SYNTHESIS AND CHARACTERIZATION OF AN INTERNAL EMISSION STANDARD AND APPLICATIONS TO FLUORESCENCE STUDIES OF PHOTOSYSTEM-II

Fluorescence measurements of photosynthetic organisms and isolated pro teins 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 properti es of frozen samples make it difficult to compare the fluorescence int ensity 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 lumine scent terbium chelate complex for use as an internal emission standard for the study of photosystem II fluorescence at cryogenic temperature s. The ligand consists of a diethylenetriaminepentaacetic acid derivat ive where pyrimidine rings sensitize the terbium luminescence, overcom ing the inherently low absorption of terbium. The chelated lanthanide remains in solution in the aqueous phase and does not interfere with p hotosystem II function. By scaling to the terbium emission, the fluore scence intensity of different samples can be readily compared. This ch elate complex could also be used as an internal emission standard for studies of other proteins. (C) 1996 John Wiley & Sons, Inc.