Crystallization of Arthrobacter sp. strain 1C N-(1-D-carboxyethyl)-L-norvaline dehydrogenase and its complex with NAD+

The novel NAD+-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 ? resolution. X-ray precession photographs have established that the crystals belong to space group P21212, with cell parameters a = 104.9, b = 80.0, c = 45.5 ? and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.     

Meerwein reaction of phosphonium ions with epoxides and thioepoxides in the gas phase

Phosphonium ions are shown to undergo a gas-phase Meerwein reaction in which epoxides (or thioepoxides) undergo three-to-five-membered ring expansion to yield dioxaphospholanium (or oxathiophospholanium) ion products. When the association reaction is followed by collision-induced dissociation (CID), the oxirane (or thiirane) is eliminated, making this ion molecule reaction/CID sequence a good method of net oxygen-by-sulfur replacement in the phosphonium ions. This replacement results in a characteristic mass shift of 16 units and provides evidence for the cyclic nature of the gas-phase Meerwein product ions, while improving selectivity for phosphonium ion detection. This reaction sequence also constitutes a gas-phase route to convert phosphonium ions into their sulfur analogs. Phosphonium and related ions are important targets since they are commonly and readily formed in mass spectrometric analysis upon dissociative electron ionization of organophosphorous esters. The Meerwein reaction should provide a new and very useful method of recognizing compounds that yield these ions, which includes a number of chemical warfare agents. The Meerwein reaction proceeds by phosphonium ion addition to the sulfur or oxygen center, followed by intramolecular nucleophilic attack with ring expansion to yield the 1,3,2-dioxaphospholanium or 1,3,2-oxathiophospholanium ion. Product ion structures were investigated by CID tandem mass spectrometry (MS(2)) experiments and corroborated by DFT/HF calculations.     

Intercalation of Toluidines into α-Zirconium Hydrogenphosphate

Intercalates of o-, m-, and p-toluidine into α-Zr(HPO₄)₂ · H₂O were prepared and characterized by powder X-ray diffraction, thermogravimetric analysis and infrared spectroscopy. As follows from IR, toludine molecules are protonated in the interlayer space. Toluidine molecules are arranged in a bimolecular way in the intercalates containing more than 1.5 toluidine molecules per Zr atom. On the other hand, a monolayer of the toluidine molecules is supposed in the intercalates with less than one toluidine molecule per Zr atom.     

Liquid chromatographic determination of urea in water-soluble urea-formaldehyde fertilizer products and in aqueous urea solutions: collaborative study

Water soluble urea-formaldehyde (UF) fertilizers, manufactured by complex reaction of urea and formaldehyde, typically contain varying amounts of unreacted urea. A liquid chromatography method for the analysis of urea in these products, and in aqueous urea solutions, was collaboratively studied. An amine chromatography column was used to separate the unreacted urea from numerous UF reaction products present in these liquid fertilizers. Unreacted urea was determined by using external urea standards with UV detection at 195 nm. The standards and test samples were prepared in the mobile phase of 85% (v/v) acetonitrile in water. Ten laboratories analyzed 5 different UF-based commercial products containing unreacted urea in the range of 6 to 17% by weight, and 5 different concentrations of urea in water equivalent to commercial products of that nature. The aqueous urea solutions contained 2-20% urea (w/w). The range of s(R) values for the 5 UF-based commercial fertilizers was 0.49-1.02 and the %RSD(R) was 1.94-6.14. The s(R) range for the 5 urea solutions was 0.10 to 0.79 and the %RSD(R) range was 2.54 to 4.88. The average recovery of urea from the aqueous urea solutions was 96-103%. Therefore, this method is capable of monitoring urea nitrogen manufacturers' label claims and total nitrogen claims in those cases where urea is the sole source of plant food nitrogen. Based on the collaborative study data, the authors recommend this method be approved for AOAC Official First Action status.     

Reversed-phase liquid chromatographic determination of riboflavin in feeds

A method for determination of riboflavin in animal feeds using liquid chromatography (LC) was developed for feed samples fortified with riboflavin at 1 mg/lb or greater (up to 10,000 mg/lb). Feed samples were extracted in 0.1 N HCl with heating on a steam bath for 30 min, followed immediately by mechanical shaking for 30 min. Sample extracts were diluted to target volume with 2% acetic acid and filtered; riboflavin was determined by LC on a reversed-phase C18 column with 2% acetic acid-acetonitrile (85 + 15) mobile phase for separation and fluorescence detection with excitation at 460 nm and emission at 530 nm. The extraction was compared with that of the AOAC Official Method for riboflavin in food and feed premixes. The 2 method extractions were not significantly different from each other at the 95% confidence level. The developed method also had good linearity over 4 orders of magnitude, recovery of 95-99% from spiked feed samples, a limit of detection of riboflavin at 0.00034 microg/mL in solution, a limit of quantitation of 0.023 mg/lb in feed, and good ruggedness.     

Single-sided membrane introduction mass spectrometry for on-line determination of semi-volatile organic compounds in air.

Construction, optimization, and testing of a novel single-sided configuration for a semi-permeable [poly(dimethylsiloxane); PDMS] membrane introduction system for mass spectrometry is described. On-line detection of semi-volatile organic compounds of environmental interest is shown, including lindane (a pesticide), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (an explosive), butylated hydroxytoluene (BHT) (an antioxidant), 1,2-dichlorobenzene, dimethylmethyl phosphonate (DMMP) (a chemical warfare agent simulant) and naphthalene. The technique has limits of detection in the sub-ppb range. with rise times of 4 to 7 s and fall times of 12 to 36 s and a response that is linear over 4 orders of magnitude (from 0.1 ppb to 1000 ppb for DMMP). The cycle time, from crude air sampling to acquisition of results, is approximately 1 min. No sample preparation is necessary.     

The free energy of a reaction coordinate at multiple constraints: a concise formulation

The free energy as a function of the reaction coordinate (rc) is the key quantity for the computation of equilibrium and kinetic quantities. When it is considered as the potential of mean force, the problem is the calculation of the mean force for given values of the rc. We reinvestigate the PMCF (potential of mean constraint force) method which applies a constraint to the rc to compute the mean force as the mean negative constraint force and a metric tensor correction. The latter allows for the constraint imposed to the rc and possible artefacts due to multiple constraints of other variables which for practical reasons are often used in numerical simulations. Two main results are obtained that are of theoretical and practical interest. First, the correction term is given a very concise and simple shape which facilitates its interpretation and evaluation. Secondly, a theorem describes various rcs and possible combinations with constraints that can be used without introducing any correction to the constraint force. The results facilitate the computation of free energy by molecular dynamics simulations.     

External interface for trap-and-release membrane introduction mass spectrometry applied to the detection of inorganic chloramines and chlorobenzenes in water.

Construction and evaluation of an external configuration trap-and-release membrane introduction system for mass spectrometry is described. This novel interface allows independent control of the temperature of the membrane and eliminates the dependence of membrane heating efficiency on its position in the ion source. The external trap-and-release MIMS configuration is successfully applied to detection of inorganic chloramines and chlorobenzenes. The method is shown to give temporal resolution of volatile vs. semi-volatile compounds, which increases its sensitivity for semi-volatiles in the presence of volatiles and provides an additional selectivity parameter. Further selectivity is provided by tandem mass spectrometry.     

A robust method to quantify low molecular weight contaminants in heparin: detection of tris(2-n-butoxyethyl) phosphate

Recently, oversulfated chondroitin sulfate (OSCS) was identified in contaminated heparin preparations, which were linked to several adverse clinical events and deaths. Orthogonal analytical techniques, namely nuclear magnetic resonance (NMR) and capillary electrophoresis (CE), have since been applied by several authors for the evaluation of heparin purity and safety. NMR identification and quantification of residual solvents and non-volatile low molecular contaminants with USP acceptance levels of toxicity was achieved 40-fold faster than the traditional GC-headspace technique, which takes ~120 min against ~3 min to obtain a (1)H NMR spectrum with a signal/noise ratio of at least 1000/1. The procedure allowed detection of Class 1 residual solvents at 2 ppm and quantification was possible above 10 ppm. 2D NMR techniques (edited-HSQC (1)H/(13)C) permitted visualization of otherwise masked EDTA signals at 3.68/59.7 ppm and 3.34/53.5 ppm, which may be overlapping mononuclear heparin signals, or those of ethanol and methanol. Detailed NMR and ESI-MS/MS studies revealed a hitherto unknown contaminant, tris(2-n-butoxyethyl) phosphate (TBEP), which has potential health risks.