| Abstract: | The interaction of the pituitary hormone corticotropin (ACTH) with bovine serum albumin (BSA) was investigated by photoaffinity labeling with 2-nitro-4-azido-phenylsulfenyl (2,4-NAPS) derivatives of aCTH and Trp-(SH)9]ACTH. Nearly 30 mol % of tritiated 2,4-NAPS-Trp9]ACTH was covalently bound to BSA at a molar ratio of hormone:BSA of 1.33. The 2,4-NAPS-Trp9] 3H]ACTH-BSA complex was isolated, and the CNBr fragments of the complex were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactivity was predominantly associated with the amino-terminal CNBr fragment corresponding to residues 1-183 in BSA. This result was confirmed by studies of the inhibition of covalent labeling of BSA by photoreactive ACTH. 8-Anilinonaphthalenesulfonic acid which binds to the amino-terminal domain of BSA strongly inhibited the photolabeling of BSA by 2,4-NAPS-Trp9]3H]ACTH. Palmitate and progesterone, known to bind to the carboxy-terminal domains of BSA, did not inhibit the incorporation of 2,4]NAPS-Trp9]3H]ACTH into BSA. The removal of ACTH from the covalent complexes was also investigated. The release of ACTH from the 2,4]NAPSS-Trp9]ACTH--BSA complex by treatment with beta-mercaptoethanol was complete in 6 h, but only 80% of ACTH was released from 2,4]NAPS-Trp9]ACTH--BSA under these conditions. |
