TY - JOUR
T1 - Competitive ion-exchange adsorption of proteins
T2 - Competitive isotherms with controlled competitor concentration
AU - Cano, Tony
AU - Offringa, Natalie D.
AU - Willson, Richard C.
N1 - Funding Information:
This work was funded by the National Science Foundation grant CTS-0004544 and by the Robert A. Welch Foundation under grant number E-1264. We would also like to thank Professor Roman Czernuszewicz for Raman spectroscopic characterization of the Manganese-protoporphyrin IX variant proteins in his laboratory. RCW wishes to acknowledge the generous support and encouragement of Cs. Horváth early in the development of his work in this area.
Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2005/6/24
Y1 - 2005/6/24
N2 - The competitive adsorption processes inevitably present in chromatographic separations of complex mixtures have not been extensively studied. This is partly due to the difficulty of measuring true competitive isotherms, in which all system parameters (including competitor concentrations) are held constant. We report a novel approach to determining competitive protein adsorption isotherms in which the competitor concentration is held constant across the entire isotherm. By using the heme prosthetic group in cytochrome b5 as a quantitative spectrophotometric label, competitive isotherms between cytochrome b5 and α-lactalbumin can be constructed. Similarly, manganese-substituted protoporphyrin IX heme replacement allows the non-perturbing labeling of individual cytochrome b5 conservative surface charge mutants by replacement of a single atom in the interior of the protein. This labeling allows the study of competition between cytochrome b 5 charge mutants of identical size and shape, which differ only in charge arrangement. Using these techniques, the effect of competing species on equilibrium behavior and the apparent heterogeneity of anion-exchange adsorbents in the presence of competitors can be quantitatively studied by fitting the data to two popular single-component binding models, the Temkin and the Langmuir-Freundlich (L-F) isotherms.
AB - The competitive adsorption processes inevitably present in chromatographic separations of complex mixtures have not been extensively studied. This is partly due to the difficulty of measuring true competitive isotherms, in which all system parameters (including competitor concentrations) are held constant. We report a novel approach to determining competitive protein adsorption isotherms in which the competitor concentration is held constant across the entire isotherm. By using the heme prosthetic group in cytochrome b5 as a quantitative spectrophotometric label, competitive isotherms between cytochrome b5 and α-lactalbumin can be constructed. Similarly, manganese-substituted protoporphyrin IX heme replacement allows the non-perturbing labeling of individual cytochrome b5 conservative surface charge mutants by replacement of a single atom in the interior of the protein. This labeling allows the study of competition between cytochrome b 5 charge mutants of identical size and shape, which differ only in charge arrangement. Using these techniques, the effect of competing species on equilibrium behavior and the apparent heterogeneity of anion-exchange adsorbents in the presence of competitors can be quantitatively studied by fitting the data to two popular single-component binding models, the Temkin and the Langmuir-Freundlich (L-F) isotherms.
KW - Anion-exchange
KW - Competitive adsorption
KW - Competitive binding
KW - Competitive isotherms
KW - Ion-exchange
KW - Langmuir-Freundlich isotherm
KW - Protein adsorption
KW - Temkin isotherm
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U2 - 10.1016/j.chroma.2005.03.120
DO - 10.1016/j.chroma.2005.03.120
M3 - Article
C2 - 16038297
AN - SCOPUS:20444432723
VL - 1079
SP - 116
EP - 126
JO - Journal of Chromatography A
JF - Journal of Chromatography A
SN - 0021-9673
IS - 1-2 SPEC. ISS.
ER -