The purification and characterization of phosphonopyruvate hydrolase, a novel carbon-phosphorus bond cleavage enzyme from Variovorax sp. Pal2.

Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell. The native enzyme had a molecular mass of 63 kDa with a subunit mass of 31.2 kDa. Activity of purified phosphonopyruvate hydrolase was Co2+-dependent and showed a pH optimum of 6.7–7.0. The enzyme had a Km of 0.53 mM for its sole substrate, phosphonopyruvate, and was inhibited by the analogues phosphonoformic acid, 3-phosphonopropionic acid, and hydroxymethylphosphonic acid. The nucleotide sequence of the phosphonopyruvate hydrolase structural gene indicated that it is a member of the phosphoenolpyruvate phosphomutase/isocitrate lyase superfamily with 41% identity at the amino acid level to the carbon-to-phosphorus bond-forming enzyme phosphoenolpyruvate phosphomutase from Tetrahymena pyriformis. Thus its apparently ancient evolutionary origins differ from those of each of the two carbon-phosphorus hydrolases that have been reported previously; phosphonoacetaldehyde hydrolase is a member of the haloacetate dehalogenase family, whereas phosphonoacetate hydrolase belongs to the alkaline phosphatase superfamily of zinc-dependent hydrolases. Phosphonopyruvate hydrolase is likely to be of considerable significance in global phosphorus cycling, because phosphonopyruvate is known to be a key intermediate in the formation of all naturally occurring compounds that contain the carbon-phosphorus bond.

Novel methodology for the preparation and purification of oligonucleotides incorporating phosphorothiolate termini

A novel phosphoramidite; N,N-diisopropylamino-2-cyanoethyl-ortho-methylbenzylphosphoramidite 1, was prepared. The reaction of 1 with DMTrT and subsequent derivatisation of the phosphite triester product under solution-phase, Michaelis–Arbuzov conditions was investigated. Coupling of 1 with the terminal hydroxyl groups of support-bound oligodeoxyribonucleotides and subsequent reaction with an activated disulfide yielded oligonucleotides bearing a terminal, phosphorothiolate-linked, lipophilic moiety. The oligomers were readily purified using RP-HPLC. Silver(I)-mediated cleavage of the phosphorothiolate linkage and desalting of the oligonucleotides were performed readily in one step to yield cleanly the corresponding phosphate monester-terminated oligomers.

The isolation and purification of tris-2,2'-bipyridine complexes of ruthenium(II) containing unsymmetrical ligands

Monomeric ruthenium(II) complexes [Ru(L)3]2+ containing unsymmetric bipyridine ligands [Where L = 5-methyl-2,2'-bipyridine (L1), 5-ethyl-2,2'-bipyridine (L2), 5-propyl-2,2'-bipyridine (L3), 5-(2-methylpropyl)-2,2'-bipyridine (L4), 5-(2,2-dimethylpropyl)-2,2'-bipyridine (L5) and 5-(carbomethoxy)-2,2'-bipyridine (L6)] have been studied and the meridional and facial isomers isolated by the use of cation-exchange column chromatography (SP Sephadex C-25) eluting with either sodium toluene-4-sulfonate or sodium hexanoate. The relative yield of the facial isomer was found to decrease with increasing steric bulk, preventing the isolation of fac-[Ru(L5)3]2+. The two isomeric forms were characterized by 1H NMR, with the complexes [Ru(L1-3)3]2+ demonstrating an unusually large coupling between the H6 and H4 protons. Crystals suitable for X-ray structural analysis of [Ru(L1)3]2+ were obtained as a mixture of the meridional and facial isomers, indicating that separation of this isomeric mixture could not be achieved by fractional crystallisation. The optical isomers of the complex [Ru(L3)3]2+ were chromatographically separated on SP Sephadex C-25 relying upon the inherent chirality of the support. It is apparent that chiral interactions can inhibit geometric isomer separation using this technique.

Purification of entangled coherent states

We suggest an entanglement purification scheme for mixed entangled coherent states using 50-50 beam splitters and photodetectors. This scheme is directly applicable for mixed entangled coherent states of the Werner type, and can be useful for general mixed states using additional nonlinear interactions. We apply our scheme to entangled coherent states decohered in a vacuum environment and find the decay time until which they can be purified.

Studies on the immunoglobulin E responses to Teladorsagia circumcincta in sheep: purification of a major high molecular weight allergen.

Studies on the immunoglobulin (Ig)E immune responses to the gastric nematode, Teladorsagia circumcincta, have demonstrated a major high molecular weight allergen (HMWTc). Cross reactive allergens of similar MW were demonstrated for Trichostrongylus colubriformis and Cooperia curticei, but not for Haemonchus contortus. Purification of HMWTc was achieved by gel-filtration chromatography, and nonreducing SDS-PAGE and Western blot analysis revealed two closely associated bands with a molecular weight of approximately 140-150?kDa. Reduction showed four IgE reactive bands of 120, 50, 45 and 30?kDa, and deglycosylation abrogated the immunoreactivity of the 120 and 30?kDa bands. Ultrastructural immunolocalization by electron microscopy revealed that the IgE reactivity was confined to the cuticular surface of the infective (L3) larvae. ELISA studies to determine the IgE anti-HMWTc responses in lambs during their first grazing season, demonstrated significantly higher IgE antibody in lambs with low accumulative faecal egg count (FEC) compared to animals with high accumulative FEC. These studies provide evidence for a protective function of IgE antibody in Teladorsagia infections in lambs.