Site accessibility and the pH dependence of the saturation capacity of a highly cross-linked matrix. Immobilized metal affinity chromatography of bovine serum albumin on chelating Superose.
Immobilized metal ion affinity chromatography has shown promise for isolating desired proteins from a mixture based on their affinity for chelated metal ions. Using frontal analysis, the pH dependence of the saturation capacity of chelating Superose matrix for bovine serum albumin (BSA) is examined over a broad pH range. A significant increase in the capacity was observed near the elution pH of BSA (pH 4.5) from a Cu-imminodiacetic acid column. The results of several experiments indicated that this apparently abnormal variation may reflect the low degree of accessibility of a large portion of copper sites inside chelating Superose. In a broader sense, these results suggest that during frontal analysis, the assumption of column saturation based on a plateau in the exit concentration that is almost at the same level as the input concentration could be misleading for highly cross-linked matrices and relatively large sized proteins. That is, the relatively less accessible copper sites may become difficult to be reached due to high levels of protein adsorption in the more accessible regions and thus give the appearance of a plateau in the breakthrough curve prior to complete column saturation. This is likely to be the case at high pH where BSA demonstrates very high affinity for immobilized copper or at high input concentrations where the equilibrium coverage is expected to be high. The results demonstrate that the estimated saturation capacity could be significantly smaller than the actual capacity.
Patwardhan
A V
AV
Department of Chemical Engineering, University of Pittsburgh, PA 15219, USA.
Ataai
M M
MM
D001665:Binding Sites; D002614:Chelating Agents; D002846:Chromatography, Affinity / Q000379:methods*; D002847:Chromatography, Agarose; D003300:Copper / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals; D012710:Serum Albumin, Bovine / Q000302:isolation & purification*
D002614:Chelating Agents; D008670:Metals; D012710:Serum Albumin, Bovine; D003300:Copper