1042 J. Sep. Sci. 2015, 38, 1042–1045 Jana Kazarjan 1 Merike Vaher 1 Th ´ er ` ese Hunter 2 Maria Kulp 1 Gary James Hunter 2 Rosalin Bonetta 2 Diane Farrugia 2 Mihkel Kaljurand 1 1 Department of Chemistry, Tallinn University of Technology, Tallinn, Estonia 2 Department of Physiology and Biochemistry, University of Malta, Msida, Malta Received August 22, 2014 Revised December 23, 2014 Accepted December 26, 2014 Short Communication Determination of metal content in superoxide dismutase enzymes by capillary electrophoresis † Superoxide dismutases are antioxidant scavenger enzymes that contain a metal cofactor (copper, zinc, iron, and manganese) in their active site. Metal content measurement is one of the essential steps to characterize enzyme biological activity. We have developed a capillary electrophoretic protocol for the determination of the metal content in superoxide dismutase enzymes. The background electrolyte containing 10 mM pyridine-2,6-dicarboxylic acid and 1 mM 1-methyl-3-tetradecylimidazolium chloride at pH 3.8 was optimized for on-column complexation of the above-mentioned metals. The minimum detectable levels of metals ranged from 0.3 to 1.2 g/mL. The reliability of the method was checked by parallel quantitative determination of the metal content in superoxide dismutase enzymes by graphite furnace or flame atomic absorption spectrophotometry methods. Keywords: Capillary electrophoresis / On-column complexation / Quantitative metal analysis / Superoxide dismutase DOI 10.1002/jssc.201400925  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 Introduction Superoxide dismutases (SOD) are metalloenzymes that protect cells against oxidative damage caused by superoxide radicals that are unavoidably formed by aerobic respiration. Three forms of SOD have been identified, each having differ- ent metal cofactors at their active site: CuZnSOD, FeSOD, and MnSOD. These metal cofactors are necessary for the redox cy- cling that results in the disproportionation of the superoxide radical into molecular oxygen and hydrogen peroxide. SOD activity, therefore, depends on the degree of metalation [1–3]. Currently, there are several methods to measure the metal content in proteins. The most common ones are graphite fur- nace or flame atomic absorption spectrophotometry (GFAAS or FAAS) and inductively coupled plasma with atomic emis- sion spectrophotometry [4, 5]. Compared to these techniques, CE is a fast and simple method that has advantages such as high separation ability of mixtures of ions, small sample, and BGE consumption and ease of automation [6]. Moreover, Correspondence: Jana Kazarjan, Department of Chemistry, Tallinn, University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia E-mail: jana.kazarjan@gmail.com Fax: +372-620-43-25 Abbreviations: AAS, atomic absorption spectrophotome- try; C 14 MImCl, 1-methyl-3-tetradecylimidazolium chloride; GF/FAAS, graphite furnace/flame atomic emission spec- trophotometry; IL, ionic liquid; PDC, pyridine-2,6-dicarboxylic acid; SOD, superoxide dismutase CE has been used for the general characterization of SODs [7, 8]. CE has been extensively used for the determination of metal ions in different matrices [9, 10]. In general, there are two possible ways to measure metals by CE [11]. The First one is the precolumn complexation, where an excess of ligand is added to the sample to ensure the complete complex forma- tion [12]. The second approach is on-column complexation, which allows direct injection of sample to CZE where a rapid complexation reaction between metal ions and ligand(s) oc- curs [13]. On-column complexation of metal ions with direct UV detection is possible when using pyridine-2,6-dicarboxylic acid (PDC) as a ligand that chelates metal ions producing an- ionic complexes [14, 15]. To get a fast CE separation of the an- ionic metal complexes, addition of a cationic surfactant is nec- essary. An ionic liquid (IL), 1-methyl-3-tetradecylimidazolium chloride (C 14 MImCl), which has a long alkyl chain on the cation, was used in this work as an additive in the BGE to re- verse the direction of the EOF, and detection of the chelated metals was performed in reverse polarity separation mode. Nowadays ILs have numerous chemical applications and are used in various analytical techniques including HPLC, GC, and MS [16–18]. ILs also serve as BGE additives in CE in the analysis of different samples [19, 20]. † This paper is included in the virtual special issue on Amino acids, proteins and peptides available at the Journal of Separation Science website. C  2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com