Studies on chicken γ-globulins and hapten-specific chicken antibodies

Abstract

1. 1. Chickens were injected with a mixture of Ars-BSA and R 4N-BSA, i.e. azoproteins prepared by coupling BSA with diazotized arsanilic acid or diazotized p-aminophenyl-N-trimethylammonium chloride, or with the doubly labeled antigen Ars-R 4N-BSA. Serotologically pure hapten-specific anti-Ars and anti-R 4N antibodies were prepared by means of immunosorbents; they were free of anti-BSA and other γ-globulins. 2. 2. Reduction of the hapten-specific antibodies with mercaptoethanol and alkylation with iodoacetamide yielded a mixture of peptide chains which on starch gel electrophoresis in urea at pH 8·2 was resolved into a diffuse band which migrated toward the cathode and 7–8 anodically migrating narrow bands. The cathodically migrating material was identified with the heavy A chains, the anodically migrating bands with the light B chains of chicken γ-globulin. No consistent significant difference was found when the band pattern of A and B chains from anti-Ars antibody was compared with the analogous bands from anti-R 4N antibody. 3. 3. Ultracentrifugation of the hapten-specific antibodies isolated from hyperimmune chickens revealed the presence of some 18 S antibodies in the 7 S population. Digestion of the isolated hapten-specific anti-Ars and anti-R 4N antibodies with papain yielded a product which on ultracentrifugation sedimented as a single peak of 3·7 S; material of the same sedimentation coefficient was obtained by the papain digestion of normal chicken γ-globulin. 4. 4. The peptide maps of the tryptic digests of anti-Ars and anti-R 4N antibodies were extremely similar if not identical. No distinct and consistent difference was found. However, distinct differences were found between fragments I and II from normal chicken γ-globulin, and also between peptide maps of anti-R 4N (or anti-Ars) antibodies from chicken and rabbit serum. 5. 5. The identity of the peptide maps of anti-Ars on the one hand and anti-R 4N on the other can be reconciled with the clear-cut serological differences between these two antibodies by the assumption that the combining sites of the antibody molecules are highly heterogeneous, that the distinct spots of the peptide maps, which are identical in anti-Ars and anti-R 4N are those of amino acid sequences which are common to both hapten-specific antibodies, and that the specific combining sites of the antibody molecules are formed by variable amino acid sequences, each of these occurring in amounts too small to produce distinct spots in the peptide maps.