A new species of bound ubisemiquinone anion in QH2: cytochrome c oxidoreductase

Using a combination of EPR and low temperature diffuse reflectance spectroscopy, a new species of semiquinone anion has been detected in QH2:cytochrome c oxidoreductase in submitochondrial particles under conditions of oxidant-induced extra reduction of cytochrome b. In contrast to the previously detected semiquinone anion, this new species is insensitive to antimycin but sensitive to treatment with 2,3-dimercaptopropanol and O2. The two species can easily be distinguished on the basis of their respective EPR properties since they differ in g-value, line width, and microwave power saturation behavior. It is concluded that the two species of semiquinone anion are bound to different domains on QH2:cytochrome c oxidoreductase. The existence of two different semiquinone anions in the enzyme strongly supports a mechanism of electron flow as proposed in the Q-cycle.     

Rapid separation of the chloroplast and cytoplasmic fractions from intact leaf protoplasts

Intact protoplasts are ruptured by rapid centrifugation through a narrow-aperture nylon mesh and the intact chloroplasts are then separated from the cytoplasm by sedimentation through a layer of silicone oil below the mesh. Within 6 to 8 s of starting the centrifuge, 90% of the chloroplasts are separated into the pellet fraction which contains only 10 to 15% contamination by mitochondria and peroxisomes and less than 5% contamination by soluble cytoplasm as judged by the distribution of marker enzymes. This technique should allow determination of the distribution of metabolites between the chloroplast and cytoplasmic compartments of intact protoplasts.     

Growth of an ice phase in frozen tissue studied by proton NMR-spectroscopy

Development of an ice phase in frozen tissue is a severe problem in cryo-ultramicrotomy. It is assumed that the growth-rate of such an ice phase is controlled by the mobility of the water molecules in the tissue. In order to verify this, wide-line nmr-spectroscopy has been performed on the protons of frozen whole blood, with and without protective agent, in the temperature range −100 °C- −40 °C. The molecular motion may be characterized by a correlation time, τ, which can be thought of as roughly the mean time between proton jumps. The value of τ can be approximately evaluated from the width of the proton resonance line. It is shown that τ varies rapidly with temperature, in a similar manner to maximum storage time for frozen blood in blood banks. Addition of glycerol increases τ at low temperature, which is consistent with its use as a protective agent in cryobiology. At high temperature, addition of glycerol reduces τ, which is in agreement with reported light-microscope observations on the growth-rate of an ice phase in the same temperature range, nmr-spectroscopy seems to be a useful tool in the further development of cryo-ultramicrotomy. However, to estimate the value in full, more data is required than is presented here.