MODULAR DESIGN OF BIOTINYLATED PHOTOAFFINITY PROBES - SYNTHESIS AND UTILIZATION OF A BIOTINYLATED PEPSTATIN PHOTOPROBE

A novel modular design is presented for the introduction of biotinylat ed photoprobes containing either 4-azidotetrafluorobenzamide, 4-(1-azi -2,2,2-trifluoroethyl)benzamide, or 4-benzoylbenzoylamide. The use of biotinylated affinity labels offers several advantages over radiolabel ed probes by virtue of their exploitation of the biotin-avidin system of detection and purification. A biotinylated benzoylbenzoyl photoprob e of pepstatin (BBB-pepstatin, 5) was synthesized in three steps from pepstatin. The photoprobe is a competitive inhibitor of porcine pepsin , with an apparent dissociation constant of 31 pM. Western blotting of BBB-pepstatin-photolabeled porcine pepsin, renin, cathepsin D and hum an renin, and cathepsin D could be detected with an avidin-horseradish peroxidase label. Routinely, 7 pM of aspartic protease could be photo labeled and detected with this system. The pepstatin photoaffinity pro be is also very selective; the probe failed to label cysteine protease (papain), metalloprotease (carboxypepitase A), and serine protease (c hymotrypsin and trypsin). To further establish the utility of the biot inylated probe, BBB-pepstain-photolabeled porcine pepsin was purified by monomeric avidin chromatography. This probe should be useful for th e identification of unknown cytosolic and membrane-bound aspartic prot eases.