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Ligand binding to a human serum albumin stationary phase: use of same‐drug competition to discriminate pharmacologically relevant interactions
Author(s) -
Ascoli Giorgio A.,
Bertucci Carlo,
Salvadori Piero
Publication year - 1998
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/(sici)1099-0801(199809/10)12:5<248::aid-bmc742>3.0.co;2-9
Subject(s) - chemistry , human serum albumin , chromatography , high performance liquid chromatography , ligand (biochemistry) , plasma protein binding , stationary phase , serum albumin , biophysics , biochemistry , receptor , biology
Abstract A technique based on a human serum albumin (HSA) stationary phase high‐pressure liquid chromatography (HPLC) has been successfully used for the past few years to characterize the interactions between HSA and new substrates. Immobilized HSA conserves the binding properties of the protein in solution, allowing fast and reliable analyses of binding interactions. Nevertheless, clear evidence that all binding mechanisms of HSA‐HPLC are pharmacologically relevant is so far lacking. In particular, non‐stoichiometric interactions of injected ligands with stationary phase components such as silica and the amino acid medium (other than protein binding areas) might interfere with the correlation between chromatographic retention and HSA binding. Here we present a quantitative method to distinguish between the molecular interactions of a ligand with binding areas of potential pharmacological interest and other, non‐saturable binding mechanisms. Such a method, based on HPLC same‐ligand displacement, is simple and reliable, as confirmed by in situ protein denaturation. Consequently, we were able to distinguish between different types of competitions detected in the co‐binding of two drugs to HSA. © 1998 John Wiley & Sons, Ltd.