Quantitative distinction between bound and free NADH in biological systems

Marina R. Kasimova; Klaas Krab; Jurga Grigiene; Peter E. Andersen; Peter Hagedorn; Henrik K. Flyvbjerg; Ian Max Moller

doi:10.1117/12.527392

1 July 2004 Quantitative distinction between bound and free NADH in biological systems

Marina R. Kasimova, Klaas Krab, Jurga Grigiene, Peter E. Andersen, Peter Hagedorn, Henrik K. Flyvbjerg, Ian Max Moller

Proceedings Volume 5322, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues II; (2004) https://doi.org/10.1117/12.527392
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

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.

Citation Download Citation

Marina R. Kasimova, Klaas Krab, Jurga Grigiene, Peter E. Andersen, Peter Hagedorn, Henrik K. Flyvbjerg, and Ian Max Moller "Quantitative distinction between bound and free NADH in biological systems", Proc. SPIE 5322, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues II, (1 July 2004); https://doi.org/10.1117/12.527392

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available