Electron-Transfer Chemistry of Ru−Linker−(Heme)-Modified Myoglobin: Rapid Intraprotein Reduction of a Photogenerated Porphyrin Cation Radical

We report the synthesis and characterization of RuC7, a complex in which a heme is covalently attached to a [Ru(bpy)_3]^(2+) complex through a −(CH_2)_7− linker. Insertion of RuC7 into horse heart apomyoglobin gives RuC7Mb, a Ru(heme)−protein conjugate in which [Ru(bpy)_3]^(2+) emission is highly quenched. The rate of photoinduced electron transfer (ET) from the resting (Ru^(2+)/Fe^(3+)) to the transient (Ru^(3+)/Fe^(2+)) state of RuC7Mb is >10^8 s^(-1); the back ET rate (to regenerate Ru^(2+)/Fe^(3+)) is 1.4 × 10^7 s^(-1). Irreversible oxidative quenching by [Co(NH_3)_5Cl]^(2+) generates Ru^(3+)/Fe^(3+):  the Ru^(3+) complex then oxidizes the porphyrin to a cation radical (P^(•+)); in a subsequent step, P^(•+) oxidizes both Fe^(3+) (to give Fe^(IV)═O) and an amino acid residue. The rate of intramolecular reduction of P^(•+) is 9.8 × 10^3 s^(-1); the rate of ferryl formation is 2.9 × 10^3 s^(-1). Strong EPR signals attributable to tyrosine and tryptophan radicals were recorded after RuC7MbM^(3+) (M = Fe, Mn) was flash-quenched/frozen

Characterization of reagent pencils for deposition of reagents onto paper-based microfluidic devices

Reagent pencils allow for solvent-free deposition of reagents onto paper-based microfluidic devices. The pencils are portable, easy to use, extend the shelf-life of reagents, and offer a platform for customizing diagnostic devices at the point of care. In this work, reagent pencils were characterized by measuring the wear resistance of pencil cores made from polyethylene glycols (PEGs) with different molecular weights and incorporating various concentrations of three different reagents using a standard pin abrasion test, as well as by measuring the efficiency of reagent delivery from the pencils to the test zones of paper-based microfluidic devices using absorption spectroscopy and digital image colorimetry. The molecular weight of the PEG, concentration of the reagent, and the molecular weight of the reagent were all found to have an inverse correlation with the wear of the pencil cores, but the amount of reagent delivered to the test zone of a device correlated most strongly with the concentration of the reagent in the pencil core. Up to 49% of the total reagent deposited on a device with a pencil was released into the test zone, compared to 58% for reagents deposited from a solution. The results suggest that reagent pencils can be prepared for a variety of reagents using PEGs with molecular weights in the range of 2000 to 6000 g/mol

Reagent pencils: A new technique for solvent-free deposition of reagents onto paper-based microfluidic devices

Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices. The technique is as simple as drawing with the reagent pencils on a device. When aqueous samples are added to the device, the reagents dissolve from the pencil matrix and become available to react with analytes in the sample. Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution. Most importantly, sensitive reagents, such as enzymes, are stable in the pencils under ambient conditions, and no significant decrease in the activity of the enzyme horseradish peroxidase stored in a pencil was observed after 63 days. Reagent pencils offer a new option for preparing and customizing diagnostic tests at the point of care without the need for specialized equipment

Validation of ED-XRF as a Reliable Method for Determining the Mineral Composition of Skim Milk Powders

Abstract of paper presented at the 2006 Joint Annual Meeting of the American Dairy Science Association & the American Society of Animal Science

Electrocatalytic Reductions of Nitrite, Nitric Oxide, and Nitrous Oxide by Thermophilic Cytochrome P450 CYP119 in Film-Modified Electrodes and An Analytical Comparison of Its Catalytic Activities with Myoglobin

Previous investigations of nitrite and nitric oxide reduction by myoglobin in surfactant film modified electrodes characterized several distinct steps in the denitrification pathway, including isolation of a nitroxyl adduct similar to that proposed in the P450nor catalytic cycle. To investigate the effect of the axial ligand on these biomimetic reductions, we report here a comparison of the electrocatalytic activity of myoglobin (Mb) with a thermophilic cytochrome P450 CYP119. Electrocatalytic nitrite reduction by CYP119 is very similar to that by Mb: two catalytic waves at analogous potentials are observed, the first corresponding to the reduction of nitric oxide, the second to the production of ammonia. CYP119 is a much more selective catalyst, giving almost exclusively ammonia during the initial half-hour of reductive electrolysis of nitrite. More careful investigations of specific steps in the catalytic cycle show comparable rates of nitrite dehydration and almost identical potentials and lifetimes for ferrous nitroxyl intermediate (FeII-NO-) in CYP119 and Mb. The catalytic efficiency of nitric oxide reduction is reduced for CYP119 as compared to Mb, attributable to both a lower affinity of the protein for NO and a decreased rate of N−N coupling. Isotopic labeling studies show ammonia incorporation into nitrous oxide produced during nitrite reduction, as has been termed co-denitrification for certain bacterial and fungal nitrite reductases. Mb has a much higher co-denitrification activity than CYP119. Conversely, CYP119 is shown to be slightly more efficient at the two-electron reduction of N2O to N2. These results suggest that thiolate ligation does not significantly alter the catalytic reactivity, but the dramatic difference in product distribution may suggest an important role for protein stability in the selectivity of biocatalysts

Electrochemistry of the Cu_A domain of Thermus thermophilus cytochrome ba _3

The electrochemistry of a water-soluble fragment from the Cu_A domain of Thermus thermophilus cytochrome ba _3 has been investigated. At 25  °C, Cu_A exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)_2Cu]^(+/0) electrode reaction were determined by variable-temperature electrochemistry (ΔS°_(rc) = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)_2Cu]^(+/0) electron transfer. An irreversible oxidation of [Cu(Cys)_2Cu]^+ at 1 V vs NHE confirms that the Cu^(II):Cu^(II) state of Cu_A is significantly destabilized relative to the Cu^(II) state of analogous blue-copper proteins

Synthesis and Electronic Properties of Fluoreno[2,1‑<i>a</i>]fluorenedione and Fluoreno[1,2‑<i>a</i>]fluorenedione

The [2,1-<i>a</i>]- and [1,2-<i>a</i>]-isomers of fluorenofluorenedione have been synthesized via intramolecular Friedel–Crafts acylations. DFT calculations indicate that the [1,2-<i>a</i>]-isomer adopts a twisted, helical <i>C</i>₂-symmetric structure and that its protonated form is the thermodynamic product of the Friedel–Crafts acylation in hot sulfuric acid. Absorption spectroscopy and cyclic voltammetry measurements provide experimental estimations of frontier molecular orbital energy levels, which are reported and discussed

Olive- and Coconut-Oil-Enriched Diets Decreased Secondary Bile Acids and Regulated Metabolic and Transcriptomic Markers of Brain Injury in the Frontal Cortexes of NAFLD Pigs

The objective of this study was to investigate the effect of dietary fatty acid (FA) saturation and carbon chain length on brain bile acid (BA) metabolism and neuronal number in a pig model of pediatric NAFLD. Thirty 20-day-old Iberian pigs, pair-housed in pens, were randomly assigned to receive one of three hypercaloric diets for 10 weeks: (1) lard-enriched (LAR; n = 5 pens), (2) olive-oil-enriched (OLI, n = 5), and (3) coconut-oil-enriched (COC; n = 5). Pig behavior and activity were analyzed throughout the study. All animals were euthanized on week 10 and frontal cortex (FC) samples were collected for immunohistochemistry, metabolomic, and transcriptomic analyses. Data were analyzed by multivariate and univariate statistics. No differences were observed in relative brain weight, neuronal number, or cognitive functioning between diets. Pig activity and FC levels of neuroprotective secondary BAs and betaine decreased in the COC and OLI groups compared with LAR, and paralleled the severity of NAFLD. In addition, OLI-fed pigs showed downregulation of genes involved in neurotransmission, synaptic transmission, and nervous tissue development. Similarly, COC-fed pigs showed upregulation of neurogenesis and myelin repair genes, which caused the accumulation of medium-chain acylcarnitines in brain tissue. In conclusion, our results indicate that secondary BA levels in the FCs of NAFLD pigs are affected by dietary FA composition and are associated with metabolic and transcriptomic markers of brain injury. Dietary interventions that aim to replace saturated FAs by medium-chain or monounsaturated FAs in high-fat hypercaloric diets may have a negative effect on brain health in NAFLD patients