The interaction of the pituitary hormone corticotropin (ACTH) with bovine serum albumin (BSA) was investigated by photoaffinity labeling with 2-nitro-4-azidophenylsulfenyl (2,4-NAPS) derivatives of ACTH and [Trp-(SH)9]ACTH. Nearly 30 mol % of tritiated [2,4-NAPSTrp9] ACTH was covalently bound to BSA at a molar ratio ofhormone:BSAof 1.33. The [2,4-NAPS-Trp9] [3H]ACTHBSA 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-NAPSTrp9] [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-NAPSSTrp9] ACTH-BSA complex by treatment with β-mercaptoethanol was complete in 6 h, but only 80% of ACTH was released from [2,4-NAPS-Trp9] ACTH-BSA under these conditions.
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