Polarized Time-Resolved Fluorescence Reveals Enzyme-Specific Anisotropy Dynamics of Bound NADPH

Ioanna A. Gorbunova; Denis A. Volkov; Maxim E. Sasin; Oleg S. Vasyutinskii

doi:10.18287/JBPE26.12.030302

Authors

Ioanna A. Gorbunova Sechenov University
Denis A. Volkov Ioffe Institute
Maxim E. Sasin Ioffe Institute
Oleg S. Vasyutinskii Ioffe Institute

DOI:

https://doi.org/10.18287/JBPE26.12.030302

Keywords:

fluorescence anisotropy, NAD(P)H, TCSPC, fluorescence lifetime, laser spectroscopy

Abstract

We used time-resolved polarized fluorescence spectroscopy for the study of nicotinamide adenine dinucleotide phosphate (NADPH) bound to isocitrate dehydrogenase (IDH) and alcohol dehydrogenase (ADH). It was shown that the fluorescence lifetimes of bound NADPH are strongly enzyme-dependent and are practically identical to those of bound NADH, indicating that fluorescence lifetime imaging microscopy (FLIM) analysis cannot universally distinguish between these two coenzymes. However, in the polarization-sensitive experiments we observed sub-nanosecond fluorescence anisotropy decay components of NADPH bound to IDH and ADH that we found to be significantly enzyme-specific. This result suggests that combined lifetime and anisotropy analysis provides increased molecular specificity for NAD(P)H.

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