Electrophoretic and chromatographic differentiation of two forms of albumin in equilibrium at neutral pH: new screening techniques for determination of ligand binding to albumin.
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 form 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 equilibrium. 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 1/4 h. It is the first time that a conformational change for albumin with such a long conversion time has been described experimentally.
Department of Chemistry, Royal Veterinary & Agricultural University, Frederiksberg C, Denmark.
O J
OJ
Bjerrum
M J
MJ
Bjerrum
N H
NH
Heegaard
D001663:Bilirubin / Q000097:blood; D002845:Chromatography / Q000379:methods*; D006207:Half-Life; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006932:Hyperbilirubinemia / Q000097:blood; D007123:Immunoelectrophoresis, Two-Dimensional / Q000379:methods*; D007231:Infant, Newborn; D007700:Kinetics; D011487:Protein Conformation; D012709:Serum Albumin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D012709:Serum Albumin; D001663:Bilirubin