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-[3H]cortisol, delta 6-[4-14C]cortisol, delta 6-[3H]corticosterone, and delta 6-[3H]progesterone. The maximal levels of specific incorporation, as estimated with tritiated photoreagents, were 0.21, 0.14, and 0.08 mol 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-[3H]cortisol and delta 6-[3H]corticosterone and of chymotryptic peptides photolabeled with delta 6-[3H]cortisol, delta 6-[3H]corticosterone, and delta 6-[3H]progesterone showed molecular masses corresponding to the addition of delta 6-steroid photoreagents to the peptide.
Grenot
C
C
Institut National de la Santé et de la Recherche Médicale, Unité INSERM U 329, Pathologie Hormonale Moléculaire, Hôpital Debrousse, Lyon, France.
Blachère
T
T
Rolland de Ravel
M
M
Mappus
E
E
Cuilleron
C Y
CY
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D003345:Corticosterone / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006854:Hydrocortisone / Q000737:chemistry*; D007202:Indicators and Reagents; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010777:Photochemistry; D011374:Progesterone / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014156:Transcortin / Q000737:chemistry* / Q000302:isolation & purification; D014357:Trypsin; D014364:Tryptophan / Q000737:chemistry*
D000345:Affinity Labels; D007202:Indicators and Reagents; D011374:Progesterone; D014364:Tryptophan; D014156:Transcortin; D014357:Trypsin; D003345:Corticosterone; D006854:Hydrocortisone