Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λ _max) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.     

Synthesis and structural characterisation of the first N-heterocyclic carbene ligand fused to a porphyrin

The functionalisation of two neighboring beta-pyrrolic positions of a porphyrin by a fused N-heterocyclic carbene ligand, the subsequent metallation of this external coordination site by palladium(II) and the structural characterisation of the resulting compounds are presented.     

Equilibrium between quenched and nonquenched conformations of the major plant light-harvesting complex studied with high-pressure time-resolved fluorescence

Nonphotochemical quenching (NPQ) of chlorophyll fluorescence plays an important role in the protection of plants against excessive light. Fluorescence quenching of the major light-harvesting complex (LHCII) provides a model system to study the mechanism of NPQ. The existence of both quenched and nonquenched states of LHCII has been postulated. We used time-resolved fluorescence and hydrostatic pressure to study differences between these states. Pressure shifts the thermodynamic equilibrium between the two states. The estimated volume difference was 5 mL/mol, indicating a local conformational switch. The estimated free energy difference was 7.0 kJ/mol: high enough to keep the quenched state population low under normal conditions, but low enough to switch in a controlled way. These properties are physiologically relevant properties, because they guarantee efficient light harvesting, while at the same time maintaining the capacity to switch to a quenched state. These results indicate that conformational changes of LHCII can play an important role in NPQ.     

Properties of the electronic density of states in TiO<Subscript>2</Subscript> nanoparticles surrounded with aqueous electrolyte

Results on the density of sates of nanostructured TiO₂ as a function of particle size and temperature are reported. In TiO₂ nanoparticles with a mean diameter 10 nm, the density of states (DOS) is strongly temperature-dependent, indicating a rearrangement of the bandgap states in which the exponential energy parameter (width of the distribution) increases from 0.080 to 50 °C. For nanoparticles with mean diameters of 20 and 30 nm the DOS is much closer to an exponential distribution, and is much less sensitive to temperature variations. It is suggested that nanometer confinement has a significant influence on the density of electronic states for 10-nm particles, while band tailing is similar to that occurring in bulk semiconductors for the larger particles.

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Unpredicted electron injection in CdS/CdSe quantum dot sensitized ZrO2 solar cells

A quantum dot sensitized solar cell based on a porous ZrO(2) film, sensitized with CdSe quantum dots using CdS as an intermediate layer is presented. We observe electron injection from photo-excited quantum dots into the ZrO(2), which is unexpected due to the much higher conduction band edge (closer to the vacuum level) of bulk ZrO(2) compared to TiO(2).     

Dynamics and energetics of Ge(001) dimers

The dynamic behavior of surface dimers on Ge(001) has been studied by positioning the tip of a scanning tunneling microscope over single flip-flopping dimers and measuring the tunneling current as a function of time. We observe that not just symmetric, but also asymmetric appearing dimers exhibit flip-flop motion. The dynamics of flip-flopping dimers can be used to sensitively gauge the local potential landscape of the surface. Through a spatial and time-resolved measurement of the flip-flop frequency of the dimers, local strain fields near surface defects can be accurately probed.     

Probe molecule chemisorption-low energy ion scattering study of surface active sites present in the orthorhombic Mo-V-(Te-Nb)-O catalysts for propane (amm)oxidation

Methanol and allyl alcohol chemisorption and surface reaction in combination with low energy ion scattering (LEIS) were employed to determine the outermost surface compositions and chemical nature of active surface sites present on the orthorhombic (M1) Mo-V-O and Mo-V-Te-Nb-O phases. These orthorhombic phases exhibited vastly different behavior in propane (amm)oxidation reactions and, therefore, represented highly promising model systems for the study of the surface active sites. The LEIS data for the Mo-V-Te-Nb-O catalyst indicated surface depletion for V (-23%) and Mo (-27%), and enrichments for Nb (+55%) and Te (+165%) with respect to its bulk composition. Only minor changes in the topmost surface composition were observed for this catalyst under the conditions of the LEIS experiments at 400 degrees C, which is a typical temperature employed in these propane transformation reactions. These findings strongly suggested that the bulk orthorhombic Mo-V-Te-Nb-O structure may function as a support for the unique active and selective surface monolayer in propane (amm)oxidation. Moreover, direct evidence was obtained that the topmost surface VO(x) sites in the orthorhombic Mo-V-Te-Nb-O catalyst were preferentially covered by chemisorbed allyloxy species, whereas methanol was a significantly less discriminating probe molecule. The surface TeO(x) and NbO(x) sites on the Mo-V-Te-Nb-O catalyst were unable to chemisorb these probe molecules to the same extent as the VO(x) and MoO(x) sites. Our findings suggested that different surface locations for V(5+) ions in the orthorhombic Mo-V-O and Mo-V-Te-Nb-O catalysts may be primarily responsible for vastly different catalytic behavior exhibited by the Mo-V-O and Mo-V-Te-Nb-O phases. Although the proposed isolated V(5+) pentagonal bipyramidal sites in the orthorhombic Mo-V-O phase may be capable of converting propane to propylene with modest selectivity, the selective 8-electron transformation of propane to acrylic acid and acrylonitrile may require the presence of several surface VO(x) redox sites lining the entrances to the hexagonal and heptagonal channels of the orthorhombic Mo-V-Te-Nb-O phase. The study of allyl alcohol oxidation over the Mo-V-O and Mo-V-Te-Nb-O catalysts further suggested that water plays a critical role during the oxidation of acrolein intermediate to acrylic acid over the orthorhombic (M1 phase) Mo-V-Te-Nb-O catalysts. Finally, the present study strongly indicated that chemical probe chemisorption combined with low energy ion scattering (LEIS) is a novel and highly promising surface characterization technique for the investigation of the active surface sites present in the bulk mixed metal oxides.