The fluorescence properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied in order to probe the conformational changes which occur in different regions of the protein with denaturation. Using time-resolved spectroscopy with a picosecond, tunable dye-laser excitation system, we could resolve the tryptophan emission into 3 components associated with 3 different lifetimes. A fluorescent photoproduct attached to the active site served as a probe for this region. The data were consistent with sequential changes in different regions of the protein as a function of time of denaturation in guanidine hydrochloride. Time-resolved anisotropy studies showed changes in the rotational mobility of the photoproduct during denaturation. The mechanism of concentration quenching of 6CF in liposomes was confirmed to be due to energy transfer to nonfluorescent dimers. Energy transfer calculations together with concentration depolarization measurements showed that this mechanism could account for all the quenching, and that any component of collisional deactivation was improbable. A study of the site of excited state protonation of serotonin is under way using ultraviolet irradiation and NMR.
