Site-directed cross-linking: a new approach to mapping antibody combining sites

The gammaA myeloma protein 315 from the mouse was affinity-labeled with m-nitrobenzene diazonium fluoroborate which, as shown previously, leads to selective modification of the tyrosine at position 34 in the light chains of this protein. The azotyrosine bond was reduced with dithionite to form 3-aminotyrosine. The aryl amino group of the aminotyrosine was selectively reacted with the bifunctional reagent 1,5-difluoro-2,4-dinitrobenzene. Cross-links were formed between the aminotyrosine and at least two residues-one on the same light chain and one in the Fd region of the heavy chain. Since affinity labeling of various antibodies with diazonium reagents has frequently led to azotyrosine formation, the approach described in this paper should have general applicability.