Inactivation of nitroalkane oxidase upon mutation of the active site base and rescue with a deprotonated substrate

Mutation of Asp402 in nitroalkane oxidase to Asn or Ala inactivates the enzyme with neutral nitroethane as substrate, but the activity can be rescued with the nitroethane anion. The V/K values of the D402N and D402A enzymes with the nitroethane anion are independent of pH, whereas the V/K values of the wild-type and D402E enzymes are pH dependent with both the protonated and the deprotonated forms of nitroethane. Moreover, although the V/K value of the D402E enzyme with neutral nitroethane is 20-fold less than that of the wild-type enzyme, there is only a 2-fold difference in the V/K values with the nitroethane anion. These results are fully consistent with a primary role for Asp402 as the active site base in nitroalkane oxidase which abstracts the substrate alpha-proton.     

Synthesis and fluorescence ion-sensory properties of the first dehydropyridoannulene-type cyclophane with enforced exotopic metal ion binding sites

The new twistophane 4 has been synthesised, which comprises a conjugated dehydropyridoannulene-type macrocyclic scaffold with outwardly projecting nitrogen-donor sites for the purpose of metal ion coordination. The macrocyclíc structure of 4 was assigned by using spectroscopic methods, and shown to exist in a twisted and chiral ground state conformation by semi-empirical theoretical calculations. A detailed spectroscopic investigation into the metal ion binding properties of 4 and precursor 11 revealed that they functioned as selective complexants, affording a fluorescence quenching output response characteristic of Pd(II) and Hg(II) ions. Furthermore, 4 also signalled the presence of Fe(II), Co(II), Ni(II) and Ag(I) ions by the precipitation of coordination polymers, and exhibited reversible proton-triggered fluorescence quenching behaviour. Macrocycle 4 thus represents a unique type of molecular sensory platform, which may find a wealth of potential applications such as the detection of heavy-metal pollutants, as well as for the fabrication of proton-switchable materials and coordination polymers with novel electronic and magnetic properties.     

The key elements of traceability in chemical measurement: agreed or still under debate?

Talking about "traceability" means talking about a "property of the result of a measurement", about "the value of a standard", about "stated references" and about an "unbroken chain of comparisons". It describes by which comparison, and to which other value, the result of a measurement has been obtained, i.e. is "traceable to". It is about the underlying structure of the measurement process of the result of a measurement and therefore about the authority of the result. Since values carried by (certified) reference materials have also been obtained by measurement, the definition of traceability equally applies. Traceability in the context of reference materials is also about the authority of the values carried by the (certified) reference materials and is, therefore, of key importance for the authority of the reference materials themselves. Hence, values of results of measurements constitute part of the traceability chain and their uncertainties are an intrinsic accompanying phenomenon. Uncertainties need a traceability chain against which they can be evaluated, and a traceability chain is an a priori requirement for evaluating the uncertainty budget of a measurement result. An attempt has been made to exemplify "traceability" chains in some types of chemical measurement and to identify the degree of international agreement on the key elements of "traceability". It is concluded that there is less than universal agreement on this issue. The debate should continue in order to arrive at the international understanding and agreement needed, as "traceability" is now being incorporated in the International Organization for Standardization (ISO), the International Laboratory Accreditation Co-operation (ILAC) and in other "guiding" or regulatory documents. It is also the reason why the Institute for Reference Materials and Measurements (IRMM) has taken up the study of the concept in its core programme on Metrology in Chemistry, and why it sponsored the Workshop in Bratislava.     

OZONE, MIDDLE ULTRAVIOLET RADIATION AND THE AQUATIC ENVIRONMENT

Marine organisms in the upper layers of the sea may be endangered by increased ultraviolet radiation resulting from declines in the thickness of stratospheric ozone. Evidence supporting this hypothesis includes the fact that wavelengths of potentially damaging ultraviolet radiation can penetrate to ecologically significant depths and laboratory findings that many marine organisms are extremely sensitive to this radiation. Estimated effects of increased ultraviolet radiation on populations of marine organisms range from insignificant to catastrophic. Direct estimation of population effects have not been made, although this is the only adequate measure of the potential impact. The extreme diminution of ozone during the Antarctic spring, coupled with the dynamics of phytoplankton production in this region, may provide conditions suitable for an environmental test of the hypotheses that marine organisms are endangered by reduced stratospheric ozone. There is an urgency to the testing of this hypothesis since these populations may be directly impacted at the present time.     

Zur Analyse des Flu?spats

Zusammenfassung Die Analyse hochprozentigen Flußspats wird in ihrer derzeitigen Ausführungsform beschrieben. Nach Beseitigung carbonatischer Begleiter durch eine Behandlung mit verd. Essigsäure wird die Kieselsäure entweder mit Flußsäure verflüchtigt und aus dem Gewichtsverlust bestimmt oder in einer gesonderten, mit Soda aufgeschlossenen Probe als Tetrapyridiniumdodecamolybdatosilicat gefällt und als SiO₂ + 12 MoO₃ ausgewogen. Der Bariumsulfatgehalt wird nach Verflüchtigung der Kieselsäure durch Abrauchen des verbleibenden Rückstandes mit Schwefelsäure und anschließendes Kochen mit verd. Salzsäure/Schwefelsäure in der üblichen Art ermittelt. Im Filtrat der Bariumsulfatfällung wird Calcium nach Abtrennung oder Maskierung der dreiwertigen Begleiter als Oxalat gefällt und zum Oxid verglüht oder direkt durch eine photometrische Titration mit ÄDTA bestimmt. Der kleine Sulfidschwefelgehalt in Flußspat wird durch Säure als Schwefelwasserstoff ausgetrieben, als Cadmiumsulfid gebunden und anschließend jodometrisch bestimmt.Für Kieselsäure, Bariumsulfat und Calciumfluorid ergibt die Fehlerrechnung Standardabweichungen von ^s SiO₂=0.04%; sBaSO₄=±0,04%; sCaF₂=±0,09%.

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Summary The present form of the analysis of high-percentage fluorspar is described. After removal of accompanying carbonates by treatment with dilute acetic acid, the silica is either volatilized with hydrofluoric acid and determined from the loss of weight or precipitated in a separate sample, decomposed with sodium carbonate, as tetrapyridinium dodecamolybdatosilicate and weighed as SiO₂ + 12 MoO₃. After volatilization of silica the remaining residue is fumed with sulphuric acid, boiled with a dilute hydrochloric/sulphuric acid mixture and the barium sulphate determined gravimetrically. In the filtrate of the barium sulphate precipitation the trivalent elements are either separated or masked. Then calcium is precipitated as oxalate and weighed after conversion to the oxide or directly determined by photometric titration with EDTA. After conversion to hydrogensulphide, the small sulphide-sulphur content in fluorspar is bound as cadmium sulphide and determined iodometrically.The error calculation results in the following standard deviations for silica, barium sulphate, and calcium fluoride: ^s SiO₂=0.04%; sBaSO₄=±0,04%; sCaF₂=±0,09%.

     

Direct determination of free methionine in soy-based infant formula

A method was developed for the direct determination of free methionine in soy-based infant formula, with analyte separation and quantitation by reversed-phase liquid chromatography (LC), and UV absorbance at 214 nm, respectively. Sample preparation required only dilution with mobile phase and syringe filtration. Using a 0.02M KH2PO4 mobile phase (pH adjusted to 2.9 with 85% o-phosphoric acid) and 0.7 mL/min flow rate, methionine eluted at approximately 8 min, and total run time was 14 min after column regeneration with acetonitrile-water. System linearity was demonstrated as peak area versus analyte concentration, ranging from 80 to 120% of the formula specification for free methionine (r > 0.999, and all residuals < 0.45%). Intermediate precision relative standard deviation values were < 1.5% for ready-to-feed and reconstituted powder samples, and recoveries ranged from 98.0 to 103.5% for inter-method comparison with an amino acid analyzer method. The limit of quantitation was 3 mg methionine/L in the "as fed" infant formula. Despite the relatively weak UV absorptivity of methionine, the 214 nm signal was sufficiently intense in the 30-65 mg/L (201-436 microM) range to afford quantitation by peak area proportionation versus a 2-point external standard calibration. This direct UV detection after reversed-phase LC separation provides a simple and accurate method for determining free methionine without derivatization.