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Electrophoretic and chromatographic differentiation of two forms of albumin in equilibrium at neutral pH: New screening techniques for determination of ligand binding to albumin
Author(s) -
Bjerrum Ole J.,
Bjerrum Morten J.,
Heegaard Niels H. H.
Publication year - 1995
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501601232
Subject(s) - albumin , chemistry , chromatography , sepharose , ligand (biochemistry) , serum albumin , kinetics , electrophoresis , plasma protein binding , bovine serum albumin , biochemistry , enzyme , receptor , physics , quantum mechanics
Analysis of normal human serum by crossed hydrophobic interaction immunoelectrophoresis with Phenyl‐Sepharose revealed a biphasic appearance of the albumin peak. The molecular mechanism behind this apparent albumin heterogeneity was investigated. Analysis of defatted purified albumin showed that a major fraction bound to the Phenyl‐Sepharose and that addition of ligands ( e.g. long‐chain fatty acids, bilirubin, sulfonamides and warfarin) before electrophoresis blocked this binding to different degrees. A quantitative relation between ligand binding and the amount of nonbinding albumin was found. Thus the technique might be suitable for screening of ligand binding to albumin. Analysis of serum samples from newborns with hyperbilirubinemia revealed a positive correlation between the fraction of the nonretarded albumin and the bilirubin concentration. By chromatography on Phenyl‐Sepharose, defatted albumin was separated into a binding and a nonbinding from and this technique was subsequently used to determine the kinetics of the intramolecular conversion. After rechromatography, each of the fractions could again be separated into two fractions, indicating the presence of an eqilibrium. By varying the passage time for albumin on the column or varying the period between the first and the second separation it was possible to calculate the conversion rates. The half‐life for the conversion was found to be as long as 1 ¼ h. It is the first time that a conformational change for albumin with such a long conversion time has been described experimentally.