AN I-125 LABELED N-6-SUBSTITUTED AZIDO ANALOG OF NAD(+) FOR THE PHOTOAFFINITY-LABELING OF NAD(+)-LINKED ENZYMES

I-125-N-6-(N-[6-N-{5-iodo-4-azidosalicyl}-aminohexyl]-aminocarbamoylmethyl)-nicotinamide adenine dinucleotide (I-125-N-6-I-ASA-AH-NAD(+)) was synthesized by coupling N-6-([6-aminohexyl]-carbamoylmethyl)-NAD(+) with 4-azidosalicylic acid N-hydroxysuccinimide eater followed by radioiodination. The utility of I-125-N-6-I-ASA-AH-NAD(+) as an effective site-directed photoprobe was demonstrated by the photolabeling of both glutamate dehydrogenase and 15-hydroxyprostaglandin dehydrogenase. Both enzymes can be saturated with labeled probe with apparent dissociation constants comparable to those reported for NAD(+). Photoincorporation of the probe into both enzymes was found to be protected specifically by NAD(+). These results indicate that I-125-N-6-I-ASA-AH-NAD(+) can be a specific photoprobe for NAD(+)-linked enzymes.