Calcium-induced aggregation of bovine caseins: effect of phosphate and citrate

The formation of colloidal particles by Ca²⁺ precipitation of whole caseinates in the presence of phosphate (Pi), citrate (Cit), or both of the anions in concentrations found to be effective in previous works was followed comparing the colloidal particle size and the ionic and proteic composition of the precipitates obtained. Ca²⁺ was incorporated to the precipitate and colloidal particles in a different way than Pi, differences which were related to the presence of Pi and/or Cit in the media. A sequential salting-out process due to progressive Ca²⁺ binding to at least two kinds of sites was observed. The precipitation curves were fitted, and the affinity constants and binding site numbers were calculated with a modification of the Farrell’s equation based on the concept of Wyman’s linked functions. Precipitates obtained at low total Ca²⁺ concentrations in different conditions varied their casein composition. Colloidal particles appeared at the beginning of the second salting-out step, in different amount, and in average size according to the presence or absence of Pi and/or Cit in the media. Consideration of these differences showed that Cit favored the formation of bigger colloidal particles, acting especially in the first steps of the casein aggregation and conditioning the mechanism of this process.     

Profiling of free and conjugated [3H]zeranol metabolites in pig plasma

Extraction and high-performance liquid chromatographic (HPLC) procedures are described that permit the complete analysis of free and conjugated zeranol metabolites in plasma from pigs implanted with [3H]zeranol. Free metabolites (9.0%) are extracted and then analysed by radio-HPLC on a reversed phase C18 column. They are distributed between three compounds that have been identified by gas chromatography-mass spectrometry as taleranol, zeranol and zeralanone. Direct radio-HPLC of the pre-extracted, deproteinated and Sep-Pak C18-purified plasma on a reversed-phase C18 column using tetrabutylammonium as an ion-pairing agent showed four main peaks: one corresponds to a weakly retained unidentified compound(s) (20%) and the other three were identified as the taleranol, zeranol and zeralanone glucuro conjugates. However, the total recovery is only about 25% owing to strong affinity of this polar material for the plasma proteins. Enzymatic deconjugation of the pre-extracted plasma followed by radio-HPLC analysis of the freed metabolites led to a good recovery of the radioactivity (81.8%) and allowed the quantitation of the different metabolites. These preliminary results indicate that zeranol is metabolized in the pig following pathways similar to those in other tested species.     

PM3-compatible zinc parameters optimized for metalloenzyme active sites

Recent studies have shown that semiempirical methods (e.g., PM3 and AM1) for zinc-containing compounds are unreliable for modeling structures containing zinc ions with ligand environments similar to those observed in zinc metalloenzymes. To correct these deficiencies a reparameterization of zinc at the PM3 level was undertaken. In this effort we included frequency corrected B3LYP/6-311G* zinc metalloenzyme ligand environments along with previously utilized experimental data. Average errors for the heats of formation have been reduced from 46.9 kcal/mol (PM3) to 14.2 kcal/mol for this new parameter set, termed ZnB for "Zinc, Biological." In addition, the new parameter sets predict geometries for the Bacillus fragilis active site model and other zinc metalloenzyme mimics that are qualitatively in agreement with high-level ab initio results, something existing parameter sets failed to do.     

Challenges encountered in extending the sensitivity of US Environmental Protection Agency Method 314.0 for perchlorate in drinking water

Concerns about the potential adverse health effects of perchlorate at concentrations below the minimum reporting level (MRL) of US Environmental Protection Agency (EPA) Method 314.0 (generally recognized as 4.0 microg/l) have led to an interest in increasing the sensitivity of the method. This work describes the use of 2 mm columns with a large-loop direct injection method, a column concentration technique and this concentration technique with a background reduction step, to increase the sensitivity for the analysis of trace levels of perchlorate in high ionic strength matrices. The concentrator columns studied were the Dionex TAC LP-1 and a new Dionex high capacity Cryptand concentrator column. The use of a surrogate to monitor trapping efficiency for the concentration technique and the use of confirmational columns to minimize the potential for false positives are also discussed. The large-loop direct injection method and the column concentration methods provided acceptable data when the samples were pre-treated with solid phase pretreatment cartridges. The background reduction technique did not provide acceptable data with either of the concentrator columns evaluated.     

Franck-Condon Factors and R-Centroids for ScF,YF, and LaF Including Rotation-Vibration Effect

We have calculated Franck-Condon factors and R-centroids for several singlet systems of ScF, YF, and LaF, taking into account the effect of rotation-vibration interaction. It has been found that this effect is far from being negligible for several cases.     

A new polymorph of bis[2,6‐bis(1H‐benzimidazol‐2‐yl‐κN3)pyridinido‐κN]zinc(II)

The title compound, [Zn(C₁₉H₁₂N₅)₂], crystallizes in the tetragonal space group P4₃2₁2, with the monomer residing on a twofold axis. The imidazole N‐bound H atoms are disordered over the two positions, with refined occupancies of 0.59 (3) and 0.41 (3). The strong similarities to, and slight differences from, a reported P4₂2₁2 polymorph which has a 50% smaller unit‐cell volume [Harvey, Baggio, Muñoz & Baggio (2003). Acta Cryst. C 59 , m283–m285], to which the present structure bears a group–subgroup relationship, are discussed.     

Nitrogen ylide-mediated cyclopropanation of lactams and lactones

Cyclopropanation of α,β-unsaturated δ-lactams and δ-lactones mediated by nitrogen ylides is described. The process tolerates different alkyl halides and gives efficiently bicyclo[4.1.0]heptanes in a totally stereoselective manner. On the other hand, ε-lactams under our experimental conditions suffer a novel process involving a skeletal reorganization to give a bicyclic[3.3.0] system.     

Insights into the acylation mechanism of class A beta-lactamases from molecular dynamics simulations of the TEM-1 enzyme complexed with benzylpenicillin

Herein, we present results from molecular dynamics MD simulations ( approximately 1 ns) of the TEM-1 beta-lactamase in aqueous solution. Both the free form of the enzyme and its complex with benzylpenicillin were studied. During the simulation of the free enzyme, the conformation of the Omega loop and the interresidue contacts defining the complex H-bond network in the active site were quite stable. Most interestingly, the water molecule connecting Glu166 and Ser70 does not exchange with bulk solvent, emphasizing its structural and catalytic relevance. In the presence of the substrate, Ser130, Ser235, and Arg244 directly interact with the beta-lactam carboxylate via H-bonds, whereas the Lys234 ammonium group has only an electrostatic influence. These interactions together with other specific contacts result in a very short distance ( approximately 3 A) between the attacking hydroxyl group of Ser70 and the beta-lactam ring carbonyl group, which is a favorable orientation for nucleophilic attack. Our simulations also gave insight into the possible pathways for proton abstraction from the Ser70 hydroxyl group. We propose that either the Glu166 carboxylate-Wat1 or the substrate carboxylate-Ser130 moieties could abstract a proton from the nucleophilic Ser70.