Interaction of human immunoglobulin G with l-histidine immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes.
L-Histidine as pseudobiospecific ligand was immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes to obtain an affinity support for immunoglobulin G (IgG) purification. The interaction of human IgG with the affinity membranes was studied by chromatography and equilibrium binding analysis. Adsorption was possible over a broad pH range and was found to depend strongly on the nature of the buffer ions rather than on ionic strength. With zwitterionic buffers like morpholinopropanesulfonic acid (Mops) and hydroxyethylpiperazineethanesulfonic acid (Hepes), much higher adsorption capacities were obtained than with other buffers like Tris-HCl and phosphate buffers. An inhibition analysis revealed that non-zwitterionic buffers competitively inhibit IgG binding, whereas Mops and Hepes in their zwitterionic form do not. By choosing the appropriate buffer system, it was possible to adsorb specifically different IgG subsets. The IgG molecules were found to adsorb on membrane immobilized histidine via their Fab part. Determination of dissociation constants at different temperatures allowed calculation of thermodynamic adsorption parameters. Decrease in KD with increasing temperature and a positive entropy value between 20 and 35 degrees C (in Mops buffer) indicated that adsorption is partially governed by hydrophobic forces in that temperature range, whereas at lower temperatures, electrostatic forces are more important for adsorption.
Haupt
K
K
Laboratoire d'Interaction Moléculaire et Technologie des Séparations (LIMTechS), Université de Technologie de Compiégne, France.
Bueno
S M
SM
Vijayalakshmi
M A
MA
D000327:Adsorption; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification*; D007526:Isoelectric Point; D008567:Membranes, Artificial; D009994:Osmolar Concentration; D011145:Polyvinyls; D013696:Temperature; D013816:Thermodynamics
D002021:Buffers; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D008567:Membranes, Artificial; D011145:Polyvinyls; C020320:ethylene-vinyl alcohol copolymer; D006639:Histidine