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By the method of affinity-labeling1 it has been possible to attach chemical tags to amino acid residues that, by a number of stringent criteria, are contact residues within the active sites of antibody (Ab) molecules.2' With IgG Ab specific to three different benzenoid haptens from four different mammalian species,3 4 it was found that tyrosine residues on both heavy (H) and light (L) chains were affinity-labeled by the specific diazonium reagents used. These results were interpreted3 to mean that structurally unique and characteristic tyrosine residues were present in all the active sites that were affinity-labeled. To identify these residues, we have recently isolated and characterized dipeptide fragments bearing the affinity label from the H and L chains of rabbit and mouse IgG Ab specific for the 2,4-dinitrophenyl (DNP) group. Our results (1) demonstrate that the labeled residues are indeed unique; (2) establish that the variable segment of the L chain' is directly involved in forming the Ab active site; (3) strongly suggest that the labeled tyrosine is residue 86 from the amino-terminal end of the L chain; and (4) lead to the suggestion that in the Fab fragment (and, in particular, in the active site) of each half of an IgG molecule, the H and L chains are structurally related by a dyad axis of pseudosymmetry.6Materials and Methods.-Rabbit anti-DNP Ab, elicited by immunization with DNPbovine 7-globulin, were purified7 and affinity-labeled with H3-m-nitrobenzenediazonium fluoborate (H3-MNBDF),8 and the labeled H and L chains were isolated,9 as in our previous studies. Mouse anti-DNP Ab were raised to DNP-keyhole limpet hemocyanin in Swiss-Webster mice in which Ehrlich ascites tumor cells were intraperitoneally implanted.'0 The mouse anti-DNP Ab were isolated from the ascitic fluid and affinity-labeled with H3-MNBDF by methods, and with results, analogous to those for rabbit Ab.The labeled chains were digested with Nagarse enzyme (Enzyme Development Corp.) at a weight ratio of enzyme to peptide of 1:50 in 0.2 M NH4HCO3 for 20 hr at 40'C. Gel filtration of the digests was carried out on P-2 Biogel in 0.05 M NH4HCO3, and the elution of radioactivity was monitored. The elution profiles obtained with rabbit-chain digests have been presented elsewhere.6 The profiles for the mouse-chain digests are shown in Figure 1. In all cases, a major fraction of radioactivity, accounting for about 50% of the original affinity label, was obtained. This major fraction, according to gel filtration experiments with calibrated P-2 Biogel columns in phenol: acetic acid: water (1:1:1), was predominantly dipeptide in size (see also below). Very little of the radioactivity was released as free m-nitrobenzeneazotyrosine."' The radioactive peptides from this major fraction were isolated in a pure state and in high yield by taking advantage of the fact that the m-nitrobenzeneazotyrosine-labeled peptides are haptens capable of specific reversible binding to the active sites of anti-DNP Ab. The labeled peptide fraction was mixed with unlabe...

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