J. Biol. Chem., Vol. 263, Issue 30, 15392-15399, 10, 1988

Biphasic denaturation of human albumin due to ligand redistribution during unfolding

A Shrake and PD Ross
Division of Blood and Blood Products, Food and Drug Administration, Bethesda, Maryland 20892.

Denaturation of defatted human albumin monomer, monitored by differential scanning calorimetry, is monophasic as reflected by the single, resulting endotherm. With low levels of various ligands, biphasic or monophasic unfolding processes are manifested as bimodal or unimodal thermograms, respectively. The greater the affinity of native protein for ligand, the greater is the tendency for biphasic denaturation. We propose that such a biphasic unfolding process arises from a substantial increase in stability (transition temperature) of remaining native protein during denaturation. This increase in stability derives from the free energy of ligand binding becoming more negative due to the release of high affinity ligand by unfolding protein. The tendency for biphasic denaturation is greatest at low (subsaturating) levels of ligand where greatest increases in stability occur. Biphasic unfolding arising from such ligand redistribution results from denaturation of different kinds of protein molecules, ligand-poor and ligand-rich species, and not from sequential unfolding of domains within the same molecule. Differentiating between these two mechanisms is necessary for the correct interpretation of biphasic denaturation data. Furthermore, biphasic unfolding due to ligand redistribution occurs independently of the means used to effect denaturation. The maximum increase in stability due to ligand binding relative to the stability of defatted albumin monomer alone occurs with the intermediate affinity ligand octanoate (22 degrees C) and not with the high affinity ligand hexadecanoate (15 degrees C). This indicates a much greater affinity of denatured albumin for hexadecanoate since increase in stability derives from the difference between free energy of ligand binding to folded and unfolded protein forms.
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