Spectroscopic investigation into the interaction of a diazacyclam-based macrocyclic copper(ii) complex with bovine serum albumin Nahid Shahabadi, a,b * Mohammad Hakimi, c Teimoor Morovati, c Saba Hadidi a,b and Keyvan Moeini c ABSTRACT: Cyclam-based ligands and their complexes are known to show antitumor activity. This study was undertaken to examine the interaction of a diazacyclam-based macrocyclic copper(II) complex with bovine serum albumin (BSA) under physiological conditions. The interactions of different metal-based drugs with blood proteins, especially those with serum albumin, may affect the concentration and deactivation of metal drugs, and thereby influence their availability and toxicity during chemotherapy. In this vein, several spectral methods including UV–vis absorption, fluorescence and circular dichroism (CD) spectroscopy techniques were used. Spectroscopic analysis of the fluorescence quenching confirmed that the Cu(II) complex quenched BSA fluorescence intensity by a dynamic mechanism. In order to further determine the quenching mechanism, an analysis of Stern–Volmer plots at various concentrations of BSA was carried out. It was found that the K SV value increased with the BSA concentration. It was suggested that the fluorescence quenching process was a dynamic quenching rather than a static quenching mechanism. Based on Förster’s theory, the average binding distance between the Cu(II) complex and BSA (r) was found to be 4.98 nm; as the binding distance was less than 8 nm, energy transfer from BSA to the Cu(II) complex had a high possibility of occurrence. Thermodynamic parameters (positive ΔH and ΔS values) and measurement of competitive fluorescence with 1-anilinonaphthalene-8-sulphonic acid (1,8-ANS) indicated that hydrophobic interaction plays a major role in the Cu(II) complex interaction with BSA. A Job’s plot of the results confirmed that there was one binding site in BSA for the Cu(II) complex (1:1 stoichiometry). The site marker competitive experiment confirmed that the Cu(II) complex was located in site I (subdomain IIA) of BSA. Finally, CD data indicated that interaction of the Cu(II) complex with BSA caused a small increase in the α-helical content. Copyright © 2016 John Wiley & Sons, Ltd. Keywords: diazacyclam-based macrocyclic copper(II) complex; bovine serum albumin; protein–metal complex interaction; multi- spectroscopic methods Introduction The field of bioinorganic chemistry is an area of highly interdisci- plinary research, which deals with inorganic chemistry and biology, and puts forth new avenues for scientific investigation into coordination compounds (1,2). Discoveries in this field pose a significant impact on modern clinical medicine and have predominantly emerged in the form of either metal-containing diagnostic imaging agents or metal-containing therapeutics (3). Copper-based drugs have been now proposed as potential anticancer substances, as they demonstrate remarkable anticancer activity and show general toxicity lower than platinum com- pounds. Copper is well known as a bioessential element. Many redox enzymes contain copper atoms bound to protein molecules such as hemocyanins, superoxide dismutase, and blue copper proteins. In addition, copper ions are found to be present in the active sites of a large number of metalloproteins, which are involved in important biological electron transfer as well as in molecular oxygen redox reactions. Copper complexes have proven to be excellent candidates for biological applications due to their binding ability and positive redox potential (4–7). Copper(II) complexes play an important role in the active sites of a large number of metalloproteins in biological systems and are a potential application for numerous catalytic processes in living organisms that involve electron transfer reactions or activation of some antitumor substances (8). Copper can bind to ligands of various types to form complexes that interact with biomolecules, mainly proteins and nucleic acid (9). The investigation of com- pounds with respect to their binding to albumins becomes important because of the pharmacokinetic and pharmacodynam- ics role of such binding reactions ((10,11)). Serum albumins are the most abundant proteins in the circulatory systems of a wide variety of organisms. They have an important role in bioregulatory functions such as maintenance of the colloidal osmotic blood pressure and blood pH. In addition, serum albumins serve both * Correspondence to: N. Shahabadi, Inorganic Chemistry Department, Faculty of Chemistry, Razi University, Kermanshah, Iran. Tel:/Fax: +98 83 34274559. E-mail: nahidshahabadi@yahoo.com a Inorganic Chemistry Department, Faculty of Chemistry, Razi University, Kermanshah, Iran b Medical Biology Research Center (MBRC), Kermanshah University of Medical Sciences, Iran c Department of Chemistry, Payame Noor University, 19395–4697 Tehran, Iran Abbreviations: 1, 8-ANS, 1-anilinonaphthalene-8-sulphonic acid; BSA, bovine serum albumin; CD, circular dichroism; Förster/FRET, fluorescence resonance energy transfer; MRE, mean residue ellipticity; UV, ultraviolet Luminescence 2016 Copyright © 2016 John Wiley & Sons, Ltd. Research article Received: 1 January 2016, Revised: 17 March 2016, Accepted: 17 March 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/bio.3146