Isolation of a peptide ligand for affinity purification of factor VIII using phage display

Polypeptides for use in affinity chromatography of factor VIII were identified using phage display technology. Phage libraries were designed to express polypeptide fusions containing five to seven residues flanked by two cysteines that form a disulfide bond. Individual bacteriophage were selected for the ability of these polypeptides to bind factor VIII, and then release the protein under mild elution conditions. Strong consensus sequences were observed that appear to be necessary for this reversible interaction. Chemically synthesized ligands identified by this screening were immobilized onto a chromatographic support and used for affinity purification of factor VIII from a complex feedstream. A chromatographic step was developed that provided a 10000-fold reduction in host cell proteins and DNA, while providing exceptional product recovery.     

Cover Picture

The cover picture shows in the background the whole cell of a methanotrophic bacterium on which are superimposed components of methane monooxygenase (the structure of the hydroxylase component (top), one of the two four-helix bundles that house the catalytic diiron centers (left)) and a schematic diagram of the catalytic cycle by which the enzyme converts dioxygen and methane into methanol and water. More about this unusual enzyme system is reported by Lippard et al. on p. 2782 ff.     

Enzymatic synthesis of neopentylpolyol esters in organic media

Utilization of lipases for synthesis of esters of hydrophilic polyols has been investigated. The choice of a suitable solvent is crucial in this type of reaction. An interesting case is fatty acid esters from neopentylpolyols, such as trimethylolpropane, which are of great interest as high temperature lubricants. Enzymatic synthesis of trimethylolpropane tricaprylate was studied as an alternative to chemical manufacturing. Triester production occurred only if the water produced by esterification was continuously removed from the medium. In these condition, kinetics of appearance and transformation of mono-, di- and triesters were determined in order to define optimal conditions.     

Dilute sulfuric acid pretreatment of corn stover at high solids concentrations

Applied Biochemistry and Biotechnology - The enzymatic digestibility of a pretreated substrate is enhanced by increasing the severity of the pretreatment conditions, apparently because a greater...     

Use of participant EQA results to assess sample homogeneity and stability for qualitative analytes

International requirements for PT and EQA state that providers must demonstrate that samples are homogeneous and stable. However, testing for homogeneity and stability can be expensive, use samples that could otherwise serve as quality-control materials, and can also fail to detect significant inhomogeneity and instability. In some situations it may be preferable to use the results from participants to identify problems with sample lots, if historic results follow predictable distributions and a statistical decision rule can be produced. An unusually high proportion of incorrect results may indicate that samples were inhomogeneous or unstable. Conditions under which this can be demonstrated are discussed, and the efficacy of the proposal is demonstrated with two examples. This procedure is especially effective when there are a large number of participants and/or a historic small proportion of incorrect results. Providers who adopt this proposal will need to retain samples for testing and assume the risk of distributing bad samples.Presented at the Eurachem PT Workshop September 2005, Portorož, Slovenia     

Convenient and High-Yielding Preparations of Mono(5-carboxy-2-ethylpentyl) Phthalate and its Ring-Deuterated Isomer – The “Third” Major Metabolite of Bis(2-ethylhexyl) Phthalate

The synthesis of an oxidative major metabolite of bis(2-ethylhexyl) phthalate is described. The target molecule and its ring-deuterated isomer were obtained via acylation of the appropriate -hydroxy benzyl ester or the corresponding carboxylate with phthalic anhydride or phthalic anhydride-d₄. All transformation steps proceed with high yields.     

Molecular Ionics of Anion Receptor Molecules: A Microcalorimetric Study

The coordination of divalent and monovalent inorganic anions to synthetic polyammonium receptors is investigated in aqueous solution around neutral pH by titration calorimetry and NMR spectroscopy. High-affinity 1:1 complexes are formed by a pyrrole type cryptand (1) with sulfate and phosphate, characterized by association constants of almost 107 M^-1. Affinities close to 105 M^-1 are found for polyazacryptands (3 and 4) exhibiting F^-/Cl^- selectivity. The binding affinities and the anion selectivities are mainly caused by the charges of ligands and anions, which is discussed on the basis of simple calculations of the electrostatic contribution to the anion/receptor interactions. The binding of all investigated anions is exothermic at 298.2 K. The contribution of the large negative ΔH values to the free energy of anion binding of the pyrrole type ligand is partially compensated by marked negative ΔS values. These unfavorable entropic contributions are attributed to the additional inclusion of water molecules in the anion/receptor complexes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stable attachment of redox groups for modified electrodes via cyanuric chloride

Methylaminopropylviologen (MAV) was covalently attached to glassy carbon electrodes via cyuranic chloride. X-ray photoelectron spectroscopy (XPS) analysis confirmed the presence of bound MAV on the electrode surfaces. Electrochemical experiments of these electrodes indicated that the bound MAV was stable and electrochemically active for extended periods of time in aqueous media. The surface coverage of MAV was in the range, of 2.0–3.0×10^?10 mol cm^?2.     

Rotational contour analysis of the electronic origin band in the emission spectrum of the orthoxylyl radical at 4683 Å

The rotational contour of the 4683 Å emission band of the o-xylyl radical was studied at high resolution. Calculations of the rotational contour of this hybrid band were made in the rigid rotor approximation for various sets of values of the excited state rotational constants and directions of the transition moment μ. Matching of computed and experimental rotational features showed that μ is oriented at +37° or ?37° with respect to the b inertial axis. The nature of the excited states of o-xylyl and the methyl-to-ring interaction are discussed with respect to these two possible assignments