Fluid shift and protein leakage corrections in protein binding equilibrium dialysis experiments |
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Authors: | Soo Peang Khor Hsiu Jean Wu Harold Boxenbaum |
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Affiliation: | School of Pharmacy, University of Connecticut, Storrs. CT 06268, U.S.A. |
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Abstract: | Conventional equilibrium dialysis methods used in the estimation of ligand-macromolecule binding parameters (affinities, capacities. unbound ligand fractions, etc.) tacitly assume and require that no ligand-macromolecule binding occurs on the buffer side of the dialysis chamber. Unfortunately, this is almost never a valid assumption, as small amounts of plasma proteins are invariably detected in the buffer chamber. Provided the extent of protein leakage is in the usual region (about 0.1%) and the extent of ligand binding does not exceed approximately 99%. errors associated with conventional free fraction estimations obtained from calculations ignoring the effect of protein leakage are usually small (about 1–10% error). As ligand binding exceeds 99%, significant errors may ensue. A generalized theoretical equilibrium dialysis method is developed which permits the estimation of association constants, the number of binding sites and free fraction determinations for a model employing any number of classes of binding sites. Application of the method requires a minimum of two experimental runs for each class of binding sites; volume shifts are automatically adjusted for by the method. |
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Keywords: | protein binding equilibrium dialysis volume shifts protein leakage |
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