Rat heart anthracycline-binding polypeptides identified by photoaffinity labeling.

A radioactive, photoactive anthracycline analogue, N-(p-azido-[3,5-3H]benzoyl)-daunorubicin (3H-NAB-daunorubicin), was synthesized and characterized by UV-visible absorption and infrared analyses. 3H-NAB-daunorubicin photoaffinity labeling of rat heart homogenates resulted in the identification of two prominently radiolabeled anthracycline-binding polypeptides of 18.3 and 31.2 kDa. Photoaffinity labeling with photoactive doxorubicin (Adriamycin), carminomycin, and nonanthracycline model compounds resulted in a clear structural dependence for binding to the 18.3-and 31.2-kDa species. In the presence of daunorubicin or N-substituted daunorubicin analogues, 3H-NAB-daunorubicin photolabeling of the 18.3-kDa polypeptide was inhibited. Photolabeling was dependent on time of UV light exposure and protein concentration and was unaffected by the presence of nitrene scavengers. The 18.3-kDa polypeptide photolabeling was saturable and reversed by greater than 90% in the presence of a 16-fold molar excess of nonradioactive analogue. Photolabeling of heart subcellular fractions demonstrates that both the 18.3- and 31.2-kDa polypeptides were localized to the inner mitochondrial membrane. Since the anthracyclines are known to have several effects on heart mitochondrial function, the identification of specific polypeptide acceptors using photoactive anthracycline analogues may elucidate biochemical mechanisms of anthracycline cellular activity.