Human serum albumin. A study of the nature of its hydrophobic binding sites

Spectroscopic labels and hydrophobic chromatography on two different supports were used to compare the size and accessibility of the hydrophobic binding sites of human serum albumin with the accessibility of non-polar residues on the surface of other globular proteins. The binding of the labels 1-alkyl-4-(3-ethoxy-4-hydroxystyryl)pyridinium bromides (HPB) with alkyl chains of different length was investigated in the ultracentrifuge and by spectrophotometry. n-Butyl (C4-HPB) and decyl (C10-HPB) labels bind to albumin with association constants of 8 . 103 and 4 . 104, respectively, at pH 5·50, and with constant of 2·6 . 105 for the C10-HPB label at pH 9·2. Whereas C4-HPB interacts with the site of local polarity of albumin not different from the bulk solution, both the C10-HPB and C16-HPB on the other hand bind to hydrophobic sites, where the solvation of the chromophore is largely reduced as evidenced by the 46 nm shift to higher wavelengths in its spectrum. For other proteins the shift was less then 5 nm. Ten molecules of C10-HPB and four molecules of C16-HPB can be attached to one molecule of albumin. The changes in the spectrum of the bound label induced by palmitate reveal that these binding sites are essentially the same as those for fatty acids. From chromatographic experiments with labeled albumin at different pH carried out on Octyl-Sepharose and Spheron the conclusion was made that the latter support interacts preferentially with the non-polar side chains on the surface of proteins. The retention and the recovery of albumin and defatted albumin was investigated as a function of salt and alcohol concentration and compared with the same parameters of other proteins. In agreement with the proposed structure of the domains of albumin evidence was obtained that the outer surface of the albumin molecule is at neutral pH predominantly hydrophilic, and that the exceptionally large hydrophobic areas are localized solely in crevices.