In this project, we apply various techniques of electron paramagnetic resonance (EPR) to studying biological systems, and also attempt to develop a new mode of applications. Results of collaborative efforts in 1987 include: (a) EPR spectra of ferricytochrome c-cardiolipin complex were analyzed to confirm Fe(III) valence state of cytochrome c in the complex, and to derive the extent of crystal field distortion around iron owing to the perturbation by cardiolipin; (b) EPR studies of a vitamin E oxidative dimer, related to gamma tocopherol, demonstrated that the dimer -O- bond undergoes at room temperature a valence tautomerism with free radical fluxional structures; (c) Germination process of c. albicans was monitored by spin label method regarding changes in membrane fluidity. Solutions to several problems facing labeling of the microbe have been worked out such as, e.g., protoplasting prior to labeling; (d) spin label studies of blood cells from canine with galactosemic cataracts have shown that the generally held notion of a decreased red cell deformability accompanying galactosemia is not supported by the EPR measurements of intracellular viscosity and flow characteristics. In our own project, a new flow EPR technique developed in this project for assessing the degree of cell deformation and orientation in flow was applied to examine the effect of hypotonic treatment of resealed ghost, which is known to cause a shift of spectrin tetramer-dimer equilibrium toward the dimers. The deformability and orientability was found dramatically decreased by such a treatment when compared with those isotonically resealed. Investigation of the motional state in cytoskeletal network by maleimide spin labeling showed an increased degree of segmental freedom in the hypotinc preparations having an altered spectrin association state, as proven also by PAGE of spectrin extracts. The results can be interpreted as evidence for a crucial role played by the spectrin network in determining red cell rheological properties.
