Chromatographic analysis of the hydrophobic interactions of rabbit IgG immunoglobulins and their papain-digested fragments by bis-(p-chlorophenyl)-acetic acid coupled to aminohexyl sepharose.
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.
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001666:Binding Sites, Antibody; D002847:Chromatography, Agarose / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D003634:DDT / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments / Q000032:analysis; D007141:Immunoglobulin Fc Fragments / Q000032:analysis; D007128:Immunoglobulin Fragments / Q000032:analysis*; D007074:Immunoglobulin G / Q000032:analysis*; D008024:Ligands; D010206:Papain; D011817:Rabbits; D012685:Sepharose / Q000031:analogs & derivatives
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D008024:Ligands; C024903:aminohexyl-sepharose; C027132:1-anilino-8-naphthalenesulfonate; C000849:bis(p-chlorophenyl)acetic acid; D012685:Sepharose; D003634:DDT; D010206:Papain