Electrostatic and hydrophobic effects in chromatography of rabbit IgG immunoglobulins on aminohexyl sepharose substituted with 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.
D000818:Animals; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D002845:Chromatography; D003634:DDT / Q000031:analogs & derivatives; D004560:Electricity; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000032:analysis; D009994:Osmolar Concentration; D011817:Rabbits; D012685:Sepharose / Q000031:analogs & derivatives; D013696:Temperature; D014867:Water
D007074:Immunoglobulin G; D014867:Water; C024903:aminohexyl-sepharose; C000849:bis(p-chlorophenyl)acetic acid; D012685:Sepharose; D003634:DDT