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Two radioactive photoaffinity reagents, based on the 2,4-dinitrophenyl group, have been used to label a homogeneous mouse myeloma protein (protein 460). After separation of the heavy and light chains, a portion of the light chains was allowed to react with maleic anhydride and then digested with trypsin while another portion was digested directly with trypsin. With one of the reagents, 2,4-[3H]dinitrophenylalanyl diazoketone, which labels the light chain predominantly, nearly all of the reagent was found attached In a previous paper (Yoshioka et al., 1973), we have reported the labeling of the combining region of protein 460 with two radioactive photoaffinity reagents based on dinitrophenyl (Dnp).* 1 *This is one of several haptens known to bind to the combining region of this protein (Jaffe et Rosenstein et al., 1972). One Dnp reagent, 2,4-[3H]dinitrophenylalanyl diazoketone ([3H]Dnp-AD), reacts predominantly with the light chain. The other photoaffinity reagent, 2,4-[3H]dinitrophenyl azide ([3H]Dnp-N3), is attached predominantly to the heavy chain. This paper deals with the isolation of light chain peptides and the location of the photoaffinity-labeled amino acid residues in the light chain of protein 460 labeled with [3H]Dnp-AD and [3H]Dnp-N3.

A mouse IgA myeloma protein, protein 460, which binds the 2,4-dinitrophenyl (Dnp) group was reacted with two photoaffinity labeling reagents, Dnp-alanyl diazoketone (which we have previously shown to label residue 54 in the light chain) and Dnp-N3. The residues that react with Dnp-N3 are located in two separate fragments derived from the heavy-chain-variable region and were obtained after cleaving partially reduced and alkylated heavy chain with cyanogen bromide. The modified amino acid residues were found both in the amino terminal ,4-Dinitrophenylalanyl diazoketone (Dnp-AD)1 (Converse and Richards, 1969) and dinitrophenyl azide (Dnp-N3) (Yoshioka et al., 1973) are photoactivated affinity labeling reagents which share the same haptenic determinant, the dinitrophenyl ring, but have different reactive groups. We have used both of these reagents to label the combining region of protein 460. Dnp-AD reacted almost exclusively with a lysine residue located at position 54 of the protein 460 light chain (Hew et al., 1973). In contrast, 85% of the Dnp-N3, attached by covalent bonds to protein 460, was found in the Fd region (Yoshioka et al., 1973). The remainder, 15%, of the covalently attached reagent was found in the light chain and was sufficient to permit its partial localization (Hew et al., 1973). This paper describes the results which allowed us to assign the positions occupied by the Dnp-N3 labeled residues in the heavy chain. Materials and Methods