Biomedical subjects

Peter Westh

Publications and source records attributed to Peter Westh.

22 records · Page 2Linked to original sources

PubMed2003

Isothermal titration calorimetric procedure to determine protein-metal ion binding parameters in the presence of excess metal ion or chelator.

Determination of binding parameters for metal ion binding to proteins usually requires preceding steps to remove protein-bound metal ions. Removal of bound metal ions from protein is often associated with decreased stability and inactivation. We present two simple isothermal titration calorimetric procedures that eliminate separate metal ion removal steps and directly monitor the exchange of metal ions between buffer, protein, and chelator. The concept is to add either excess chelator or metal ion to the protein under investigation and subsequently titrate with metal ion or chelator, respectively. It is thereby possible in the same experimental trial to obtain both chelator-metal ion and protein-metal ion binding parameters due to the different thermodynamic "fingerprints" of chelator and protein. The binding models and regression routines necessary to analyze the corresponding binding isotherms have been constructed. Verifications of the models have been done by titrations of mixtures of calcium chelators (BAPTA, HEDTA, and EGTA) and calcium ions and they were both able to account satisfactorily for the observed binding isotherms. Therefore, it was possible to determine stoichiometric and thermodynamic binding parameters. In addition, the concept has been tested on a recombinant alpha-amylase from Bacillus halmapalus where it proved to be a consistent procedure to obtain calcium binding parameters.

Bacillus↗

PubMed2003

Unilamellar DMPC vesicles in aqueous glycerol: preferential interactions and thermochemistry.

Glycerol is accumulated in response to environmental stresses in a diverse range of organisms. Understanding of favorable in vivo effects of this solute requires insight into its interactions with biological macromolecules, and one access to this information is the quantification of so-called preferential interactions in glycerol-biopolymer solutions. For model membrane systems, preferential interactions have been discussed, but not directly measured. Hence, we have applied a new differential vapor pressure equipment to quantify the isoosmotic preferential binding parameter, Gamma( micro 1), for systems of unilamellar vesicles of DMPC in aqueous glycerol. It is found that Gamma( micro 1) decreases linearly with the glycerol concentration with a slope of -0.14 +/- 0.014 per molal. This implies that glycerol is preferentially excluded from the membrane-solvent interface. Calorimetric investigations of the same systems showed that the glycerol-DMPC interactions are weakly endothermic, and the temperature of the main phase transition increases slightly (0.16 degrees C per molal) with the glycerol concentration. The results are discussed with respect to a molecular picture which takes into account both the partitioning of glycerol into the membrane and the preferential exclusion from the hydration layer, and it is concluded that the latter effect contributes about four times stronger than the former to the net interaction.

Calorimetry, Differential Scanning↗

PubMed2003

Solvent accessible surface area (ASA) of simulated phospholipid membranes.

The membrane-solvent interface has been investigated through calculations of the solvent accessible surface area (ASA) for simulated membranes of DPPC and POPE. For DPPC at 52 degrees C we found an ASA of 126+/-8 A(2) per lipid molecule, equivalent to twice the projected lateral area. The ASA was dominated by the contribution from the quaternary ammonium ion which accounts for about two thirds of the total value. The remaining ASA is primarily the phosphate moiety (22+/-1 A(2)), and this contribution almost entirely represents the two 'out-of-chain' oxygen atoms. Interestingly, even the most exposed parts of the PC head-group show average ASAs of less than half of its maximal or 'fully hydrated' value. The average ASA of a simulated POPE membrane was 96+/-7 A(2) per lipid. The smaller value than for DPPC reflects much lower ASA of the ammonium ion, which is partially compensated by increased exposure of the ethylene and phosphate moieties. The ASA of the polar moieties of (PO(4), NH(3) and COO) constitutes 65% of the total accessible area for POPE, making this interface more polar than that of DPPC. It is suggested that ASA information can be valuable in attempts to rationalize experimental investigations, particularly molecular interpretations of thermodynamic information.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

A differential vapor-pressure equipment for investigations of biopolymer interactions.

The design and performance of an equipment for the measurement of vapor pressures over liquid or solid samples is presented. The equilibrium pressure difference, DeltaP, between a sample and a reference of known vapor pressure is recorded as a function of composition and/or temperature. Through the use of high-accuracy capacitance manometers and a leak-tight system of stainless steel pipes, below-sealed valves and metal-gasket fittings, DeltaP can be measured with a resolution of about 0.5 micro bar (0.05 Pa) in some applications. This sensitivity level, along with other features of the equipment, particularly a "gas-phase titration" routine for changing the cell composition, makes it effective for the investigations of several types of biopolymer interactions. These include isothermal studies of net affinities such as the adsorption of water to proteins or membranes, the preferential interaction of biopolymers with the components of a mixed solvent, the partitioning of solutes between a membrane and the aqueous bulk and the weak, specific binding of ligands to macromolecules. Furthermore, a temperature-scanning mode allows real-time elucidation of such interactions at thermally induced conformational changes in biopolymers. Selected examples of these applications are presented and discussed.

1-Butanol↗