Determination of the concentration of potential efflux pump inhibitors, pheophorbide a and pyropheophorbide a, in the feces of animals by fluorescence spectroscopy.

Efflux pumps are vital bacterial components, and research has demonstrated that some plant compounds such as pheophorbide a (php) possess efflux pump inhibitor (EPI) activity. This study determined the quantity of php present in feces as an indicator of EPI activity. Feces were collected from different species of animals fed a variety of feeds. The chlorophyll metabolites php and pyropheophorbide a (pyp) were determined using fluorescense spectroscopy. The average concentrations [µg/g dry matter (DM) feces] of pyp/php in feces were as follows: guinea pig, 180; goat, 150; rabbit, 150; dairy cow, 120; feedlot cattle, 60; rat, <1; pig, <1; chicken, <1. These data indicate that animals consuming "green" diets will excrete feces with concentrations of php/pyp that exceed levels demonstrated to be inhibitory to bacterial efflux pumps (0.5 µg/mL). The natural presence EPIs in the gastrointestinal tract may modulate the activity of microbial efflux pumps and exert selection pressure upon resident microbial populations.

Enhanced stability and activity of cellulase in an ionic liquid and the effect of pretreatment on cellulose hydrolysis.

We discuss the hydrolysis of cellulose using a pure cellulase: endo-1,4-ß-D-glucanase (EG) from the fungus, Aspergillus niger, in buffer, the pure ionic liquid (IL), tris-(2-hydroxyethyl)-methylammonium methylsulfate (HEMA), and various mixtures of the two at different temperatures. Steady-state fluorescence and absorbance studies were performed to monitor the stability and activity of EG using cellulose azure as the substrate. EG attains its highest activity at 45°C in buffer and denatures at ∼55°C. On the other hand, HEMA imparts substantial stability to the enzyme, permitting the activity to peak at 75°C. The relative roles of temperature, viscosity, pH, polarity, and the constituent ions of the ILs on the hydrolysis reaction are examined. It is demonstrated that pretreatment of cellulose with ILs such as BMIM Cl, MIM Cl, and HEMA results in more rapid conversion to glucose than hydrolysis with cellulose that is not pretreated. The percent conversion to glucose from pretreated cellulose is increased when the temperature is increased from 45 to 60°C. Two different ILs are used to increase the efficiency of cellulose conversion to glucose. Cellulose is pretreated with BMIM Cl. Subsequent hydrolysis of the pretreated cellulose in 10-20% solutions of HEMA in buffer provides higher yields of glucose at 60°C. Finally, to our knowledge, this is the first study dealing with a pure endoglucanase from commercial A. niger. This enzyme not only shows higher tolerance to ILs, such as HEMA, but also has enhanced thermostability in the presence of the IL.

Photoinduced trans-to-cis isomerization of cyclocurcumin.

The photophysical properties of cyclocurcumin in various solvents are studied for the first time to shed light on the nonradiative processes of the parent compound, curcumin, which has a range of medicinal properties. Steady-state fluorescence and fluorescence-excitation spectra of cyclocurcumin in polar aprotic solvents are strongly dependent on excitation (λ(ex)) and emission (λ(em)) wavelength, respectively. The fluorescence quantum yield also depends on λ(ex) and increases with the viscosity of the medium. Time-resolved studies show nonexponential fluorescence decay in all solvents studied. The two fluorescence decay components of cyclocurcumin in alcohols exhibit a strong dependence on viscosity and temperature. NMR spectroscopy indicates that cyclocurcumin is entirely in the trans form with respect to the C6-C7 double bond in methanol, chloroform, and acetone. It is suggested that at least two conformational isomers about another single bond (C5-C6 or C7-C1″ or both) and that trans-to-cis isomerization about the C6-C7 double bond of these isomers provide the most likely means of rationalizing the steady-state spectra and the nonradiative decay mechanisms in both protic and aprotic polar solvents.

Comparison of the dielectric response obtained from fluorescence upconversion measurements and molecular dynamics simulations for coumarin 153-apomyoglobin complexes and structural analysis of the complexes by NMR and fluorescence methods.

We present a comparison of the dielectric response obtained from fluorescence upconversion experiments and from molecular dynamics simulations of the complexes of coumarin 153 with five apomyoglobins (apoMbs): wild-type horse heart (HH-WT) and those of wild-type sperm whale (SW-WT); its two triple mutants, L29F/H64Q/V68F and H64L/V68F/P88A; and its double mutant, L29F/V68L. Comparisons between experimental and simulated solvation relaxation functions, C(t)s, for the wild-type proteins range from very good to excellent. For the three mutants we investigated, however, agreement between experiment and simulation was considerably inferior. Thus, an NMR study of the complex of the HH-WT complex apoMb, and fluorescence energy transfer and anisotropy studies of the five complexes, were performed to investigate the structures upon which the simulations were based. The NMR measurements confirm our earlier conclusions that the C153 lies in the heme pocket of the HH-WT apoMb. For the wild-type complexes, fluorescence energy transfer measurements provide two rise times, suggesting a definite spatial relationship between the two Trp donors and the C153 acceptor. These results confirm the structural integrity of the wild-type complexes and validate the initial structures used for the molecular dynamics simulations. On the other hand, the three mutants provided single exponential rise times for energy transfer, suggesting that the position of the C153 used in the simulations may have been in error or that the C153 is mobile on the time scale of the energy transfer experiment. Fluorescence anisotropy studies also suggest that the double mutant was not structurally intact. Furthermore, examination of these systems demonstrates the sensitivity of C153 to its environment and permits the observation of differences in the heme pockets. These results point to the importance of structural characterization of modified proteins used in studies of the dielectric response and suggest strategies for performing molecular dynamics simulations of modified proteins.

Solvation dynamics of the fluorescent probe PRODAN in heterogeneous environments: contributions from the locally excited and charge-transferred states.

The coexistence of different excited states with different properties of the same chromophores could have significant consequences for the accurate characterization of solvation dynamics in a heterogeneous environment, such as a protein. The purpose of this work is to study the contributions of the locally excited (LE) and charge-transferred (CT) states of the fluorescent probe molecule 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) to its solvation dynamics in the heterogeneous environment provided by reverse micelles formed by sodium 1,4-bis-(2-ethylhexyl) sulfosuccinate (AOT)/n-heptane/water. We have found that the LE and CT states of PRODAN solvate on different time scales in reverse micelles (2 and approximately 0.4 ns, respectively), consistent with results suggested in the literature, and have concluded that PRODAN's use as a probe of heterogeneous environments must be used with caution and that, more importantly, the same caution must be exercised with any chromophore capable of emitting from different excited states.