Structural heterogeneity of the binding sites of HSA for phenyl-groups and medium-chain fatty acids. Demonstration of equilibrium between different binding conformations.
A new facet of the very heterogeneous albumin molecule is described. Chromatography at pH 6-9 of human serum albumin (HSA) on a phenyl-sepharose column separates it into two nonconvertible conformations that are, in turn, in equilibrium with its binding and nonbinding forms. The hydrophobic interaction of HSA with phenyl-sepharose depends on ionic strength, pH, and time of contact with the immobilized ligand. Binding as a function of pH shows a minimum at pH 6.5, and the binding profile at pH 7-9 fits the titration of a weak monoprotic acid with a pKa of 7.3. There was no observable difference in the CD spectra or the masses of the two forms. The equilibrium between the albumin forms was examined under defined conditions and cannot be explained by a simple two-state model. Thus rechromatography of the nonbinding fraction derived from a sample in which 50% of the protein was originally retained resulted only in 10-20% bound protein. Correspondingly only 70-80% of the binding form was retained. A model explaining the observations can be derived if two species, I and II, exist in the solution, both being in an equilibrium with a binding and a nonbinding form, but in which I is not in equilibrium with II. The rate of conversion between the binding and nonbinding conformations was determined to be faster than 15 s at room temperature.
Lund
M
M
Department of Chemistry, Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
Bjerrum
O J
OJ
Bjerrum
M J
MJ
D001665:Binding Sites; D002942:Circular Dichroism; D003652:Decanoic Acids / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D012685:Sepharose / Q000031:analogs & derivatives* / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism*
D003652:Decanoic Acids; D005227:Fatty Acids; D012709:Serum Albumin; C031071:decanoic acid; C020927:phenyl-sepharose; D012685:Sepharose