The purpose of this research is to develop methodology that enchances the usefulness of nitroxide radical spin-labels as probes of structure and function of biological systems using electron paramagnetic resonance (EPR) spectroscopy. The proposal contains two main subject areas: saturation transfer spectroscopy and spin-label oximetry, these being the subjects of greatest significane in research under this grant in the preceding eight years. In saturation transfer, five new experimental methods are proposed - with intensive applications to measurements of motion and transport in liposomal and cellular membranes. A particularly novel experiment involves electron-electron double resonance (ELDOR) between 14N and 15N substituted spin labels, thereby permitting a direct measurement of the collision frequency between labels in different parts of the membrane. Many of the saturation transfer experiments involve time domain or pulse EPR. The second subject area, spin-label oximetry, is primarily concerned with studies of cellular respiration based on measurement of collisions between oxygen and spin-labels. Experiments are proposed to determine the role of diffusion limitation in respiration, which is thought to be critical in oxygen depleted regions of solid tumors. Also, for the first time it is believed, a way has been found to measure the enthalpy and entropy of repiration -- fundamental thermodynamic quantities that contribute to our understanding of the respiratory process at the most basic level. Related grants held by the P.I.: National Biomedial ESR Center (RR01008) and Development of Biomedical ESR Instrumentation (GM27665) provide funds for the development of new ESR instruments used in some of the research of the present proposal and make all instrumental and methodological advances available at a national level.
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