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Proceedings Paper

Quantitative distinction between bound and free NADH in biological systems
Author(s): Marina R. Kasimova; Klaas Krab; Jurga Grigiene; Peter E. Andersen; Peter Hagedorn; Henrik K. Flyvbjerg; Ian Max Moller
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Paper Abstract

Without exceptions, in all living cells NADH is a key metabolite linking a large number of metabolic pathways. Flux rates through such pathways are an essential component in the understanding of the functioning of living cells. Knowledge about the way these fluxes depend on the concentrations of the metabolites involved (including NADH/NAD+) allows calculation of these fluxes. Therefore, a method to determine the concentration of free NADH is necessary. A distinction between the free and protein-bound NADH can be made on the basis of fluorescence emission spectra and fluorescence lifetimes. A method for such measurements using a microscopic set-up for time-gated fluorescence spectroscopy has been introduced by Schneckenburger and co-workers (Paul RJ, Schneckenburger H. Naturwissenschaften 83, pp. 32-35, 1996). We further improve this method by first characterizing NADH binding to model proteins by isothermal titration calorimetry and fluorescence. This allows a precise calculation of bound and free NADH and their respective spectra. An analysis of experimental data is advanced by applying two-component deconvolution and subsequent fitting. Using this method we can detect a significant proportion of free NADH in isolated potato tuber mitochondria respiring malate. Taken together these improvements allow a more accurate characterization of the NADH turnover in biological systems.

Paper Details

Date Published: 1 July 2004
PDF: 10 pages
Proc. SPIE 5322, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues II, (1 July 2004); doi: 10.1117/12.527392
Show Author Affiliations
Marina R. Kasimova, Riso National Lab. (Denmark)
Klaas Krab, Vrije Univ. Amsterdam (Netherlands)
Jurga Grigiene, Kaunas Univ. of Medicine (Lithuania)
Peter E. Andersen, Riso National Lab. (Denmark)
Peter Hagedorn, Riso National Lab. (Denmark)
Henrik K. Flyvbjerg, Riso National Lab. (Denmark)
Ian Max Moller, Riso National Lab. (Denmark)

Published in SPIE Proceedings Vol. 5322:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues II
Dan V. Nicolau; Joerg Enderlein; Robert C. Leif; Daniel L. Farkas, Editor(s)

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