Thermal stability of a flavoprotein assessed from associative analysis of polarized time-resolved fluorescence spectroscopy

Upon gradually heating a particular mutant of the flavoprotein NADH peroxid ase, it was found from the peculiar time-resolved fluorescence anisotropy p attern of the flavin prosthetic group (FAD) that, at elevated temperature, FAD is released from the tetrameric enzyme. Since in this case a mixture of free and enzyme-bound FAD contributes to the time-dependent fluorescence a nisotropy, its analysis can only be accomplished by an associative fitting model, in which specific fluorescence lifetimes of both species are linked to specific correlation times. In this letter the general approach to the a ssociative polarized fluorescence decay analysis is described. The procedur e can be used for other flavoproteins to determine the temperature at which the onset of thermal denaturation will start, leading to release of the fl avin prosthetic group.