Georg Holtermann

Water contributes actively to the rapid crossing of a protein unfolding barrier

The cold-shock protein CspB folds rapidly in a N <= => U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive (Delta V(++)(NU)=16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions.

Effects of cobalt on haem proteins of erythropoietin-producing HepG2 cells in multicellular spheroid culture.

The hypoxia‐induced increase of spectrophotometrically measured light absorption at 560 nm, considered as reduced cytochrome b, in HepG2 cells is diminished after exposure to cobalt chloride (50 or 100 μM) for 18–36h. The redox state of cytochrome c and cytochrome aa 3, however, remains stable, indicating a particular affinity of cytochrome b for cobalt. Erythropoietin production of HepG2 cells increases after application of cobalt chloride, whereas H2O2 production, as measured by the dihydrorhodamine technique, decreases. It is concluded that cobalt stimulates a signal cascade with cytochrome b as receptor and H2O2 as second messenger for regulating erythropoietin production.

Effects of hydrostatic pressure on the conformational equilibrium of tryptophan synthase from Salmonella typhimurium.

A wide range of parameters influence allosteric communications between the α‐ and β‐subunits of the Trp synthase α2β2 multienzyme complex with L‐Ser, including monovalent cations, pH, temperature, ligands, organic solvents, and hydrostatic pressure. The conformational change from closed to open can be monitored either by absorbance at 423 nm or fluorescence at 495 nm from the pyridoxal‐5′‐phosphate‐L‐Ser complex. Pressure perturbation was used to quantify the effects of monovalent cations, ligands, and mutations on the conformational equilibrium of Trp synthase. P‐jump kinetics in the presence of Na+, NH4+, and Na+ together with benzimidazole were also examined. The plots of lnk versus P are nonlinear and require a compressibility (β‡o) term to obtain a good fit. β‡o is positive for the Na+ enzyme but negative for NH4+ and Na+ with benzimidazole. These results suggest that there is a large contribution of solvation to the kinetics of the conformational change of Trp synthase. The relaxation kinetics are also different if the P‐jumps are made by increasing or decreasing pressure, suggesting that the enzyme conformations are ensembles of microstates.

Local determination of oxygenation and cell vitality in multicellular spheroids by microelectrodes and photometry

Multicellular spheroids are nearly spherical aggregates of cultured tumor cells. They develop radial proliferation gradients and are used as models to study radiation as well as drug effects for improving therapeutic strategies. As the knowledge of oxygenation as well as cell vitality in the spheroids is of importance to understand antiproliferative measures methods have to be applied which are able to resolve the three-dimensional architecture of the spheroids without being destructive. Microelectrodes sensitive to oxygen or ph and specifically designed to record extracellularly symmetrical PO2- and pH-gradients give information about the relationship between oxygen consumption and lactate production in different depths of the spheroids. The quotient delta PO2/delta pH can be related to radiation sensitivity and growth rate of the tumor spheroids. Combination of measurements with PO2-sensitive microelectrodes and photometry of the respiratory chain (Cytochromes, NADH, FAD) give information about the critical PO2 in different depths of the spheroids or regulation of oxygen consumption by glucose. After staining of tumor cells in spheroids with vital/letal fluorescence dyes for characterisation of areas with proliferative or necrotic cells fluorescence intensity measurements in depths up to 200 mm can be obtained by confocal laser microscopy. Symmetrical distribution of FDA (Fluoresceindiacetate) showed a decline of viably stained cells in the centre at a diameter of 40–100 mm with an appearance of Lucifer Yellow VS stained cells in the centre, indicating starting necrosis.

Spectrophotometric analysis of heme proteins in oxygen sensing cell systems.

The mechanism by which specialized cells in the body are able to sense the microenvironmental PO2 and to form a corresponding signal for avoiding hypoxic cell damages is not elucidated. There are, however, many indications that part of the oxygen sensing mechanism is brought about by heme proteins. We have, therefore, carried out spectral analysis of carotid body cells as well as hepatoma cells (HepG2) to characterize their inherent heme proteins. Both cell systems are very well known for their capability to transduce changes of the oxygen pressure into a biological signal like the chemoreceptor discharge stimulating respiration or circulation or the hormone erythropoietin stimulating proliferation and maturation of the erythrocytic progenitors in the bone marrow.