Luminol Oxidation by Hydrogen Peroxide Catalyzed by Tobacco Anionic Peroxidase: Steady-State Luminescent and Transient Kinetic Studies

The properties of a newly isolated anionic tobacco peroxidase from transgenic tobacco plants overexpressing the enzyme have been studied with respect to the chemiluminescent reaction of luminol oxidation. These were compared to the properties of horseradish peroxidase in the cooxidation of luminol and p -iodophenol, the enhanced chemiluminescence reaction. The pH, luminol and hydrogen peroxide concentrations were optimized for maximum sensitivity using the tobacco enzyme. The detection limit for the latter under the optimal conditions (2.5 m M luminol, 2 m M hydrogen peroxide, 100 m M Naborate buffer, pH 9.3) was about 0.1 p M , which is at least five times lower than that for horseradish peroxidase in enhanced chemiluminescence with p -iodophenol. The rate constants for the elementary steps of the enzyme-catalyzed reaction have been determined: k ₁= 4.9 × 10⁶ M ⁻¹ s¹, k ₂= 7.3 × 10⁶ M ⁻¹ s⁻¹, k ³= 2.1 × 10⁶ M ⁻¹ s⁻¹ (pH 9.3). The similarity of these rate constants is unusual for plant peroxidases. The high catalytic activity of tobacco peroxidase in the luminescent reaction is explained by the high reactivity of its Compound II toward luminol and the high stability of the holoenzyme with respect to heme dissociation. This seems to be a unique property of this particular enzyme among other plant peroxidases.     

Relaxation dynamics in glycerol-water mixtures: I. Glycerol-rich mixtures

We discuss the relaxation dynamics of glycerol-water mixtures, as studied by dielectric spectroscopy in the frequency range from 1 Hz to 250 MHz and at temperatures between 173 and 323 K. The experimental results obtained for the glycerol-rich mixtures suggest that the main dielectric relaxation process, as well as the so-called high-frequency "excess wing" (EW) and dc conductivity, follow the same temperature dependence. This result indicates that all of these processes are induced by the same molecular origin. A new phenomenological function is proposed to describe the whole dielectric spectrum in the covered frequency range, and some possible mechanisms of dielectric behaviors through the dc conductivity, the main relaxation process, and the EW are discussed.     

Photoisomerization and photodissociation of aniline and 4-methylpyridine

Photoisomerization and photodissociation of aniline and 4-methylpyridine at 193 nm were studied separately using multimass ion imaging techniques. Photofragment translational energy distributions and dissociation rates were measured. Our results demonstrate that more than 23% of the ground electronic state aniline and 10% of 4-methylpyridine produced from the excitation by 193 nm photons after internal conversion isomerize to seven-membered ring isomers, followed by the H atom migration in the seven-membered ring, and then rearomatize to both methylpyridine and aniline prior to dissociation. The significance of this isomerization is that the carbon, nitrogen, and hydrogen atoms belonging to the alkyl or amino groups are involved in the exchange with those atoms in the aromatic ring during the isomerization.     

Mycosamine orientation of amphotericin B controlling interaction with ergosterol: sterol-dependent activity of conformation-restricted derivatives with an amino-carbonyl bridge

Amphotericin B (AmB 1) is known to assemble together and form an ion channel across biomembranes. The antibiotic consists of mycosamine and macrolactone moieties, whose relative geometry is speculated to be determinant for the drug's channel activity and sterol selectivity. To better understand the relationship between the amino-sugar orientation and drug's activity, we prepared conformation-restricted derivatives 2-4, in which the amino and carboxyl groups were bridged together with various lengths of alkyl chains. K+ influx assays across the lipid-bilayer membrane revealed that ergosterol selectivity was markedly different among derivatives; short-bridged derivative 2 almost lost the selectivity, while 3 showed higher ergosterol preference than AmB itself. Monte Carlo conformational analysis of 2-4 based on NOE-derived distances indicated that the amino-sugar moiety of 2 comes close to C41 because of the short bridge, whereas those of 3 and 4 are pointing outward. The mutual orientation of the amino-sugar moiety and macrolide ring is so rigid in derivatives 2 and 3 that these conformations should be unchanged upon complex formation in lipid membranes. These results strongly suggest that the large difference in sterol preference between derivatives 2 and 3 is ascribed to the different orientation of amino-sugar moieties. These findings allowed us to propose a simple model accounting for AmB-sterol interactions, in which hydrogen bonding between 2'-OH of AmB and 3beta-OH of ergosterol plays an important role.     

Controlling the agglomeration of anisotropic Ru nanoparticles by the microwave-polyol process

A rapid polyol process for the synthesis of ruthenium nanoparticles was developed using microwave irradiation. A colloidal solution of monodispersed anisotropic Ru metal nanoparticles (mean particle size 2-6 nm) with different aspect ratios was obtained first. Particles with different degrees of agglomeration have also been synthesized using monodisperse particles as seeds and PVP (poly-N-vinyl-2-pyrrolidone) as the stabilization reagent. The stabilization mechanisms of different protective reagents were studied by UV-vis spectra. The HRTEM images indicated that dendritic particles formed by controlling agglomeration consisted of single-crystal domains with a random crystalline orientation.     

Age-dependent variations of cell response to oxidative stress: proteomic approach to protein expression and phosphorylation

We investigated the protein profiles of variously aged rat astrocytes in response to oxidative stress. After H2O2-exposure of cells at 100 microM for 30 min, the relative intensity of ten protein spots changed on two-dimensional (2-D) gels compared with control gels after silver staining. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis after in-gel digestion revealed that six of these spots corresponded to three kinds of proteins, each of which was composed of a protein and its modified form with a different isoelectric point (pI). These three proteins were identified as peroxiredoxins (PRDXs) II and III, and calpactin I light chain (p11). H2O2-exposure increased the intensity of the spot with lower pI and simultaneously decreased that of the spot with higher pI for both PRDXs II and III. In addition, the expression of annexin VII, S-adenosyl-L-homocysteine hydrolase, elongation factor II fragment (EF-II), and adenosine deaminase was increased by H2O2-exposure in astrocytes from variously aged rats. Using the Pro-Q Diamond staining, heat shock protein 60 kDa (Hsp 60) and alpha-tubulin were observed to be phosphorylated upon H2O2-exposure. While phosphorylation of alpha-tubulin was correlated positively with age, the changes in abundance of ten protein spots as described above were independent of age. These results suggest that aging does not suppress the responses aimed at limiting injury and promoting repair brought about by severe oxidative stress, and might affect cell dynamics including the formation of microtubules.     

Self-assembly of phosphate fluorosurfactants in carbon dioxide

Anionic phosphodiester surfactants, possessing either two fluorinated chains (F/F) or one hydrocarbon chain and one fluorinated chain (H/F), were synthesized and evaluated for solubility and self-assembly in liquid and supercritical carbon dioxide. Several surfactants, of both F/F and H/F types and having varied counterions, were found to be capable of solubilizing water-in-CO2 (W/C), via the formation of microemulsions, expanding upon the family of phosphate fluorosurfactants already found to stabilize W/C microemulsions. Small-angle neutron scatteringwas used to directly characterize the microemulsion particles at varied temperatures, pressures, and water loadings, revealing behavior consistent with previous results on W/C microemulsions.     

The Hund paradox and stabilization of molecular chiral states

The “paradox of optical isomers” revealed by Hund in 1927 is re-examined taking into account the interaction of a chiral molecule with phonons of optically inactive solids below the Debye temperature θ_D. It has been shown, that in the strong coupling limit, where the dephasing rate is much higher than the energy splitting frequency, the interaction can lead to a complete loss of coherence between mirror-antipodal states of the molecule due to the phonon-induced dephasing. This, in turn, suppresses tunnelling oscillations between them, so that transitions, which are reversible in time for an isolated chiral molecule become irreversible for the same molecule placed in a low-temperature matrix. In order to propose a particular mechanism of such stabilization, contributions of a single- and multi-phonon processes in the dephasing rate have been evaluated. It has been established that only the two-phonon scattering provides the sufficiently strong dephasing in low-temperature solids. Such a two-phonon mechanism is responsible for the stabilization of molecular chiral states as long as the temperature of the medium is lower θ_D, but exceeds a few Kelvin degrees. These findings are discussed in the context of the evolutionary concept known as “the cold prehistory of life”.     

Excluded volume effect for large and small solutes in water

The cavitation effect, i.e., the process of the creation of a void of excluded volume in bulk solvent (a cavity), is considered. The cavitation free energy is treated in terms of the information theory (IT) approach [Hummer, G.; Garde, S.; Garcia, A. E.; Paulaitis, M. E.; Pratt, L. R. J. Phys. Chem. B 1998, 102, 10469]. The binomial cell model suggested earlier is applied as the IT default distribution p(m) for the number m of solute (water) particles occupying a cavity of given size and shape. In the present work, this model is extended to cover the entire range of cavity size between small ordinary molecular solutes and bulky biomolecular structures. The resulting distribution consists of two binomial peaks responsible for producing the free energy contributions, which are proportional respectively to the volume and to the surface area of a cavity. The surface peak dominates in the large cavity limit, when the two peaks are well separated. The volume effects become decisive in the opposite limit of small cavities, when the two peaks reduce to a single-peak distribution as considered in our earlier work. With a proper interpolation procedure connecting these two regimes, the MC simulation results for model spherical solutes with radii increasing up to R = 10 A [Huang, D. H.; Geissler, P. L.; Chandler, D. J. Phys. Chem. B 2001, 105, 6704] are well reproduced. The large cavity limit conforms to macroscopic properties of bulk water solvent, such as surface tension, isothermal compressibility and Tolman length. The computations are extended to include nonspherical solutes (hydrocarbons C1-C6).