Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10 fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.
The Imaging Probe Development Center (IPDC) has been set up under the auspices of the National Institutes of Health (NIH) Roadmap as part of the molecular libraries and imaging initiatives. It comprises a core synthesis facility dedicated to the preparation of imaging probes, initially for intramural NIH scientists, and later, for the extramural scientific community. The facility opened fully in late 2006, in refurbished laboratories in Rockville, Maryland, and a staff of around a dozen was recruited into place by early 2007; the director was hired in late 2005. The IPDC provides a mechanism for the production of sensitive probes for use by imaging scientists who cannot obtain such probes commercially. The probes to be made will encompass all major imaging modalities including radionuclide, magnetic resonance, and optical. The operation of the IPDC is outlined, together with the results of interim achievements while the IPDC maintained a small temporary laboratory in Bethesda. As of December 2006, a total of eleven probe compositions had been made, and several of these are described with particular mention of those probes intended for use in optical applications.
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