Binding of Indole Analogues to Human Serum Albumin

In earlier studies on the binding of indole analogues with defatted human serum albumin (l), the intrinsic association constants for both a neutral ligand and a ligand bearing a negative charge were found to be invariant in the pH region of 5 to 10, implying that the structure of the binding site on the albumin did not change in this region. As the pH increased or decreased from this region the transition from binding to nonbinding took place within a very small pH interval-an interval smaller than one that could be assigned to a normal ionization process. Since conformational changes are known to occur in the albumin structure at pH 4 to 5 and at pH 10 to 11 (2-6), the rapid loss of binding in these regions was correlated with changes in gross protein structure. It was also demonstrated that the conformational changes in both pH regions involved a common part of the protein molecule, i.e. the binding site. These studies further showed the importance of considering various parameters: electrostatic effects, ion inhibitor effects, and salting out effects in an evaluation of the binding profiles. The purpose of the present investigation was to examine the binding profiles of indole analogues and albumin from which the fatty acids had not been removed. Some preliminary investigation of albumin in the alkaline pH region had shown that there were marked differences between the defatted and nondefatted albumin preparations, both in the magnitude of the respective association constants and in the shape of the pH-binding profiles (7). It therefore appeared worthwhile to investigate in greater detail the manner in which fatty acids affect the association properties of albumin. In view of the rather extensive binding studies already reported for defatted albumin (l), the present studies are concerned primarily with the association properties of nondefatted albumin, or of defatted albumin which has been reconstituted with fatty acid.