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Analytical Biochemistry 397 (2010) 218–226

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Analytical Biochemistry
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A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon
Crystal E. Säbel, Joseph M. Neureuther, Stefan Siemann *
Department of Chemistry and Biochemistry, LaurentianUniversity, Sudbury, Ont., Canada P3E 2C6

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Zincon (2-carboxy-20 -hydroxy-50 -sulfoformazylbenzene) has long been known as an excellent colorimetric reagent for the detection of zinc and copper ions in aqueous solution. To extend the chelator’s versatility to the quantification of metal ions in metalloproteins, the spectral properties of Zincon and itscomplexes with Zn2+, Cu2+, and Co2+ were investigated in the presence of guanidine hydrochloride and urea, two common denaturants used to labilize metal ions in proteins. These studies revealed the detection of metals to be generally more sensitive with urea. In addition, pH profiles recorded for these metals indicated the optimal pH for complex formation and stability to be 9.0. As a consequence, anoptimized method that allows the facile determination of Zn2+, Cu2+, and Co2+ with detection limits in the high nanomolar range is presented. Furthermore, a simple two-step procedure for the quantification of both Zn2+ and Cu2+ within the same sample is described. Using the prototypical Cu2+/Zn2+–protein superoxide dismutase as an example, the effectiveness of this method of dual metal quantificationin metalloproteins is demonstrated. Thus, the spectrophotometric determination of metal ions with Zincon can be exploited as a rapid and inexpensive means of assessing the metal contents of zinc-, copper-, cobalt-, and zinc/copper-containing proteins. Ó 2009 Elsevier Inc. All rights reserved.

Article history: Received 18 September 2009 Received in revised form 9 October 2009 Accepted 21 October2009 Available online 23 October 2009 Keywords: Zincon Spectrophotometry Zinc Copper Cobalt Metalloproteins Superoxide dismutase

Introduction Metals are indispensable constituents of approximately onethird of all proteins [1]. As such, metals are involved in virtually all biological processes, including metabolism, energy transduction, gene expression, cell signaling, formation of endo- andexoskeletons, and electron and information transfer [1,2]. Among the techniques suitable for the quantification of metal ions in metalloproteins, inductively coupled plasma mass spectrometry and atomic absorption and emission spectroscopies are likely to be the most widely employed [3]. However, although these techniques are reliable and sensitive, they suffer from the limitation of being rathercostly (considering instrument acquisition and maintenance), time-consuming (with respect to sample preparation), and not always readily available [4,5]. Thus, simple spectrophotometric (or spectrofluorometric) techniques, which tend to be less costly and labor-intensive, are viable alternatives to those methods requiring more sophisticated instrumentation.

* Corresponding author. Fax: +1 705 6754844. E-mail address: (S. Siemann). 1 Abbreviations used: UV–Vis, ultraviolet–visible; GdnHCl, guanidine hydrochloride; SOD, superoxide dismutase; Mes, 2-(N-morpholino)ethanesulfonic acid; Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetraacetic acid; CyDTA, 1,2-cyclohexanediamine-N,N,N’,N’-tetraacetic acid; PAR, 4-(2-pyridylazo)resorcinol. 0003-2697/$ - seefront matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.10.037

The determination of trace metals by ultraviolet–visible (UV– Vis)1 spectroscopy typically relies on alterations in the absorption spectrum of a chromophoric chelator on binding the desired metal ion [6]. Among the many reagents eliciting complexation-induced spectral changes, the formazan dye Zincon (see Fig. 1)...
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