Journal of Inorganic Biochemistry 89 (2002) 45–53 www.elsevier.com / locate / jinorgbio Metal binding by melanins: studies of colloidal dihydroxyindole-melanin, and its complexation by Cu(II) and Zn(II) ions b a a a, * Bruno Szpoganicz , Shirley Gidanian , Philip Kong , Patrick Farmer a Department of Chemistry, University of California, Irvine, Irvine, CA 92697-2025, USA b ´ Departamento de Quımica, Universidade Federal de Santa Catarina, Florianopolis, SC 88040-900, Brazil Received 30 July 2001; received in revised form 17 October 2001; accepted 5 November 2001 Abstract Melanins are colloidal pigments known to have a high affinity for metal ions. In this work, the nature of the metal-binding sites are determined and the binding affinities are quantified. Initial potentiometric titrations have been performed on synthetic dihydroxyindole (DHI) melanin solutions to determine the chemical speciation of quinole / quinone subunits. Two types of acidic functionalities are assignable: catechol groups, with pK between 9 and 13, and quinone imines (QI), with pK of 6.3. The presence of the quinone-imine a a tautomer has, to our knowledge, never been assessed in polymeric melanins. Melanin solutions obtained from N-methylated DHI lack the pK 6.3 buffer, consistent with its inability to form the quinone-imine tautomer. EPR spectroscopy of the DHI-melanin samples a demonstrates that the semiquinone radical is in too low a concentration to contribute to the bulk binding of metals. Changes in the titration curves after addition of Cu(II) and Zn(II) ions were analyzed to obtain the binding constants and stoichiometry of the metal-melanin complexes, using the BEST7 program. UV–Vis spectra at neutral and high pH are used to identify absorbances due to Cu-bound quinone imine and catechol groups. The derived binding constants were used to determine speciation of the Cu(II) and Zn(II) ions coordinated to 2 2 the quinone imine and catechol groups at various pH. The mixed complexes, Zn(QI)(Cat) and Cu(QI)(Cat) are shown to dominate at physiological pH.  2001 Elsevier Science Inc. All rights reserved. Keywords: Melanin; Dihydroxyindole (DHI); Quinone imine; Potentiometric titration; Metal-binding constants 1. Introduction ability of melanins to act as buffers against oxidative and photochemical stress in vivo, and has been identified as a Melanins are ubiquitous black pigments in nature, possible target for chemotherapy [11]. formed from tyrosine oxidations [1–4]. In melanocytes, a specific enzyme tyrosinase catalyzes two successive re- actions, hydroxylation of tyrosine and oxidation of the product, L-dopa. The oxidation of dopa produces a cyclized 5,6-dihydroxyindole intermediate, which is highly reactive, and upon further oxidation gives rise to eumelanic poly- mers by a radical-coupling pathway. Natural melanins contain a wide variety of bound metals in vivo [5–8]. They are unusual among the biopolymers in that they are highly polyionic, cross-linked, and heterogeneous polymers, whose bonding patterns likely vary in subtle ways. Also, they are unique in that they contain a stable population of organic free radicals indicative of semiquinone monomers, intermediate between the quinol and quinone oxidation states [2,7,9,10]. This redox reactivity is inherent in the The binding of metals to melanins also affects the ability of native melanins to protect cells from harmful redox- *Corresponding author. Fax: 11-949-824-2210. E-mail address: pfarmer@uci.edu (P. Farmer). active species such as reactive oxygen species (ROS) and 0162-0134 / 01 / $ – see front matter  2001 Elsevier Science Inc. All rights reserved. PII: S0162-0134(01)00406-8