Specific photoaffinity-labeling of Tyr-50 on heavy chain and of Tyr-32 on the light chain in the steroid combining site of a mouse monoclonal anti-estradiol antibody using C3-, C6-, and C7-linked 5-azido-2-nitrobenzoylamidoestradiol photoreagents

A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiol antibody 9D3, r aised against the same immunogen as that employed for generating the report ed anti-estradiol antibody 15H11 [Rousselot, P., et al. (1997) Biochemistry 36, 7860-7868], was found to exhibit an opposite specificity profile with a much stronger recognition of the D-ring than of the A-ring extremity of t he steroid, but a similar lack of specificity for both 6- and 7-positions o f the B-ring. This antibody was photoaffinity-labeled with five (5-azido-2- nitrobenzoyl)amido (ANBA) derivatives of [17 alpha-H-3]estradiol, synthesiz ed from 3-aminoethyloxy, 3-(aniinoethylamido)carboxymethyloxy, 6 alpha- and 6 beta -amino, and 7-[O-(aminoethylamido)carboxymethyl]oximino precursors. After tryptic digestion, the radioactive peptides on L and H chains were i mmunopurified with the immobilized antibody 9D3, separated by reversed-phas e liquid chromatography, sequenced, and characterized by mass spectrometry, including post-source decay-matrix-assisted laser desorption/ionization ti me-of-flight mass spectrometry. The long 3-(ANBA-ethylamido)carboxymethyl e ther photoreagent was found to label TyrL-32 (on CDR L1), whereas no labeli ng was observed with the shorter 3-derivative, a result in agreement with a binding pocket large enough to explain the high crossreactivity with estra diol 3-conjugates. The two 6 alpha- and 6 beta -ANBA-estradiol isomers, as well as the 7-[O-(ANBA-ethylamido)carboxymethyl]oximinoestradioI photoreage nt derived from the steroid hapten, labeled the same TyrL-32 residue. The 6 beta -ANBA epimer also labeled TyrH-50 (at the basis of CDR H2). These exp eriments indicate that TyrL-32 is freely accessible from the three C3, C6, and C7 positions, all presumed to be exposed to solvent, while TyrH-50 is p robably located on the beta -face of estradiol. These results, obtained in solution, provide experimental data useful for molecular modeling of the st eroid-antibody complex.