Allan L. Grossberg

Number of binding sites of rabbit macroglobulin antibody and its subunits

Abstract The IgM (γ1- or s2-macroglobulin) antibody was purified from rabbit antisera against the p-azobenzenearsonate group. The preparations obtained were shown to be of high purity by ultracentrifugation, radioimmunoelectrophoresis and absorption with a specific immunoadsorbent. Equilibrium dialysis experiments with p-iodobenzenearsonate showed that six binding sites are present per molecule of IgM antibody on the basis of 1,000,000 molecular weight. Binding constants for IgM antibodies were similar to those for IgG (7Sγ-globulin) antibodies. Essentially no change in the number of binding sites nor in binding constant was observed upon dissociation of the IgM antibody by reduction and alkylation at pH 8. Radioimmunoelectrophoresis also demonstrated the antigen-binding activity of the dissociated IgM antibody subunits. A small but significant difference between the amino acid composition of IgM and IgG antibodies indicated a difference of the primary structure of these antibodies.

Imnunoglobulin M Antibodies with Ten Combining Sites

Immunoglobulin M rabbit antibodies to a hapten are shown to have ten binding sites per molecule. The affinity for the specific hapten is approximately 100 times greater for one-half of the sites than for the other half. All sites are retained in the five 7S subunits produced by reduction and alkylation of the immunoglobulin M. Each of the 7S subunits of the IgM molecule apparently has one strong and one weak site.

Separation of antibodies into fractions with different binding properties

Abstract Rabbit antibodies against p -azobenzoate were fractionated by successive adsorption of sera with small amounts of specific immunoadsorbents followed by elution of antibodies with propionic acid. The binding constants for p -iodobenzoate differed among antibody fractions as much as 100-fold for one antiserum and 20-fold for another while the fractions from the third antiserum showed no difference. Antibodies of higher affinity were obtained in the first adsorptions. The last fractions reflect the contribution of the carrier protein to the combining site.

Antibody-ligand interactions studied by fluorescence enhancement methods_I: Properties of the ligands 4-anilinonaphthalene-1-sulfonate and 6-anilinonaphthalene-2-sulfonate☆

Abstract The arylnaphthalenesulfonates, 4-anilinonaphthalene-1-sulfonate and 6-anilnonaphthalene-2-sulfonate, are shown to be suitable ligands for measuring specific binding to antibodies against the respective azonaphtthalenesulfonates,4-azonaphthalene-1-sulfonate and 6-azonaphthalene-2-sulfonate, by the method of fluorescence enhancement. Antibodies against the latter structure have not been studied previously. Each ligand, which is essentially non-fluorescence in aqueous solution shows enhanced fluorescence when bound to antibody against the homologous azonaphthalenesulfonate. The maximum fluorescence of each ligand when occupying sites of the homologous antibody was about 600 times the fluorescence shown by the ligand in the presence of normal rabbit γ-globulin. The enhanced fluorescence of each ligand when bound to antibody appears to depend in part on the hydrophobic character of its environment so that these ligands may serve to map out the hydrophobic region of an antibody site. However the contribution of other factors to the observed fluorescence enhancement must be first evaluated. The fluorescent properties of the ligands and the corresponding toluidinyl analogues were also studied when these compounds were in solutions of organic solvents and when bound to serum albumin.

The disulfide bonds of rabbit γ-globulin and its fragments☆

Abstract The number and reactivity of the free sulfhydryl groups and disulfide bonds has been determined for rabbit γ-globulin and its fragments. Rabbit γ-globulin has somewhat over two groups which react with silver ions in the presence of Duponol C. They exhibit an unusually low reactivity toward reagents regarded as specific for SH groups. It is concluded that the anomalous behavior is due to interactions with neighboring groups rather than to a hidden position in the molecule. They are probably located in the portion of the molecule which corresponds to papain fraction III. The number of “available” disulfide bonds (reducible with 2-mercaptoethylamine (MEA) in the absence of urea or detergent at pH 7.5, 37 °C.) as well as the total disulfide content (reducible by MEA in 10 M urea, pH 7.5, 37 °C.) has been determined for intact γ-globulin, unfractionated pepsin digest of the same, isolated 5 S (pepsin) fragment, and isolated papain fractions I, II, and III. The sum of the number of available bonds in the individual fragments does not exceed that found in the intact γ-globulin, indicating that splitting of the molecule into fragments does not expose new disulfide bonds for the action of the reducing agent. This observation along with others in the literature suggests that proteolytic action on γ-globulin proceeds along lines which mark preformed subdivisions in the molecule. The fragments produced by proteolysis are probably not identical with the recently discovered individual peptide chains derived by reduction in urea without the use of proteolytic enzymes (15–17). The total number of SH groups obtained by reduction with the MEA-urea mixture is in good agreement with the 1 2 - cystine content of rabbit γ-globulin (1).

Limited heterogeneity of antibodies resolution of hapten binding curves into linear components

Abstract The hapten bindign curve for a heterogeneous antibody preparation was resolved by a graphical procedure into two linear binding curves. The antibody, obtained from the earlier bleedings of an individual rabbit, consisted principally of two homogeneous components on the basis of light chain disc electrophoresis and specific fractionation. Production of one of the two components subsequently ceased and the other component persisted. The measured binding constant for the persistant component, 104M−1, was used in the resolution as K for one of the components of the heterogeneous preparation; this component was found to amount to 40 per cent of the mixture. The binding constant of the other component was found to be 2·2 × 105M−1 and amounted to 60 per cent of the mixture. The resolution procedure should be useful in describing other antibody preparations.

Specificity fractionation of antibodies from individual animals against the 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP) determinant☆

Abstract Antibodies against the 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP) determinant were specifically purified by adsorption and elution from an immunoadsorbent column. Such antibodies from individual rabbits showed up to 70-fold differences in degree of cross-reactivity with the cross-reactive haptens picrate and diidotyrosine. Antibodies were fractionated by elution from the immunoadsorbent with picrate, diiodohydroxybenzoate and acid. Individual fractions from a single serum showed as much as a 700-fold difference in relative combining constant for a given cross-reactive hapten, while having the same binding constant for NIP-acid. Rabbits were found to vary significantly in the amount of the antibodies produces of each cross-reactivity. The fractions showed heterogeneous than the unfractioned antibodies. Antibody tended to become less cross-reactive at 6 months than at 1 month after immunization. Assays for binding activity were developed for microgram amounts of antibody.

Antibody-ligand interactions studied by fluorescence enhancement methods—II: Binding properties of anti-azonaphthalenesulfonate antibodies

Abstract Spscially purified antibodies directed toward the 4-azonaphthelene-1-sulfonate group and toward the 6-azonaphthalene-2-sulfonate group from individual animals have been investigated by the method of flourescence enhancement. The fluorescent ligands used in the these studies were 4-anilinonaphtalene-1-sulfonate (4, 1-ANS), 6-anilinonaphthalene-2-sulfonate (6 2-ANS) and the corresponding toluidinylnaphthalene sulfonates, 4, 1-TNS and 6,2-TNS. The degree of fluorescent enhancement was found to be greater for the ligands when bound by their homologous antibody than when bound by the heterologous antibody. There was some variation in the fluorescent enhancement shown by a given ligand when bound by different individual antibody preparations of the same specificity. This variation was not related to the strength of binding of ligand by the different antibody preparations. Binding constants were calculated from fluorescence enhancement measurement at different total ligand concentrations. Relative binding constants for nonfluorescent ligands were measured by displacing a fluorescent indicator ligand from antibody. This method allowed determination of the contribution of the component parts of the naphthalenesulfonate structures to the binding, and provided information on the steric configuration of the binding sites in the antibody systems.

Arginine and lysine in binding sites of anti-4-azophthalate antibodies

Abstract Antibodies from individual rabbits, directed against 5-azoisophthalate have been examined for the presence of lysine in their combining sites by complete maleylation of amino groups in the absence and presence of hapten, and for arginine by glyoxalation of guanidinium groups in the absence and presence of hapten. By each procedure, antibody sites were lost when hapten was absent and this loss could be partially prevented when hapten was present during the chemical modification procedure. The results indicate that a large proportion of the sites of anti-5-azoisophthalate antibodies studied contain arginine and that a small proportion contain lysine.

Structural Basis of Antibody Specificity

Immunologic methods for steroid determination depend on the ability of antibodies directed against a steroid determinant group to react preferentially with that particular steroid. The specificity of the reaction depends on a complementary fit of the combining site of the antibody with the steroid of interest.