Identification of the corticotropin binding domain of bovine serum albumin by photoaffinity labeling.
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.