Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio Molar absorption coefficients and stability constants of Zincon metal complexes for determination of metal ions and bioinorganic applications Anna Kocyła, Adam Pomorski, Artur Krężel ⁎ Department of Chemical Biology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland ARTICLE INFO Keywords: Zincon Molar absorption coefficient Stability constant Zinc protein Copper Metal ions ABSTRACT Zincon (ZI) is one of the most common chromophoric chelating probes for the determination of Zn 2+ and Cu 2+ ions. It is also known to bind other metal ions. However, literature data on its binding properties and molar absorption coefficients are rather poor, varying among publications or determined only in certain conditions. There are no systematic studies on Zn 2+ and Cu 2+ affinities towards ZI performed under various conditions. However, this widely commercially available and inexpensive agent is frequently the first choice probe for the measurement of metal binding and release as well as determination of affinity constants of other ligands/ macromolecules of interest. Here, we establish the spectral properties and the stability of ZI and its complexes with Zn 2+ , Cu 2+ , Cd 2+ , Hg 2+ , Co 2+ , Ni 2+ and Pb 2+ at multiple pH values from 6 to 9.9. The obtained results show that in water solution the MZI complex is predominant, but in the case of Co 2+ and Ni 2+ , M(ZI) 2 com- plexes are also formed. The molar absorption coefficient at 618 nm for ZnZI and 599 nm for CuZI complexes at pH 7.4 in buffered (I = 0.1 M) water solutions are 24,200 and 26,100 M -1 cm -1 , respectively. Dissociation constants of those complexes are 2.09 × 10 -6 and 4.68 × 10 -17 M. We also characterized the metal-assisted Zincon decomposition. Our results provide new and reassessed optical and stability data that are applicable to a wide range of chemical and bioinorganic applications including metal ion detection, and quantification and affinity studies of ligands of interest. Synopsis: Accurate values of molar absorption coefficients of Zincon complex with Zn 2+ , Cd 2+ , Hg 2+ , Co 2+ , Ni 2+ , Cu 2+ , and Pb 2+ for rapid metal ion quantification are provided. Zincon stability constants with Zn 2+ and Cu 2+ in a wide pH range were determined. 1. Introduction The determination of trace metal ions has long been an important subject in environmental analysis, many types of industrial processes, as well as chemical and biochemical reactions. Among analytical methods, UV–vis spectroscopy is the most widely used technique for metal ion determination due to its speed and simplicity [1–3]. Although most metal ions from the d and p blocks demonstrate characteristic spectroscopic properties in the UV–vis range, they are not used in trace analysis due to low molar absorption coefficients and possible over- lapping of bands. Other metal ions, such as Zn 2+ , are spectroscopically silent. Therefore, there is a need for selective metal chelators that de- monstrate much more visible spectral changes - metallochromic in- dicators. They change their spectroscopic properties upon metal ion binding and possess suitable complex affinities. Chronologically, one of the best known metal ion chelating chromophores is dithizone, which is a sensitive but not specific probe reacting with over 20 different metal ions, mostly in organic media [4]. Other examples of chromophoric chelators include Eriochrome blue SE, Eriochrome red B, Naphthyla- zoxine 6S, sodium salt of 7-(4-sulfo-1-naphthylazo)-8-hydroxyquino- line-5-sulfonic acid (SNAZOXS) and 4-(2-pyridylazo)resorcinol (PAR) [5–7]. The main limitation of metallochromic indicators is their lack of specificity and water solubility. They often require organic solvents, which is not desirable in most of the metalloprotein-based applications [8,9]. The sensitivity and selectivity of metal ion indicators was defi- nitely improved in the group of fluorogenic chelators, e.g. zinc probes: N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ), 2-[2-[2-[2-[bis (carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-4-(2,7-di- fluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3), ZnAF and the Zinpyr family [10–13]. Unfortunately, their high cost and poor market availability frequently make them impossible to use in routine analysis. One of the most common classical chelating chromophore is Zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene monosodium salt). It was first used for Zn 2+ and Cu 2+ determination by Yoe and Rush in 1952 [14,15]. This ligand has also been used for the spectroscopic http://dx.doi.org/10.1016/j.jinorgbio.2017.08.006 Received 31 May 2017; Received in revised form 27 July 2017; Accepted 21 August 2017 ⁎ Corresponding author. E-mail address: artur.krezel@uwr.edu.pl (A. Krężel). Journal of Inorganic Biochemistry 176 (2017) 53–65 Available online 24 August 2017 0162-0134/ © 2017 Elsevier Inc. All rights reserved. T