NAD+ analogs in formation of new fluorophores on ultraviolet irradiation with D-glyceraldehyde-3-phosphate dehydrogenase.

A number of NAD+ analogs have been tested in their ability to form fluorescent derivatives when UV irradiated with the active site Cys-149 carboxymethylated GAPDH and this has been compared with their properties of acting as hydrogen acceptors and forming the Racker band. Among the analogs tested, NHD+, NGD+, APAD+ and epsilon NAD+ give positive results in all the above-mentioned reactions whereas alpha NAD+, NMN+ and CPAD+ are all negative. FPAD+ forms a fluorescent derivative on UV irradiation with the carboxymethylated enzyme but is inactive as a hydrogen acceptor and does not form the Racker band. This is probably due to thiohemiacetal formation of the pyridine 3-aldehyde of this derivative with the active site SH group required for both the latter 2 reactions. TPAD+, although active active as a hydrogen acceptor, does not form either a fluorescent derivative or a Racker band. The fact that for the great majority of the analogs, the property of forming fluorescent derivatives is in parallel with their hydrogen acceptor activity seems to show that the formation of the fluorescent derivative is indeed at the active site, and hence can be used as an intrinsic probe for the study of the conformation of the active site of this enzyme.