2-STAGE PHOTOSENSITIVE LABEL FOR ANTIBODY COMBING SITES

Dinitrophenylglycine diazoketone, dinitrophenyl alanine diazoketone, and their tritiated analogs were synthesized and used to label, in two steps, the combining sites of rabbit antidinitrophenyl antibodies. Diazoketones are unreactive with proteins, but when photolyzed, they yield carbenes and by rearrangement, ketenes. Such derivatives are reactive with most amino acid side chains. The dinitrophenylamino acid diazoketones enter the antidinitrophenyl site. Excess reagent may then be removed. The diazoketones are then photolyzed within the site, using light of 300-400 mμ. Up to 47% of the sites are covalently labeled. The label is chiefly on the γ-globulin heavy chains, and is limited to the Fab fragments. © 1969, American Chemical Society. All rights reserved.