Cellular energy ultimately derives form oxidation, with molecular oxygen being the predominate electron acceptor. This research project is directed toward the development of near infrared phosphorescence for determining oxygen concentrations in tissues. The specific goals are: 1. Phosphorescent probe molecules with optimal properties for oxygen measurements in biological systems will be designed. Probes that absorb light and phosphoresce in spectral regions where there is minimal absorption by biological pigments will be evaluated. Probes that remain in the circulatory system and are non-toxic to animals upon injection and/or are rapidly excreted to minimize the possibility of long term toxicity will be selected. Other probes will be designed to be taken up by cells and trapped internally, allowing direct measure of intracellular oxygen concentration. 2. The possible role of oxygen diffusion gradients in mitochondrial respiration will be examined. By comparing the oxygen dependencies of quenching of probes bound to respiring and non-respiring mitochondria, the effective oxygen depletion at the mitochondria can be determined. 3. Trans-plasma membrane oxygen differences will be examined in hepatocytes. To measure intra- and extra-cellular oxygen concentration two oxygen probe molecules will be used, one in the extracellular medium and the other trapped in the cellular cytoplasm. From differences in the oxygen dependence of the two, it will be possible to determine the barrier for oxygen diffusion imposed by the cell membrane and its associated unstirred layer. 4. Instrumentation will be developed for imaging the oxygen distribution in tissue. This will involve real time video imaging of phosphorescence intensity. Data processing will be used to obtain three dimensional images of the oxygen distribution in tissues. Eye lens, a transparent tissue where light scattering is not a problem, will be first examined. Later, rat kidney, a small but highly scattering tissue will be used.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036393-06
Application #
3290278
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1986-04-01
Project End
1994-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
6
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Vanderkooi, J M; Wright, W W; Erecinska, M (1994) Nitric oxide diffusion coefficients in solutions, proteins and membranes determined by phosphorescence. Biochim Biophys Acta 1207:249-54
Papp, S; King, T E; Vanderkooi, J M (1991) Intrinsic tryptophan phosphorescence as a marker of conformation and oxygen diffusion in purified cytochrome oxidase. FEBS Lett 283:113-6
Wilson, D F; Rumsey, W L; Vanderkooi, J M (1989) Oxygen distribution in isolated perfused liver observed by phosphorescence imaging. Adv Exp Med Biol 248:109-15
Vanderkooi, J M; Berger, J W (1989) Excited triplet states used to study biological macromolecules at room temperature. Biochim Biophys Acta 976:1-27
Papp, S; Vanderkooi, J M (1989) Tryptophan phosphorescence at room temperature as a tool to study protein structure and dynamics. Photochem Photobiol 49:775-84
Green, T J; Wilson, D F; Vanderkooi, J M et al. (1988) Phosphorimeters for analysis of decay profiles and real time monitoring of exponential decay and oxygen concentrations. Anal Biochem 174:73-9
Wilson, D F; Rumsey, W L; Green, T J et al. (1988) The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration. J Biol Chem 263:2712-8
Kanagy, C; Vanderkooi, J M; Bonner Jr, W D (1988) Luminescence from the carbon monoxide derivative of Agaricus bispora tyrosinase. Arch Biochem Biophys 267:668-75
Rumsey, W L; Vanderkooi, J M; Wilson, D F (1988) Imaging of phosphorescence: a novel method for measuring oxygen distribution in perfused tissue. Science 241:1649-51
Koloczek, H; Vanderkooi, J M (1987) Domain structural flexibility in rhodanese examined by quenching of a phosphorescent probe. Biochim Biophys Acta 916:236-44

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