Specific Photoaffinity-Labeling of Tyr-50 on the 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-nitrobenzoylamidestradiol Photoreadgents

摘要

A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiolantibody 9D3, raised against the same immunogen as that employed for generating the reported 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 the steroid, but a similar lack of specificity for both 6-and 7-positions of the B-ring. This antibody was photoaffinity-labeled with five (5-azido-2-nitrobenzoyl)amido (ANBA) derivatives of [17#alpha#-~3H]estradiol, synthesized from 3-aminoethyloxy, 3-(aminoethylamido)carboxymethyloxy, 6#alpha#- and 6#beta#-amino, and 7-[O(aminoethylmido)carboxymethyl]oximino precursors. After tryptic digestion, the radioactive peptides on L and H chains were immunopurified with the immobilized antibody 9D3, separated by reversed-phase liquid chromatography, sequenced, andcharacterized by mass spectrometry, including post-source decay-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The long 3-(ANBA-ethylamido)carboxymethyl ether photoreagent was found to label TyrL-32 (on CDR L1), whereas no labelingwas observed with the shorter 3-derivative, a result in agreement with a binding pocket large enough to explain the high crossreactivity with estradiol 3-conjugates. The two 6#alpha#- and 6#beta#-ANBA-estradiol isomers, as well as the 7-[O-(ANBA-ethylamido)carboxymethyl]oximinosestradiol photoreadgent 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 experiments indicte that TyrL-32 is freely acessible from the three C3, C6, and C7 positions, all presumed to be exposed to solvent, while TyrH-50 is probably located on the #beta#-face of estradiol. These results, obtained in solution, provide experimental data useful for molecular modeling of the steroid-antibody complex.