Tryptophan determination in proteins and feedstuffs by ion exchange chromatography

A chromatographic method was developed for the determination of tryptophan content in food and feed proteins. The method involves separation and quantitation of tryptophan (released from protein by alkaline hydrolysis with NaOH) by isocratic ion-exchange chromatography with O-phthalaldehyde derivatization followed by fluorescence detection. In this procedure, chromatographic separation of the tryptophan and alpha-methyl tryptophan, the internal standard, was complete in 15 min, without any interference from other compounds. The precision of the method was 1-4%, relative standard deviation. Accuracy was validated by agreement with the value for chicken egg white lysozyme, a sequenced protein, and by quantitative recoveries after spiking with lysozyme. The method allows determination in a range of feed proteins, containing varied concentrations of tryptophan, and is applicable to systems used for routine amino acid analysis by ion-exchange chromatography. (C) 2004 Elsevier Ltd. All rights reserved.

Controlled oxidative protein refolding using an ion-exchange column

Column-based refolding of complex and highly disulfide-bonded proteins simplifies protein renaturation at both preparative and process scale by integrating and automating a number of operations commonly used in dilution refolding. Bovine serum albumin (BSA) was used as a model protein for refolding and oxido-shuffling on an ion-exchange column to give a refolding yield of 55 % after 40 Ih incubation. Successful on-column refolding was conducted at protein concentrations of up to 10 mg/ml and refolded protein, purified from misfolded forms, was eluted directly from the column at a concentration of 3 mg/ml. This technique integrates the dithiothreitol removal, refolding, concentration and purification steps, achieving a high level of process simplification and automation, and a significant saving in reagent costs when scaled. Importantly, the current result suggests that it is possible to controllably refold disulfide-bonded proteins using common and inexpensive matrices, and that it is not always necessary to control protein-surface interactions using affinity tags and expensive chromatographic matrices. Moreover, it is possible to strictly control the oxidative refolding environment once denatured protein is bound to the ion-exchange column, thus allowing precisely controlled oxido-shuffling. (c) 2005 Elsevier B.V. All rights reserved.

Use of anion clay hydrotalcite to remove coloured organics from aqueous solutions

Anion clay hydrotalcite sorbents were prepared to investigate their adsorption capabilities in the removal of coloured organic substances from various aqueous systems. Anion clay hydrotalcite was found to be particularly effective at removing negatively charged species. Its excellent uptake levels of anionic species can be accounted for by its high surface area and anion exchange ability. That is, coloured substances can be adsorbed on the surface or enter the interlayer region of the clay by anion exchange. In the adsorption of Acid Blue 29 on the anion clay hydrotalcite, an equilibrium time of 1 h with dye removal exceeding 99% was obtained. The hydrotalcite was found to have an adsorption capacity marginally below that of commercial activated carbon. It should be noted that the spent sorbents can be regenerated easily by heating at 723 K to remove all adsorbed organics. The reused sorbents displayed greater adsorption capabilities than the newly prepared hydrotalcite. Hence, the anion clay hydrotalcite is easily recoverable and reusable such that it is a promising sorbent for environmental and purification purposes. (C) 2002 Elsevier Science B.V. All rights reserved.

Heterogeneity of physico-chemical properties in structured soils and its consequences

Structured soils are characterized by the presence of inter- and intra-aggregate pore systems and aggregates, which show varying chemical, physical, and biological properties depending on the aggregate type and land use system. How far these aspects also affect the ion exchange processes and to what extent the interaction between the carbon distribution and kind of organic substances affect the internal soil strength as well as hydraulic properties like wettability are still under discussion. Thus, the objective of this research was to clarify the effect of soil aggregation on physical and chemical properties of structured soils at two scales: homogenized material and single aggregates. Data obtained by sequentially peeling off soil aggregates layers revealed gradients in the chemical composition from the aggregate surface to the aggregate core. In aggregates from long term untreated forest soils we found lower amounts of carbon in the external layer, while in arable soils the differentiation was not pronounced. However, soil aggregates originating from these sites exhibited a higher concentration of microbial activity in the outer aggregate layer and declined towards the interior. Furthermore, soil depth and the vegetation type affected the wettability. Aggregate strength depended. on water suction and differences in tillage treatments.

Crystallization and preliminary x-ray diffraction analysis of the unliganded human growth hormone receptor

The crystal structure of the extracellular domain of growth hormone receptor complexed to its ligand, growth hormone, has been known since 1992. However, no information exists for the unliganded form of the receptor. The human growth hormone receptor's extracellular ligand-binding domain, encompassing amino-acid residues 1 - 238, has been expressed in Escherichia coli, purified by anion ion-exchange chromatography and crystallized in its unliganded state by the hanging-drop vapour-diffusion method in 100 mM HEPES pH 7.0 containing 27.5%(w/v) PEG 5000 monomethyl ether and 200 mM ammonium sulfate as the co-precipitants. The crystals belong to the othorhombic space group C222(1), have unit-cell parameters a = 99.7, b = 112.2, c = 93.2 Angstrom and diffract to 2.5 Angstrom resolution using synchrotron radiation. The crystal structure will shed light on the nature of any conformation changes that occur upon ligand binding and will provide information to develop potential low-molecular-weight agonists/antagonists to treat clinical diseases in which the growth hormone receptor is implicated.

Purification and partial characterisation of recombinant human hepcidin

Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays. (C) 2005 Elsevier SAS. All rights reserved.