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

Biochemistry. 1994 Aug 2;33(30):8969-81. doi: 10.1021/bi00196a015.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels
  • Amino Acid Sequence
  • Binding Sites
  • Corticosterone / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Hydrocortisone / chemistry*
  • Indicators and Reagents
  • Kinetics
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptide Mapping
  • Photochemistry
  • Progesterone / chemistry*
  • Spectrometry, Fluorescence
  • Transcortin / chemistry*
  • Transcortin / isolation & purification
  • Trypsin
  • Tryptophan / chemistry*


  • Affinity Labels
  • Indicators and Reagents
  • Progesterone
  • Tryptophan
  • Transcortin
  • Trypsin
  • Corticosterone
  • Hydrocortisone