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

yl}-aminohexyl]-aminocarbamoylmethyl)-nicotinamide adenine dinucleotid e (I-125-N-6-I-ASA-AH-NAD(+)) was synthesized by coupling N-6-([6-amin ohexyl]-carbamoylmethyl)-NAD(+) with 4-azidosalicylic acid N-hydroxysu ccinimide eater followed by radioiodination. The utility of I-125-N-6- I-ASA-AH-NAD(+) as an effective site-directed photoprobe was demonstra ted by the photolabeling of both glutamate dehydrogenase and 15-hydrox yprostaglandin dehydrogenase. Both enzymes can be saturated with label ed probe with apparent dissociation constants comparable to those repo rted 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.