Photoaffinity labeling of lactate dehydrogenase by the carbene derived from the 3-diazirino analogue of nicotinamide adenine dinucleotide.

The 3-diazirino analogue of NAD+, DAD+, in which the diazirine group replaces the carboxamide in an almost isosteric substitution, has been synthesized as a photoaffinity reagent for dehydrogenases. With lactate dehydrogenase, the Kdiss is 4 mM (compare the NAD+ Kdiss of 0.6 mM). On photolysis, three types of interaction can be discerned and separately quantitiated by using [3H]DAD+: noncovalent binding of the photoproducts of DAD+, which is removed by protein denaturation; nonspecific covalent labeling, which is eliminated by the presence of a scavenger such as glutathione; specific covalent labeling at the active site, which is prevented by competition from the natural ligands. The photolabeling efficiency (sites covalently labeled/sites initially occupied) is approximately 0.3. It is evident that the carbene generated from DAD+ is more effective than that from the more bulky 3-diazoacetate or than the less reactive nitrene derived from the 3-azido-NAD+ analogue.