- 1The ‘blue-shift’ of the fluorescence maximum of NAD(P)H bound to many proteins offers a means of studying coenzyme binding in whole cells. In earlier work there was a discrepancy between the fluorescence maximum seen in whole tissues (liver, kidney, heart-maximum at 460–470 nm, characteristic of NAD(P)H in solution) and in mitochondria isolated from them (maximum at 440 nm).
- 2Study of the fluorescence-emission spectra and absolute absorption spectra of some types of isolated cell suspensions and tissue slices, now suggests that a blue shift of NAD(P)H fluorescence in cells may be disguised by an inner-absorption effect, since there is a marked change of absorbance from 410 to 460 nm.
- 3The change of absorbance between 410 and 460 nm, and hence the distorting effect of inner absorption, varies considerably in different cell types.
- 4The influence of inner-absorption on the fluoresence also varies with the concentration of cell suspensions, or thickness of slices; working at low cell concentrations, or extrapolation to zero thickness of slices, allows estimation of the wavelength of maximal NAD(P)H fluorescence, undistorted by inner-absorption.
- 5Undistorted fluorescence maxima were thus estimated to be at approximately 440 nm for suspensions of bakers' yeast and rat-liver cells, and for liver slices. Maxima close to 460 nm were given by Ehrlich tumor cells and slices of hepatoma 3924 A. Fluorescence of NAD(P)H in cytosolic and mitochondrial compartments can be distinguished with appropriate substrates and inhibitors. In a given cell type, the fluorescence maximum in the two compartments was indistinguishable.
- 6The difference between the fluorescence maxima of NAD(P)H in liver and hepatoma 3924 A is considered in the light of the known differences of dehydrogenase enzyme activity in the two tissues.