a modelocked dye laser and time‐correlated single photon counting. Information about energy transport and functional organization of the chloroplasts is revealed by such time‐resolved fluorescence studies. Quenching experiments using treatment with UV light or the chemical agent dibromothymoquinone are consistent with the notion that there is heterogeneity associated with PS II units and that such heterogeneity is reflected over the entire time range of fluorescence decay, not just in a single component. Phosphorylation experiments were also carried out which permit us to relate these kinetic studies to previous steady state observations.

Photoselection with intense laser pulses

The method of photoselection uses a polarized photon beam to create a preferentially oriented population of excited state molecules. This population is examined using polarized absorption or emission techniques in order to obtain information about the symmetries of transition moments. Prelaser photoselection was limited in application to exceptionally long‐lived states (or stable photoproducts) in perfectly rigid matrices or to strongly fluorescent states in moderately viscous media. With lasers, extension is possible to almost any state with a lifetime of picoseconds or longer, making photoselection a very powerful, general technique. This paper rederives the expected experimental behavior and extends the calculations to include saturation effects, which are prominent in practical laser experiments but were not important in classical studies. Furthermore, it shows that in the nonlinear regime, information on transition moment symmetry is available even from a single beam experiment. One very simple exper...

KINETIC MODELING OF TIME‐RESOLVED FLUORESCENCE IN SPINACH CHLOROPLASTS

Abstract— Time‐resolved fluorescence decay profiles following picosecond excitation of plant chloro‐plasts or algae are now being measured with high precision and reproducibility. A number of suggestions have been put forward to explain the complex decay kinetics which are observed. In this paper three models are defined explicitly, solved rigorously, and compared in detail with experimental data. The complete description of energy transport in chloroplasts is no doubt very complicated, and only such quantitative comparisons will make it possible to decide which models are consistent with experiments. Of the three models examined, only the heterogeneous bipartite model has solutions which agree with commonly agreed upon features of the data. It offers, therefore, one starting point for more elaborate models and a guide to the design of further experiments.

Kinetics of CO and NO Ligation with the Cys331 → Ala Mutant of Neuronal Nitric-oxide Synthase

Nitric-oxide synthases (NOS) catalyze the conversion of l-arginine to NO, which then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine motif (CX 4C) that participates in a ZnS4center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys331→ Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding ofl-Arg and tetrahydrobiopterin (BH4). Because binding l-Arg and BH4 to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding l-Arg and BH4 has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS.