Affinity Labeling of the Active Sites of Antibodies to the 2,4-Dinitrophenyl Hapten^*

Abstract

The method of afkity labeling provides a general method for attaching a covalently bound label to the active sites of antibody and enzyme molecules. Previous studies with antibodies directed to the benzenearsonate hapten were entirely in accord with the predictions of the method. In the present studies, antibodies to the 2,4dinitrophenyl (DNP) hapten were investigated. The labeling reagent p-nitrophenyldiawnium fluoborate was found to react irreversibly with an excess of the unproteded antibody: (a) at a much more rapid rate than with the antibody whose active sites were protected with an excess of N-DNP-e-aminocaproic acid; (b) to give a product with stentially a pure azotyrosine spectrum; (c) at a rate which was first-order in the concentration of the reversible complex formed initially between the reagent and the antibody s i t s , in accord with the mechanism proposed for affinity labeling; and (d) to produce a loss of antibody binding sites corresponding closely to the number of azotyrosine groups formed. Related but less extensive studies were carried out with DNP-diazonium and p(carboxy)-benzenediazonium fluoborates and with other reagents. The results closely follow the predictions of the method, and provide strong evidence that a tyrosine residue is present in anti-DNP antibody sites. The results also indicate that there is a broad distribution of rates a t which the antibody sites are labeled, and therefore that the sites are heterogeneous. These studies provide the basis for an attempt to isolate and analyze labeled peptide

On the location and structure of the active sites of antibody molecules.

“…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.6…”

Affinity labeling of a mouse myeloma protein which binds nitrophenyl ligands. Kinetics of labeling and isolation of a labeled peptide

“…In these experiments, the protein was precipitated at 0" by the addition of absolute ethanol to achieve a final concentration of 70%. The labeled protein was then processed as previously described (Wofsy et al, 1962;Metzger et al, 1963). Counting and ODBso m u measurements were performed after dissolving the precipitates in 0.5% sodium dodecyl sulfate in 0.02 M sodium phosphate buffer (pH 6.2) at 37".…”

Eureka! And Other Pleasures

“…In my case it was to continue studies on affinity labeling that Wofsy, Singer, and I had pursued in La Jolla (11,12). I planned to use that approach to examine IgM antibodies, but a number of circumstances soon prompted me first of all to learn something more about the structure per se of these "macroglobulins."…”