The Electronic State of Flavoproteins: Investigations with Proton Electron–Nuclear Double Resonance

Electron–nuclear double resonance (ENDOR) spectroscopy provides useful information on hyperfine interactions between nuclear magnetic moments and the magnetic moment of an unpaired electron spin. Because the hyperfine coupling constant reacts quite sensitively to polarity changes in the direct vicinity of the nucleus under consideration, ENDOR spectroscopy can be favorably used for the detection of subtle protein–cofactor interactions. A number of pulsed ENDOR studies on flavoproteins have been published during the past few years; most of them were designed to characterize the flavin cofactor by means of its protonation state, or to detect individual protein–cofactor interactions. The aim of this study is to compare the pulsed ENDOR spectra from different flavoproteins in terms of variations of characteristic proton hyperfine values. The general concept is to observe limits of possible influences on the cofactor’s electronic state by surrounding amino acids. Furthermore, we compare ENDOR data obtained from in vivo experiments with in vitro data to emphasize the potential of the method for gaining molecular information in complex media.     

Sulfated glycopolymer thin films - preparation, characterization, and biological activity

The impact of heparinoid characteristics on model surfaces obtained from immobilization of sole sulfate groups as well as sulfated glycosides, sulfated cellulose, and definite heparin has been investigated. The obtained layers were physico-chemically characterized regarding film thickness, chemical composition, wettability, and surface morphology. Antithrombin adsorption, studied by fluorescence labeling, revealed a strong dependence on the presence of glycosidic structures and on the molecular weight of the grafted saccharide. On contact with whole blood, the coatings resulted in a diminished plasmatic and cellular coagulation in vitro, which did not reflect well the antithrombin binding. Therefore, more complex activating pathways are discussed.     

GA/GV measured in the decay of polarized neutrons

A measurement of [G_A/G_V] in the decay of polarized neutrons, basically quite similar to our 1968 one, gives a value of 1.258±0.015. The asymmetry coefficient, A, is ?0.113±0.006. These results include the 1968 data.     

Single Amino Acid Substitution Reveals Latent Photolyase Activity in Arabidopsis cry1

A quick switch: A single amino acid substitution at a conserved residue (D396N) of Arabidopsis cryptochrome-1 (Atcry1) confers single-stranded DNA repair activity in?vitro, conferring photolyase activity onto the cryptochrome. The mutant protein undergoes photoreduction of flavin to the fully reduced anionic form, similar to photolyases and unlike wild-type cryptochromes.     

Production and characterization of antibodies against crosslinked gelatin nanoparticles and first steps toward developing an ELISA screening kit

Nanotechnologies are finding a growing range of applications in the food sector. Nanoparticles are used notably to add vitamins and other nutrients to foods and beverages without affecting taste and color. They are also used to develop new tastes, preserve food texture, control the release of flavors, improve the bioavailability of compounds such as antioxidants and vitamins, and monitor freshness with nanosensors. Crosslinked gelatin nanoparticles are a component of nano-sized carriers for nutrient and supplement delivery in foods and related products. This paper describes the production and characterization of polyclonal antibodies against gelatin nanoparticles. Two immunization schemes were investigated: subcutaneous injection with and without a first intravenous injection. Two enzyme-linked immunosorbent assay formats were used to characterize the antibodies: an inhibition format with an antigen-coated plate for detection of the immune response and a sandwich format for development of the method. The antibodies showed good sensitivity with an IC50 equal to 0.11 ng mL(-1) using indirect ELISA format and a good specificity for the nanomaterials, without significant cross-reactivity against native gelatin. The limit of detection was determined-0.42, 0.27, 0.26, and 0.24 μg mL(-1) for apple, orange juice, milk, and soft drink matrices, respectively. ELISA technology offers rapid, low-cost assays for screening foods, feeds, and beverages. We have studied a prototype ELISA for detection of gelatin-based nanocarrier systems. Fruit juices, milk, and a soft drink were the matrices selected for assay development.     

Production of reference materials for the detection and size determination of silica nanoparticles in tomato soup

A set of four reference materials for the detection and quantification of silica nanoparticles (NPs) in food was produced as a proof of principle exercise. Neat silica suspensions were ampouled, tested for homogeneity and stability, and characterized for total silica content as well as particle diameter by dynamic light scattering (DLS), electron microscopy (EM), gas-phase electrophoretic molecular mobility analysis (GEMMA), and field-flow fractionation coupled with an inductively coupled mass spectrometer (FFF-ICPMS). Tomato soup was prepared from ingredients free of engineered nanoparticles and was spiked at two concentration levels with the silica NP suspension. Homogeneity of these materials was found sufficient to act as reference materials and the materials are sufficiently stable to allow long-term storage and distribution at ambient temperature, providing proof of principle of the feasibility of producing liquid food reference materials for the detection of nanoparticles. The spiked soups were characterized for particle diameter by EM and FFF-ICPMS (one material only), as well as for the total silica content. Although questions regarding the trueness of the results from EM and FFF-ICPMS procedures remain, the data obtained indicate that even assigning values should eventually be feasible. The materials can therefore be regarded as the first step towards certified reference materials for silica nanoparticles in a food matrix.     

Pressure-controlled high-performance liquid chromatographic study on the influence of rim chemistry on partial molar volume differences between free and complexed cyclodextrins

Pragmatic comparison of pressure dependent retention for differing cyclodextrin rim chemistries is assessed using controlled-pressure HPLC. For pressure differences of <300 bar, systematic shifts in solute capacity factor are observed for both native and methylated beta-cyclodextrin stationary phases. In addition to the importance of this observation for the practice of liquid chromatography, this technique can also be implemented in the fundamental determination of the influence of rim chemistry on the cyclodextrin partial molar volume both with and without solute inclusion. That is, pressure-controlled measurements provide a direct comparison between the partial molar volumes for native cyclodextrin (CD) and methylated cyclodextrin (MCD) in the presence and absence of the complexing solute (comp). Surprisingly, direct comparison of the measured partial molar volumes for the two rim chemistries indicates that the presence of neutral solutes does not contribute significantly to the volumetric component of complexation, V(comp,CD) - V(comp,MCD) approximately V(CD) - V(MCD). In contrast, their ionized counterparts are shown to exhibit marked rim chemistry differences in the partial molar volume of cyclodextrins with and without anion inclusion, V(comp(-),CD) - V(comp(-),MCD) < V(CD) - V(MCD). Not previously demonstrated by direct chromatographic measurement, these results have interesting implications for advancing the fundamental understanding of host-guest solvation properties.