High-performance ion-exchange chromatography and adsorption of plasma proteins.
Resolution and recovery are primary concerns in protein chromatography. Separations are often by size, ion exchange, or hydrophobic-hydrophilic properties of the support, eluent and protein. Adsorption of a protein to a synthetic surface plays an essential role in this complex process. In this study, we examined the adsorption properties of three representative plasma proteins (albumin, fibrinogen, and immunoglobulin G) on nonporous column materials containing either quaternary amine or sulfopropyl functional groups. The adsorption properties were studied at 37 degrees C and pH 7.4. Salt gradients were used to examine the adsorption/desorption properties of each of the proteins on each type of surface. The salt concentrations at desorption were measured and compared to the protein isoelectric points. In addition, we examined protein recoveries as a function of desorption time. Our results suggest that protein recoveries depend not only on the protein, eluent and surface, but also the residence time and overall charge concentration during the initial adsorption process. Finally, we correlated the number of charge sites on a molecule with the width of a chromatographic band at half height. The data produced as a result of this study may be used to determine the actual unfolding time of a protein, given a certain set of conditions. The data may also help in understanding the chemistry and dynamics of the protein adsorption processes in ion-exchange chromatography as well as provide key structural information about the proteins.
Goheen
S C
SC
Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Hilsenbeck
J L
JL
D000327:Adsorption; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002138:Calibration; D002417:Cattle; D002852:Chromatography, Ion Exchange; D005340:Fibrinogen / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification; D007202:Indicators and Reagents; D008970:Molecular Weight; D016062:Porosity; D012709:Serum Albumin / Q000737:chemistry / Q000302:isolation & purification
D001798:Blood Proteins; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D012709:Serum Albumin; D005340:Fibrinogen