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-nitrobenzoylamidoestradiol photoreagents

Biochemistry. 2001 Dec 11;40(49):14907-20. doi: 10.1021/bi011174b.


A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiol antibody 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 [17alpha-(3)H]estradiol, synthesized from 3-aminoethyloxy, 3-(aminoethylamido)carboxymethyloxy, 6alpha- and 6beta-amino, and 7-[O-(aminoethylamido)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, and characterized 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 labeling was observed with the shorter 3-derivative, a result in agreement with a binding pocket large enough to explain the high cross-reactivity with estradiol 3-conjugates. The two 6alpha- and 6beta-ANBA-estradiol isomers, as well as the 7-[O-(ANBA-ethylamido)carboxymethyl]oximinoestradiol photoreagent derived from the steroid hapten, labeled the same TyrL-32 residue. The 6beta-ANBA epimer also labeled TyrH-50 (at the basis of CDR H2). These experiments 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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / isolation & purification
  • Antibody Specificity
  • Base Sequence
  • Binding Sites
  • Estradiol / analogs & derivatives*
  • Estradiol / chemistry
  • Estradiol / immunology*
  • Immunoglobulin Heavy Chains / chemistry
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Heavy Chains / metabolism
  • Immunoglobulin Light Chains / chemistry
  • Immunoglobulin Light Chains / genetics
  • Immunoglobulin Light Chains / metabolism
  • Mice
  • Molecular Sequence Data
  • Molecular Structure
  • Photoaffinity Labels / chemistry*
  • Photoaffinity Labels / metabolism
  • Protein Conformation
  • Sequence Alignment
  • Tyrosine / chemistry*
  • Tyrosine / metabolism


  • 6-(5-azido-2-nitrobenzoyl)amidoestradiol
  • Antibodies, Monoclonal
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Light Chains
  • Photoaffinity Labels
  • Tyrosine
  • Estradiol