Kinetically controlled refolding of a heat denatured hyperthermostable protein
Koutsopoulos, S. ; Oost, J. van der; Norde, W. - \ 2007
FEBS Journal 274 (2007)22. - ISSN 1742-464X - p. 5915 - 5923.
archaeon pyrococcus-furiosus - crystal-structure - thermodynamic properties - thermal-denaturation - citrate synthase - stability - temperature - enzyme - water - dehydrogenase
The thermal denaturation of endo-ß-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144¿°C, corresponding to protein denaturation and complete unfolding, respectively, as shown by circular dichroism and fluorescence spectroscopy data. Calorimetric studies also showed that the denatured state did not refold to the native state unless the cooling temperature rate was very slow. Furthermore, previously denatured protein samples gave well-resolved denaturation transition peaks and showed enzymatic activity after 3 and 9¿months of storage, indicating slow refolding to the native conformation over time.
Lutetium speciation and toxicity in a microbial bioassay: Testing the free-ion model for lanthanides
Weltje, L. ; Verhoof, L.R.C.W. ; Verweij, W. ; Hamers, T.H.M. - \ 2004
Environmental Science and Technology 38 (2004)24. - ISSN 0013-936X - p. 6597 - 6604.
rare-earth-elements - metal toxicity - thermodynamic properties - stability-constants - complexes - water - seawater - copper - assay - bioavailability
The validity of the free-ion model (FIM) for the element lutetium (Lu), a member of the lanthanides, was assessed in experiments with the bacterium Vibrio fischeri. The FIM is mainly based on divalent metals and synthetic ligands and has not yet been validated for the trivalent lanthanides. The bioluminescence response of V fischeri was studied at different Lu concentrations in the presence and absence of natural and synthetic organic ligands [citrate, malate, oxalate, acetate, ethylenediaminetetraacetate (EDTA), and nitrilotriacetate (NTA)]. All ligands were tested separately to ensure that their concentrations would not cause adverse effects themselves. Free Lu3+ concentrations were calculated with a speciation program, after extension of its database with the relevant Lu equilibria. The results confirmed the FIM for Lu: that is, in contrast to total dissolved Lu concentrations, free Lu3+ concentrations had an apparent relationship with the response of V fischeri. However, a contribution of minor inorganic Lu complexes cannot be ruled out. In the presence of malate and oxalate, the EC50 for Lu3+ decreased faster in time than for the other ligands, indicating lower elimination rates. With an EC50 Of 1.57 muM, Lu3+ is more toxic than La3+, Cd2+, or Zn2+ and approximately equally as toxic as Cu2+. Although the pH increased slightly during the experiments, it was shown that the influence of pH on Lu speciation was limited.