INTERACTION OF HUMAN SERUM ALBUMIN WITH HEMATOPORPHYRIN AND ITS ZN2+‐AND FE3+‐DERIVATIVES

Abstract

Human serum albumin at pH values above 6.8 has one strong binding site for hematoporphyrin; the stability constant of the 1:1 complex is about 10⁶ M^-1 as determined by Scatchard plot after estimation of the bound hematoporphyrin-induced quenching of the fluorescence emitted by the single tryptophanyl residue of the protein. Determination of the tryptophan-to-hematoporphyrin energy transfer efficiency yields a Förster parameter R₀ of 6.2–6.9nm, depending on the value chosen to represent the donor-acceptor mutual orientation, and a tryptophan-to-hematoporphyrin distance of about 1.7nm. Zn²⁺- and Fe³⁺- hematoporphyrin also give a 1:1 complex with albumin, probably binding at the same site as hematoporphyrin, as shown by the identity of the energy transfer parameters; however, the metal ions do not appear to be involved in the formation of the albumin-porphyrin complex. The albumin-hematoporphyrin interaction is drastically affected by the pH of the medium; below pH 6.5 we find a large number of binding sites with weak affinity for hematoporphyrin, which disappear upon increasing the pH. The main site, below pH 6.5, has an affinity comparable with that of the secondary sites. Circular dichroism studies show that the pH effect is due to a change in the protein conformation leading to different interactions between bound porphyrin and specific amino acid side chains.