sepharose and bis(p-chlorophenyl)acetic-acid

The binding interactions of rabbit IgG immunoglobulins with bis-(p-chlorophenyl)-acetic acid (DDA) substituted aminohexyl Sepharose (AHS) (DDA-AHS) at low ionic strength are under the influence of pH, temp, ionic strength and their antibody specificity. The IgG molecules held on a DDA-AHS column in the presence of 0.01 M phosphate buffer (pH 6.8) at 4 degrees C could be stepwise eluted by the addition of 0.01 M acetate buffers (pH 5.5, 5.0 and 4.5) followed by 3 M NaClO4, a chaotropic reagent. The adsorbability of IgG molecules by the DDA-AHS column was reinforced at temps higher than 4 degrees C and at increasing NaCl concns ranging from 0.05 to 0.2 M. The antibody specificity and, perhaps, binding affinity greatly affected this binding system. On the basis of these results, it was evident that both the DDA ligand and charged groups (omega-amino and isourea groups) took part in the binding interactions between IgG molecules and the agarose derivative at low ionic strength. Thus the binding interactions of rabbit IgG immunoglobulins with DDA-AHS at low ionic strength are assumed to be due to hydrophobic plus electrostatic interactions. It was also observed that the binding site on the surface of IgG molecules at low ionic strength was located only in the Fab region.

Topics: Animals; Antibody Specificity; Binding Sites, Antibody; Chromatography; DDT; Electricity; Hydrogen-Ion Concentration; Immunoglobulin G; Osmolar Concentration; Rabbits; Sepharose; Temperature; Water

The present study revealed that the IgG immunoglobulins of normal or non-immune rabbit IgG and anti-bovine serum albumin, anti-ovalbumin, anti-bovine IgG and anti-p-chlorobenzoic acid antibodies could non-specifically bind to bis-(p-chlorophenyl)-acetic acid (DDA) coupled to omega-amino-hexyl Sepharose (AHS) by the use of strengthened hydrophobic interactions dependent on the concentration of NaCl. The main binding site on the adsorbent of DDA-substituted AHS (DDA-AHS) was found to be the DDA ligand. The hydrophobic potency of the DDA ligand was thought to be more effective than that of p-chlorobenzoic acid coupled to AHS. Out study also demonstrated that two different binding sites capable of interacting the DDA ligand were contained in IgG molecules. One was located in the Fc region and the other in the Fab region. The former had an ability to adhere to the DDA-AHS adsorbent in the presence of NaCl of 3 M or over, while the latter showed heterogeneous binding behavior depending upon its antibody specificity. Differences in the chromatographic distribution among the whole IgG immunoglobulins including anti-DDA antibody were found by a hydrophobic salting-out chromatography (HSOC) method on a DDA-AHS column. It was therefore assumed that when whole IgG proteins were subjected to HSOC on a DDA-AHS column, the hydrophobic binding site in the Fc region played a decisive role at high salt concentrations of 3 M or over, while the hydrophobic binding site in the Fab region played a major role at intermediate and low salt concentrations of 2 M or below. Thus, by taking advantage of this HSOC method, whole IgG or its Fab molecules possessing very strongly hydrophobic binding sites to promote high quantum yields of 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence can be easily separated. We concluded that the ligand of DDA is a probe for the hydrophobic regions in IgG immunoglobulins.

Topics: Anilino Naphthalenesulfonates; Animals; Binding Sites, Antibody; Chromatography, Agarose; Chromatography, Gel; DDT; Fluorescent Dyes; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin Fragments; Immunoglobulin G; Ligands; Papain; Rabbits; Sepharose