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A number of NAD~+ analogs have been tested in their ability to form fluorescent deriva-tives when UV irradiated with the active site Cys-149 carboxymethylated GAPDH and this hasbeen compared with their properties of acting as hydrogen acceptors and forming the Rackerband. Among the analogs tested, NHD~+, NGD~+, APAD~+ and ∈NAD~+ give positive results inall the above-mentioned reactions whereas αNAD~+, NMN~+ and CPAD~+ are all negative.FPAD~+ forms a fluorescent derivative on UV irradiation with the carboxymethylated enzymebut is inactive as a hydrogon aceeptor and does not form the Racker band. This is probablydue to thiohemiacetal formation of the pyridine 3-aldehyde of this derivative with the activesite SH group required for both the latter 2 reactions. TPAD~+, although active as a hydrogenacceptor, does not form either a fluorescent derivative or a Racker band. The fact that forthe great majority of the analogs, the property of forming fluorescent derivatives is is parallelwith their hydrogen acceptor activity seems to show that the formation of the fluorescentderivative is indeed at the active site, and hence can be used as an intrinsic probe for thestudy of the conformation of the active site of this enzyme.
A number of NAD ~ + analogs have been tested in their ability to form fluorescent deriva-tives when UV irradiated with the active site Cys-149 carboxymethylated GAPDH and this hasbeen compared with their properties of acting as hydrogen acceptors and forming the Rackerband. Among the Analogs tested, NHD ~ +, NGD ~ +, APAD ~ + and ∈NAD ~ + give positive results inall the above mentioned-mentioned reactionsAnalNAD ~ +, NMN ~ + and CPAD ~ + are all negative.FPAD ~ + forms a fluorescent derivative on UV irradiation with the carboxymethylated enzyme but is inactive as a hydrogon aceeptor 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 as a hydrogenacceptor, does not form either a fluorescent derivative or a Racker band. The fact that forthe great majority of the analogs, the property of forming fluorescent derivatives is parallelwith their hydrogen acceptor activity seems to show that the formation of the fluorescent derivative is indeed at the active site, and therefore can be used as an intrinsic probe for the study of the conformation of the active site of this enzyme.