The modification of affinity labeled antibodies

Abstract

We previously showed that antibodies which had been affinity labeled at a tyrosyl residue with a diazonium labeling reagent could be further modified usefully. By reductively cleaving the azotyrosine bond, 3-aminotyrosine (3-NH 2 Tyr) was generated in situ , and this residue could then serve as an initiation point for further modifications - for example with the bifunctional reagent difluoro-dinitrobenzene (F 2 DNB). We now present the results of further experiments utilizing this approach. The affinity labeled nitrophenyl binding protein of the plasma cell tumor MOPC 315 was easily reduced with small amounts of dithionite (Na 2 S 2 O 4 ). When F 2 DNB was added it reacted initially at the 3-NH 2 Tyr at position 34 on the light chain and then subsequently with other amino acid residues leading to the formation of light-heavy and intra-light chain cross-links. Although under suitable conditions F 2 DNB also reacted with the native protein of MOPC 315, the reaction occurred largely on the heavy chain. These results testify to the high specificity obtainable using these procedures. Experiments involving the reaction of F 2 DNB with either labeled-unreduced protein, or labeled-reduced protein in the presence of the ligand dinitrophenyl-aminocaproate, suggests that the F 2 DNB may in part have functioned as an affinity labeling reagent. Parallel studies were performed with the phosphorylcholine binding protein of plasma cell tumor TEPC 15. This protein affinity labeled at two alternative tyrosines on the light chains. The azo bonds were much more difficult to reduce wwith dithionite. When they were cleaved some of the sites remained completely inactive whereas others bound normally. The stoichiometry suggests that those sites with a 3-NH 2 Tyr at position 92 were inactivated while those with 3-NH 2 Tyr at position 34 were unaffected. Native protein-15 reacted only minimally with F 2 DNB under the conditions used whereas the labeled-reduced protein became modified on the light chains exclusively.