IDENTIFICATION OF TRP-371 AS THE MAIN SITE OF SPECIFIC PHOTOAFFINITY-LABELING OF CORTICOSTEROID-BINDING GLOBULIN USING DELTA-6 DERIVATIVES OF CORTISOL, CORTICOSTERONE, AND PROGESTERONE AS UNSUBSTITUTED PHOTOREAGENTS

Immunopurified human corticosteroid binding globulin (CBG) was photolabeled with Delta(6)-[H-3]-cortisol, Delta 6-[4-C-14]cortisol, Delta 6-[H-3]corticosterone, and Delta(6)- [H-3]progesterone. The maximal levels of specific incorporation, as estimated with tritiated photoreagents, were 0.21,0.14, and 0.08 mel of label/mol of CBG, respectively. Tryptic cleavage of photolabeled CBG gave in all cases a major radioactive peptide that was no longer detectable when a 100-fold molar excess of cortisol was added to the photoreagents. Edman sequencing of tryptic peptides photolabeled with Delta(6)-[3H]cortisol or Delta(6)-[3H]corticosterone showed that these peptides correspond to residues 357-378 of the human CBG sequence. The major peak of radioactivity of these peptides was eluted at the 15th cycle (Trp-371). The radioactive tryptic peptides photolabeled with the four steroid photoreagents were subcleaved with alpha-chymotrypsin. The major part of radioactivity was recovered in the T-[*X]-S-S-L-F hexapeptide 370-375 (major peptide) and in the D-H-F-T-[*X]-S-S-L-F nonapeptide 367-375, at the second and fifth Edman cycles, respectively, whereas no PTH derivative could be identified at these cycles, thus suggesting Trp-371 as the main site of photolabeling for all tested photoreagents. Mass spectrometry of tryptic peptides photolabeled with Delta(6)-[H-3]cortisol and Delta(6)-[H-3]-corticosterone and of chymotryptic peptides photolabeled with Delta(6)-[H-3]cortisol, Delta(6)-[H-3]corticosterone, and Delta(6)-[3H]progesterone showed molecular masses corresponding to the addition of Delta(6)-steroid photoreagents to the peptide.