Synthesis and characterization of an internal emission standard and applications to fluorescence studies of photosystem II

Fluorescence measurements of photosynthetic organisms and isolated proteins at ambient and low temperature have played an important role in understanding their function. When comparing fluorescence measurements at cryogenic temperatures, the differences in the scattering properties of frozen samples make it difficult to compare the fluorescence intensity of these samples. An internal emission standard can be used to scale the fluorescence intensity, compensating for these differences. We report the synthesis, purification and characterization of a luminescent terbium chelate complex for use as an internal emission standard for the study of photosystem II fluorescence at cryogenic temperatures. The ligand consists of a diethylenetriaminepentaacetic acid derivative where pyrimidine rings sensitize the terbium luminescence, overcoming the inherently low absorption of terbium. The chelated lanthanide remains in solution in the aqueous phase and does not interfere with photosystem II function. By scaling to the terbium emission, the fluorescence intensity of different samples can be readily compared. This chelate complex could also be used as an internal emission standard for studies of other proteins. © 1996 John Wiley & Sons, Inc.