Characterization of Met-139 as the photolabeled amino acid residue in the steroid binding site of sex hormone binding globulin using delta 6 derivatives of either testosterone or estradiol as unsubstituted photoaffinity labeling reagents.
Immunopurified human sex hormone binding globulin (SHBG) was photoinactivated and photolabeled by radioinert and radioactive photoaffinity labeling steroids delta 6-testosterone (delta 6-T) and delta 6-estradiol (delta 6-E2). The maximal levels of specific incorporation of these two reagents were 0.50 and 0.33 mol of label/mol of SHBG, respectively. Covalently labeled SHBG fractions were citraconylated, reduced, carboxymethylated, and cleaved by trypsin. Separation of tryptic digests by reverse-phase liquid chromatography gave single radioactive peaks at the same retention times with both steroid reagents. However, the two labeled peptidic fractions could be distinguished by capillary electrophoresis and immunodetection with anti-steroid antibodies, whereas the covalent attachment of radioactivity was confirmed by thin-layer chromatography on silica gel. Edman degradation of the two labeled peptides showed a single sequence His-Pro-Ile-([3H]X)-Arg corresponding to the pentapeptide His-Pro-Ile-Met-Arg 136-140 of SHBG sequence. The coincidence, in both cases, of the absence of an identifiable amino acid residue and of the elution of the most intense peak of radioactivity at the fourth cycle of Edman degradation suggests that the same Met-139 residue was labeled by delta 6-[1,2-3H2]T or by delta 6-[17 alpha-3H]E2. Liquid secondary ion mass spectrometry of the two peptides showed [M+H]+ ions at m/z 939.8 or 923.8, corresponding respectively to the addition of delta 6-T or delta 6-E2 to the pentapeptide. The presence of the steroid molecule in the delta 6-[3H]T-pentapeptide conjugate was confirmed by the difference of 2 mass units with the [M+H]+ peak of the delta 6-[4-14C]T-pentapeptide conjugate.
Grenot
C
C
Institut National de la Santé et de la Recherche Médicale, Unité INSERM U 329, Hôpital Debrousse, Lyon, France.
de Montard
A
A
Blachère
T
T
de Ravel
M R
MR
Mappus
E
E
Cuilleron
C Y
CY
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D013196:Dihydrotestosterone / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D004963:Estrenes / Q000138:chemical synthesis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008715:Methionine; D008969:Molecular Sequence Data; D010782:Photolysis; D012738:Sex Hormone-Binding Globulin / Q000302:isolation & purification / Q000378:metabolism*; D013739:Testosterone / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*
D000345:Affinity Labels; D004963:Estrenes; D012738:Sex Hormone-Binding Globulin; D013196:Dihydrotestosterone; C076348:3-hydroxyestra-1,3,5(10),6-tetraen-17-one; C023630:6-dehydrotestosterone; D013739:Testosterone; D004958:Estradiol; C076347:estra-1,3,5(10),6-tetraene-3,17-diol; D008715:Methionine