The application of capillary electrophoresis, mass spectrometry and Brdicka reaction in human and rabbit metallothioneins analysis

DOI 10.17219/acem/98916 Copyright © 2018 byWroclaw Medical University This isanarticle distributed under theterms ofthe Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc-nd/4.0/) Address for correspondence Marta Kepinska E-mail: zalew[email protected] Funding sources Study was supported bygrant No. Pbmn178 from Wroclaw Medical University and by the AZV (Czech Agency for Healthcare Research) AZV CR grant No. 14-28334a (Prague, Czech Republic). Conflict ofinterest None declared Received onJune 6, 2018 Reviewed onAugust 3, 2018 Accepted onOctober 11, 2018 Abstract Background. Metallothioneins (MTs) constitute afamily ofevolutionary conserved low molecular weight proteins with small variations intheir amino acid sequences. They play arole inthe regulation oftrace metals metabolism, inthe detoxification ofheavy metal ions and inmechanisms controlling growth, differentiation and proliferation ofcells. Objectives. Theaim ofthis study was toevaluate the human and rabbit MTs purity and characterization using advanced analytical approaches. Due tothe common use ofMT from rabbit liver as amodel protein, the properties ofthe rabbit and human MTs were compared. Material and methods. Capillary electrophoresis (CE), matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Brdicka reaction were used for human and rabbit MTs characterization. Results. Inchip CE analysis, changes inthe range of5–8 kDa corresponding tothe MT monomer, as well as some peaks of13–14 kDa corresponding todimers inboth species, were observed. Using MALDI-MS, rabbit (MT-2D) and human (MT-1A, MT-1G, MT-1G + Cd and MT-2A) MTs were identified. Inthe Brdicka reaction analysis, alower concentration ofMTs from both organisms coincided with adecrease inthe signal corresponding toMT level (Cat2). However, human MT gave higher Cat2 peak than the same concentration (0.025mg/mL) ofrabbit MT. Conclusions. Theapplied methods allowed for the characterization ofMTs and gave complementary information about MT isoforms. Altered electrochemical activity ofhuman and rabbit MTs, despite the same number of–sulfhydryl (−SH) groups, was observed, which may be due todifferent availability ofMT cysteinyl groups. Key words: metallothionein, mass spectrometry, capillary electrophoresis, Brdicka reaction Original papers Theapplication ofcapillary electrophoresis, mass spectrometry andBrdicka reaction inhuman and rabbit metallothioneins analysis Marta Kepinska1,A–F, Sona Krizkova2,3,B,F, Ewelina Guszpit1,B,F, MiguelA. Merlos Rodrigo2,3,B,F, Halina Milnerowicz1,E,F 1 Department of Biomedical and Environmental Analysis, Faculty of Pharmacy with Division of Laboratory Medicine, Wroclaw Medical University, Poland 2 Department ofChemistry and Biochemistry, Faculty ofAgronomy, Mendel University inBrno, Czech Republic 3 Central European Institute ofTechnology, Brno University ofTechnology, Czech Republic A– research concept and design; B – collection and/orassembly ofdata; C – data analysis and interpretation; D – writing thearticle; E – critical revision ofthearticle; F – final approval ofthearticle Advances inClinical and Experimental Medicine, ISSN 1899-5276 (print), ISSN 2451-2680 (online) Adv Clin Exp Med. 2018;27(11):1601–1608 M. Kepinska, etal. Metallothioneins characterization 1602 Introduction Metallothioneins (MTs) make up alarge family ofevolutionarily conserved low molecular weight, thermostable proteins, found inpractically all life forms: invertebrates, invertebrates, fungi, and even inplants. Their sizes range from 25 (fungal) to84 (plant) amino acids.1 Asingle polypeptide chain ofmammalian MT iscomposed of61–68 mostly non-aromatic amino acids and contains 20 cysteinyl residues inhighly conserved locations along the peptide chain. High number ofcysteines inMTs explains their high capacity for binding metal ions through sulfhydryl groups, forming metal–thiolate complexes. Thus, their main proposed functions were derived from their role inthe homeostatic control ofanumber ofessential metals (copper –Cu, zinc –Zn), in detoxification oftoxic ones (e.g., cadmium –Cd, mercury –Hg), and inprotective actions against oxidative stress conditions.2–4 This known fact forms the basis ofMTs being used as possible biomarkers for metal exposure.5 Four isoforms ofmammalian MT (MT-1, MT-2, MT3, and MT-4) have been identified. Isoforms with minor differences, such as amino acid residue, have been detected as subgroups ofthe 2major isoforms (MT-1 and MT-2) and are termed subisoforms. These 2 main groups ofMT isoforms have been classified according tohuman MT elution order byanionic-exchange chromatography: MT-1 and MT-2.6 They are the most widely distributed MT isoforms, expressed inmany cell types indifferent tissues and organs. They can be induced bythe administration ofmetals such as Cd, Zn and Cu; byoxidative stress; byglucocorticoids; and bycytokines.7 MT-1 and MT-2 are present innearly all tissues and have been isolated from organs such as the liver, kidney and brain. MT-3 isexpressed mostly innervous tissue8 and MT-4has been detected inepithelial cells.9 Since various subisoforms are biosynthesized differentially, they could play aspecific biological role, but this aspect isstill being investigated. Theinteresting properties ofMTs are closely related to their structure. Inaddition toslight differences inamino acid sequences, MTs exist as amixture ofvarying metallic content.5 Itisnot a simple task todetect and quantify MT, also due toits high cysteine content and relatively low molecular mass. Thecharacteristics ofhuman MTs are not widely described because rabbit MTs are more often used instudies as amodel protein; thus, itisso valuable tocompare MTs from these organisms. Toanalyze MTs, many methods can be used that differ inthe analytical approach, usually based onthe metal ions detection, free thiol moieties or enzyme-linked immunosorbent assay (ELISA). Human and rabbit MTs have previously been analyzed bywestern blot, ELISA, capillary electrophoresis (CE), and high-performance liquid chromatography.10–15 Therefore, the purpose ofthe research was the comprehensive characterization ofhuman and rabbit MTs. Modern electrochemical and advanced analytical approaches such as the Brdicka reaction, chip CE and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used tocharacterize and compare human and rabbit MTs. Material and methods Material All chemicals used were commercially available. Rabbit liver MT-2 (M-5392), 2-mercaptoethanol, ammonia solution, ammonium bicarbonate, trifluoroacetic acid (TFA), and formic acid were purchased from Sigma–Aldrich (St. Louis, USA). Acetonitrile (ACN) and methanol were purchased from Fluka (Seelze, Germany). Acidic MALDI matrices α-cyano4-hydroxycinnamic acid (HCCA), 2,5-dihydroxobenzoic acid (DHB) and peptide calibration standard mixture were purchased from Bruker Daltonics (Bremen, Germany). All reagents were ofthe highest available purity. Doubly distilled deionized water was used throughout all the experiments. Preparation ofmetallothioneins from human liver Isoforms MT-1 and MT-2 were purified from human liver earlier at the Department ofBiomedical and Environmental Analysis, Faculty of Pharmacy with Division of Laboratory Medicine, Wroclaw Medical University, Poland, and the presence ofMTs was confirmed bysodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) and western blot.10 Chip capillary electrophoresis Capillary electrophoresis experiments were performed onanautomated microfluidic Experion electrophoresis system (Bio-Rad, Hercules, USA), according tothe manufacturer’s instructions and with supplied chemicals (Experion Pro260 analysis kit, Cat. No.700-7101, Bio-Rad). Metallothioneins inaquantity of4µL (0.1 or 0.18µg/µl) were mixed with 2µL ofreducing sample buffer (30µL ofanonreducing sample buffer and 1µL ofβ-mercaptoethanol); after 4min ofboiling, 84μL ofwater were added. Theladder was also prepared inreducing conditions. After the priming ofthe chip with the gel and gel-staining solution inthe diluted priming station sample, 6µL ofthe mixture were loaded into the sample wells. ThePro260 Ladder included inthe kit was used as astandard. For operation and standard data analysis, Experion software v. 3.10 (Bio-Rad) was used. Mass spectrometry analysis Mass spectrometry analysis was performed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Working standard Adv Clin Exp Med. 2018;27(11):1601–1608 1603 solutions were prepared daily bydiluting the stock solutions. Amixture ofpeptide calibration standard (Bruker Daltonics) was used tocalibrate the instrument. All measurements were performed onthe UltrafleXtreme MALDITOF/TOF mass spectrometer (Bruker Daltonics) equipped with alaser operating at awavelength of355nm with anaccelerating voltage of25 kV, cooled with liquid nitrogen, at amaximum energy of36µJ with arepetition rate of2000Hz inreflector-positive ionization mode, and with software for data acquisition and processing ofmass spectra flexControl v. 3.4 and flexAnalysis v. 2.2. Thespectra were measured inthe range of0–20,000mass-to-change ratio (m/z). Laser power intensity was set to5% above the threshold, which determines the minimal intensity for suitable spectrum; itisset experimentally bymeasuring spectra with increasing laser power intensity. AMALDI MTP 384 target plate (polished steel) was used. Before MT analysis, acut-off filter (Amicon 3K; Merck Millipore, Burlington, USA) for desalinization ofthe MT sample and buffer exchange towater was used. Then, the samples were mixed with DHB or HCCA matrix solution in1:1 volume ratio and then 1µL ofthe mixture was deposited onthe target plate (dried-droplet method) and dried under atmospheric pressure and ambient temperature. Saturated solutions ofDHB or HCCA were prepared inTA30 (30% ACN and 0.1% TFA). Mass spectra were typically acquired byaveraging 20 subspectra from atotal of500 shots ofthe laser (Smartbeam 2 v. 1_0_38.5; Bruker Daltonics). Differential pulse voltammetry Differential pulse voltammetry (DPV) measurements were performed with a747 VA Stand instrument connected toa746 VA Trace Analyzer and a695 Autosampler (Metrohm, Herisau, Switzerland) using astandard cell with 3 electrodes and acooled sample holder (4°C). Ahanging mercury drop electrode (HMDE) with adrop area of0.4mm2 was used as the working electrode. An Ag/AgCl/3M KCl was the reference electrode and aglassy carbon electrode was the auxiliary electrode. TheGPES v. 4.9 software supplied byEcoChemie (Utrecht, the Netherlands) was employed for smoothing and baseline correction ofthe raw data. TheBrdicka supporting electrolyte containing 1mM Co(NH3)6Cl3 and 1 Mammonia buffer (NH3(aq) + NH4Cl, pH=9.6) was used. Theparameters ofthe measurement were as follows: initial potential of−0.7 V, end potential of−1.75 V, modulation time 0.057s, time interval 0.2s, step potential 2 mV, and modulation amplitude −250 mV. All experiments were carried out at 4°C employing aJulabo F12 thermostat (Julabo Labortechnik GmbH, Seelbach, Germany). Results and discussion Sequence differences inhuman and rabbit metallothioneins Most studies concerning mammalian MTs usually associate MT-1 with MT-2. These 2 isoforms are examined as equal peptides, mostly because they are the closest paralogs that have been found and purified together.16 They are commonly referred as MT-1/MT-2. However, the availability oftranscriptomic and proteomic techniques highlights differences inMT-1 and MT-2 that cannot be ignored, suggesting some degree offunctional differentiation between these isoforms both within the same species as well as between humans and rabbits. Amino acid sequences ofrabbit MT isoforms show differences when compared tothe sequences ofhuman MTs (Fig.1). Human MTs are controlled by17 genes located onchromosome 16, 10 of which are functional. Todate, 10 subisoforms ofhuman liver MT-1/2have been reported (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1M, MT-1L, MT-1X, and MT-2A)2; their amino acid sequences are shown inFig.1A. Subisoform MT-1G contains 62 amino acids, whereas the others display astructure of61 amino acids. Rabbit MT genes are located onchromosome 5. Six subisoforms ofrabbit liver MT-1/2have been reported so far (MT-1A, MT-2A, MT-2B, MT-2C, MT-2D, and MT-2E)17; their amino acid sequences are presented inFig.1B. Subisoforms MT-2A and MT-2C contain 62 amino acids, whereas the others display astructure of61 amino acids. Thereasons for the high diversity ofMT isoforms and their specific functional roles are still unclear. Thepotential biological significance ofeach isoform should not be neglected but the analytical techniques available cannot easily separate them.18 By ion-exchange chromatography, human MTs were separated into 2 charge-separable isoforms, designated as MT fractions 1 and 2.17 However, MT fractions from rabbit liver obtained withthe same ion-exchange chromatography cannot be separated into MT-1 and MT-2 subfractions. This conventional nomenclature division based oncharge differences refers tothe absence (MT-1) or presence (MT-2) ofanaspartic acid residue at position 11 or 12 (10 or 11) ofthe sequence (Fig.1) and itisnot based ontheir global charge.19 Amino acid sequences of6 rabbit MT isoforms show some differences compared tothe sequences ofhuman MTs (Fig.1). Owing tothe insertion ofanadditional amino acid at position 10, MT-2A and MT-2C sequences have atotal chain length of62 amino acids. MT-1G, having the same length and the same insertion, has also been identified inhuman liver. Thepresence ofdifferential expression ofMT-1G1 (with anadditional alanine) and MT-1G2 (without additional alanine) suggests tissueand cell-specific alternative splicing for the MT-1G isoform.20 Theinsertion ofthe alanine residue at position 10 ofthe sequence relates tothe location ofthe connection ofexons 1 and 2 ofthe rabbit M. Kepinska, etal. Metallothioneins characterization 1604 MT-2A and 2C gene and human MT-1G gene. Thepresence ofthe additional alanine does not seem toinfluence neither the tertiary structure nor the metal-binding properties ofthe protein.17,21 Thebiological consequence ofthat insertion remains undefined but its presence inmore than 1 species suggests acommon mechanism leading tothe same phenotype. Inrabbit MT-2D and MT-2E, there isasubstitution ofarginine (R) residue inposition 10/11 (Fig.1B). Theadditional positive charge may be involved incompensating for the net negative charge ofthe 3-metal-cluster and thus can increase the stability ofthe β-cluster.17 Even minor changes inthe sequence ofMT may induce structural changes that can alter the metal-binding properties or stability ofthe 3-metal-cluster. Theflexibility ofthe polypeptide chain as well as the charge environment ofthe coordinating sulfur atoms may greatly influence the feasibility and stability ofthe metal-thiolate bonds, and as aresult, ofthe final metal–MT complex. Aras etal. noticed aconserved PKC phosphorylation site at serine-32 (S32) ofMT, which was important inmodulating Zn2+-regulated gene expression.22 Itispresent inall rabbit and human MT isoforms shown inFig.1, except human MT-1B. Thepresence ofcysteine inplace ofserine can change the tertiary structure ofthe protein as well as change the function ofthe protein dependent onphosphorylation. Different expression patterns ofMT subisoforms are observed invarious types ofhuman malignancies; this fact can be useful inthe diagnosis and therapy oftumors.3 Theexact biological significance ofthe differences inamino acid sequences inhuman and rabbit MTs still remains unclear. Detection ofmetallothioneins usingcapillary gel electrophoresis Capillary electrophoresis isoften used toanalyze and separate MT isoforms from different organisms and tissues.11,23 Itcan also be used for monitoring MT concentration inbiological samples.23 Křížková etal. were the first topropose achip CE technique for the analysis ofMT, i.e., of the effect ofoxidation onthe structure ofMT and its ability toform aggregates and polymers.24 Unfortunately, due tothe presence ofsystem peaks, anaccurate analysis ofthe MT monomer isdifficult.12,25 Inthis study, changes inthe range of5–8 kDa corresponding tothe MT monomer were observed for both human and rabbit MTs. However, the monomers co-migrated with asystem peak, which made itdifficult tomeasure their correct size. InFig.2, the peak obtained for rabbit MT shifted toabigger mass (8.2 kDa) incomparison tohuman MTs (6.0 and 7.7 kDa for human MT-1, and 5.3 and 6.1 kDa for human MT-2). Anadditional peak was observed at about 23–24min, which may indicate the presence of13.4 and 14.0 kDa oligomers. Peaks corresponding toMT dimers were observed both for human and rabbit MTs. Mass spectrometry analysis Inthe majority ofMALDI-MS studies, DHB and HCCA were the constituents ofthe matrix used for the appropriate determination ofMTs.26 Inour study, utilization ofboth matrixes resulted indifferent types ofcrystals. While HCCA produced uniform crystals, DHB produced Fig.1. Comparison ofhuman and rabbit metallothionein (MT) isoforms 1 and 2 sequences (MT-1 and MT-2). Sequence comparison ofMT-1/2 from human (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1M, MT-1L, and MT-1X, MT-2A) and from rabbit (MT-1A, MT-2A, MT-2B, MT-2C, MT-2D, and MT-2E) was performed using ClustulX program.37 Thesequences were derived from the database SWISS PROT (http://www.expasy.org/sprot/) * indicates positions which have asingle, fully conserved residue; : indicates conservation between groups ofstrongly similar properties –scoring>0.5; indicates conservation between groups ofweakly similar properties –scoring≤0.5. Thered color indicates negative amino acid residues; the color green indicates positive amino acid residues; the pink color indicates cysteine residues; the blue color marks alanine –anadditional amino acid at position 10; substitution ofanaspartic acid residue at position 11/12 isindicated bythe frame. Adv Clin Exp Med. 2018;27(11):1601–1608 1605 heterogeneous and robust crystals (Fig.3A). Furthermore, DHB exhibited asignificant rabbit MT-2 concentrationdependent increase inobserved signal intensity (expressed in a.u.), higher when compared tothat of HCCA (Fig.3B). Themain observed signal for rabbit MT-2 was 6211.07 m/z. Moreover, itwas estimated that the isolated MT-2 was highly pure, because each spectrum showed only 1 peak ofhigh intensity for the relevant protein. This peak showed amass similar torabbit MT-2D (6215.40 m/z).27 Theoccurrence ofthe MT-2D form has been also observed inMT-2 from Sigma lots27,28 and inMT-1 sample, as aremaining contaminant.28 Thesize of12342.11 Da corresponds tothe MT-2 dimer (Fig.3C inset). TheDHB was chosen as the most appropriate matrix interms ofsensitivity and reproducibility for human MTs; at given conditions (20mg/mL ofDHB in30% ACN and 1% TFA), homogeneous spots applicable for automated MALDI-TOF-MS were formed. Further, toverify the proper isolation ofMT-1 and MT-2 from humans, we analyzed their mass distribution. Themain observed signals for human MTs (hMTs) shown inFig.4 were quasimolecular ions assigned as follows: [hMT-1]+ (6145.23 m/z, 6176.46 m/z and 6220.12 m/z) (Fig.4A), and [hMT-2]+ (6066.24 m/z) (Fig.4B). Thesizes of12523.89 Da and 12329.89 Da correspond toMT-1 and MT-2 dimers. Themass spectra ofthe human MT-1 sample yielded amain peak, the most intense at 6176.46 Da. Peaks observed inthe spectra ofthese MT-1 samples can be attributed topotential subisoforms: MT-1A (6145.23 m/z), MT-1G (6176.46 m/z) and MT-1G+Cd (6240.12 m/z).29,30 Thetheoretical mass ofhuman MT-2A is6042.16 m/z and our results showed main signals at 6066.24 m/z, corresponding to[hMT-2A+Na]+. Theexposure ofMTs toacidic pH values causes metal depletion and unfolding ofthe protein structure, generating apothioneins or metal-free forms. Using these matrixes caused the coordinated metals ofthe proteins tobe removed, and the peaks obtained should be apothioneins, i.e., subisoforms without metal content. Metallothionein dimers were first detected as transient intermediate species during interprotein metal exchange, and longer-lived dimers have been isolated that were stabilized byintermolecular disulfide bonds.31 No transient intermediate dimers could be detected with mass spectrometry. However, astable dimeric complex ofMTs has been detected inthis work byMALDI-TOF-MS and chip CE, and itappears tobe stabilized bydisulfide bridging. Metallothioneins can dimerize through the oxidation oftheir cysteinyl residues toform aninterprotein Cys– Cys dithiol bridge; the experimental mass would decrease by2 Da for every oxidative thiol bridge formation event. Metallothionein analysis using the Brdicka reaction Metallothionein exhibits significant electrochemical activity owing toits high content ofsulfhydryl (SH) moieties. Themethod ofdetermining proteins which contain –SH groups inanammonia-buffered cobalt (III) solution was first described byBrdička.32 Brdicka reaction isone ofthe mostoften-used electrochemical methods for MT determination Fig.2. Metallothionein (MT) separation using chip capillary gel electrophoresis (CE). Electropherograms of: A) inred, MT-1 from human liver; inblue, MT-2 from human liver; and ingray, MT-2 from rabbit liver are shown; B) chip CE virtual gel output ofPro260 ladder, MT-2 from rabbit liver, MT-1 and MT-2 from human liver –10 10 30 50 70 90 23 23.524 24.525 me [min] fluorescence [a.u.] – 500 –250 0 250 500 750 1000 1250 1500 1750 2000 15 20 25 30 35 me [s] fluorescence [a.u.] MT-2, human, C = 0.18 mg/mL MT-2, rabbit, C = 0.1 mg/mL MT-1, human, C = 0.1 mg/mL lower marker 1.2 kDa system peak 6.1 kDa 7.6 kDa 5.3 kDa 6.0 kDa 7.7 kDa 8.2 kDa 14.0 kDa ladder MT-2, rabbit MT-1, human MT-2, human 1.2 kDa 10 20 25 37 50 75 AB 13.4 kDa M. Kepinska, etal. Metallothioneins characterization 1606 Fig.4. Mass spectrum ofhuman metallothionein (MT). Spectrum ofMT-1 (A) and MT-2 (B) from human measured bymatrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with the 2,5-dihydroxybenzoic acid (DHB) matrix inlinear positive mode inamass range of4–16 kDa prepared inTA30 with repetition rate of2000Hz Fig.3. Mass spectrum ofrabbit metallothionein (MT). A) Spectrum ofMT-2 from rabbit liver measured bymatrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with (A.1) α-cyano-4-hydroxycinnamic (HCCA) and (A.2) 2,5-dihydroxybenzoic acid (DHB) matrix. B) Graphs ofsignal intensity ofdifferent concentrations ofrabbit MT-2 inDHB and HCCA matrices. 1–2µL ofsample/matrix mixture (1:1) deposited onMALDI plate and dried at room temperature (dried-droplet method). Analysis byaMALDI-TOF-MS mass spectrometer was performed inlinear and reflector mode. Dimerization ofMT analyzed byMALDI-TOF-MS isshown inthe inset ofFig. C) Spectrum ofrabbit MT-2 measured byMALDI-TOF-MS with the DHB matrix inlinear positive mode inamass range of4–17 kDa prepared inTA30 with repetition rate of2000Hz B A + 1.00 4 ×1025,000 DHB A.1 0.25 0.50 0.75 intensity [a.u.] 15,000 20,000 HCCA [M+H]+ 0.00 6,211.492 1.5 5 ×10 10,000 A.2 0.5 1.05,000 0.0 4,0006,000 [m/z]7,0005,000 0 1.5 3.5612 24 µg of MT 6,211.072 6,000 8,000 12,342.10 Da dimer_MT-2 [M+H]+ C rabbit MT-2 4,000 0 2,000 4,0006,000 8,000 10,000 12,000 14,000 16,000 [m/z] 6,211.021 intensity [a.u.] intensity [a.u.] intensity [a.u.] [M+H] 2.0 4 ×10 12,523.89 Da 6,176.465 A human MT-1 1.5 dimer_MT-1 6,145.236,220.12 0.5 1.0 6,066.238 4 4 ×10 Bhuman MT-2 3 412,329.89 Da dimer_MT-2 1 2 intensity [a.u.] intensity [a.u.] 04,000 6,000 8,00010,000 12,000 14,00016,000 [m/z] Adv Clin Exp Med. 2018;27(11):1601–1608 1607 inbiological samples.33 Itisbased onthe reduction ofhydrogen at the mercury electrode catalyzed bythe –SH groups inthe ammonia-buffered cobalt(III) solution.34 TheBrdicka reaction has potential for monitoring the differences ofvarious MT isoforms, both rabbit and human.26 During MT analysis, changes inCat2 signal (−1.55 V, which represents the current response ofthe MT complex with components ofthe Brdicka electrolyte), Co2+ (−1.25 V) and Cat1 (−1.40 V) corresponding tohydrogen evolution from the supporting electrolyte catalyzed bythe MT were observed.35 Thepresence ofthe Cat2 peak isclosely connected with the quantification of–SH groups occurring inthe Brdicka solution,36 thus corresponding tothe MT level. Thevoltammograms ofhuman and rabbit MT are shown inFig.5. Itcan be observed that the character ofthe mentioned MT signals changes with different MT concentration both for rabbit and human MT (Fig.5). Concentrations ofMT 0.025 and 0.05mg/mL were analyzed. With alower concentration ofrabbit MT, the Co2+ signal decreases and shifts toaless negative potential, whereas itslightly increases with adecreasing concentration ofhuman MT (Fig.5). Cat1 and Cat2 signals are more exposed inalower concentration ofMTs. With alower rabbit MT concentration, Cat1 and Cat2 signals decreased and shifted toamore positive potential. These signals inthe lower concentration ofhuman MT had anegligible potential shift. Thecharacter ofthe mentioned MT signals changed with MTs concentrations. Alower concentration ofMTs from both organisms coincided with adecrease inthe Cat2 signal. Lower, but more distinct signals were observed for human and rabbit MTs when alower concentration was used (0.025mg/mL). Thesame concentration ofhuman MT gave higher Cat2 peak than rabbit MT. Metallothioneins isolated inboth organisms behave differentially despite the same number of–SH groups. Conclusions Thestructure ofMTs isconstantly being studied due totheir dynamic nature, lack ofsecondary structural features when metals are not present, and the absence ofspectroscopically active aromatic residues. Thus, different methods have been used for the analysis ofhuman MT-1 and MT-2 isoforms and rabbit MT-2 isoforms. Both inrabbit and human MTs, mainly monomers were detected with chip CE and MALDI-TOF-MS. Additionally, distinctly lower signals from the dimers were observed. When MALDI-TOF-MS isemployed, the main observed signal for rabbit MT-2 corresponds tothe mass ofMT-2D. Inhuman, MT-1A, MT-1G, MT-1G+Cd and MT-2A were detected. Metallothionein subisoforms, except inthe case ofhuman MT-1G+Cd, have been identified without metals, which can be due tothe use ofacidic matrixes that cause arelease ofless bound metals. Inthe Brdicka reaction, signals are different for MTs isolated from rabbit and human liver, meaning that they behave differentially inelectrochemistry despite the same Fig.5. Metallothioneins (MTs) from rabbit and human liver analysis using the Brdicka reaction. Different concentrations ofMT were used: 0.025mg/mL and 0.05mg/mL. –1100 rabbit MT-2, C = 0.05 mg/mL A Cat 1 Cat 2 human MT-2, C = 0.05 mg/mL C 900 700 500 Cat 1 Cat 2 500 nA Cat1 Cat2 300 100 100 current [nA] Co2+ scan peak 100 Co2+ scan T peak 100 300 –1.70–1.50–1.30–1.10–0.90–0.70 MT 100 300 M T D B –300 human MT-2, C = 0.025 mg/mL D Cat1 Cat2 –300 - rabbit MT-2, C = 0.025 mg/mL B 0 Co2+ –100 0 Cat1 Cat2 Co2+ 0 100 200 scan 0 100 200 scan M T peak M T peak 200 200 –1.70–1.50–1.30–1.10–0.90–0.70 M M potenal [V] – – – – – –200 –200 –100 –1.70–1.50–1.30–1.10–0.90–0.70 –1.70–1.50–1.30–1.10–0.90–0.70 current [nA] current [nA] current [nA] –1100 900 700 500 300 100 – – – – – potenal [V] potenal [V] potenal [V] M. Kepinska, etal. Metallothioneins characterization 1608 number of–SH groups. This may be due todifferent availability ofMT cysteinyl groups indifferent organisms. These methods used together allow for the identification and characterization ofMT and give complementary information about MT forms. Furthermore, MALDI-TOF-MS iscapable ofdirectly resolving more than just the main MT isoforms inthe sample, which makes itthe best ofthese techniques toidentify isoforms ofMT. References 1. Osobová M, Urban V, Jedelský PL, etal. Three metallothionein isoforms and sequestration ofintracellular silver inthe hyperaccumulator Amanita strobiliformis. New Phytol. 2011;190(4):916–926. 2. Zalewska M, Trefon J, MilnerowiczH. Therole ofmetallothionein interactions with other proteins. Proteomics. 2014;14(11):1343–1356. 3. Křížková S, Kepinska M, Emri G, etal. Aninsight into the complex roles ofmetallothioneins inmalignant diseases with emphasis on(sub)isoforms/isoforms and epigenetics phenomena. Pharmacol Ther. 2017; 183:90–117. 4. Torreggiani A, Chatgilialoglu C, Ferreri C, Melchiorre M, Atrian S, CapdevilaM. Non-enzymatic modifications inmetallothioneins connected tolipid membrane damages: Structural and biomimetic studies under reductive radical stress. JProteomics. 2013;92:204–215. 5. Macirella R, BrunelliE. Morphofunctional alterations inzebrafish (Danio rerio) gills after exposure tomercury chloride. Int JMol Sci. 2017;18(4):824. 6. Sanz-Nebot V, Andón B, BarbosaJ. Characterization ofmetallothionein isoforms from rabbit liver byliquid chromatography coupled toelectrospray mass spectrometry. JChromatogr BAnalyt Technol Biomed Life Sci. 2003;796(2):379–393. 7. Ruttkay-Nedecky B, Nejdl L, Gumulec J, etal. Therole ofmetallothionein inoxidative stress. Int JMol Sci. 2013;14(3):6044–6066. 8. Artells E, Palacios O, Capdevila M, AtrianS. Invivo-folded metalmetallothionein 3 complexes reveal the Cu-thionein rather than Znthionein character ofthis brain-specific mammalian metallothionein. FEBS J. 2014;281(6):1659–1678. 9. Thirumoorthy N, Shyam Sunder A, Manisenthil Kumar K, Senthil Kumar M, Ganesh G, ChatterjeeM. Areview ofmetallothionein isoforms and their role inpathophysiology. World JSurg Oncol. 2011;9:54. 10. Milnerowicz H, BizońA. Determination ofmetallothionein inbiological fluids using enzyme-linked immunoassay with commercial antibody. Acta Biochim Pol. 2010;57(1):99–104. 11. Zalewska M, Bizoń A, MilnerowiczH. Comparison ofcapillary electrophoretic techniques for analysis and characterization ofmetallothioneins. JSep Sci. 2011;34(21):3061–3069. 12. Guszpit E, Křížková S, Kepinska M, etal. Fluorescence-tagged metallothionein with CdTe quantum dots analyzed bythe chip-CE technique. JNanopart Res. 2015;17(11):423. 13. Guszpit E, Krejčová L, Křížková S, etal. Kinetic analysis ofhuman metallothionein and CdTe quantum dot complexes using fluorescence and voltammetry techniques. Colloids Surf BBiointerfaces. 2017; 160:381–389. 14. Dabrio M, Virtanen V, Bordin G, Rodriguez AR. Contribution tothe study ofcomplexing properties ofZn-metallothioneins byCZE-DAD. Talanta. 2000;53(3):587–598. 15. Mounicou S, Ouerdane L, L’Azou B, etal. Identification ofmetallothionein subisoforms inHPLC using accurate mass and online sequencing byelectrospray hybrid linear ion trap-orbital ion trap mass spectrometry. Anal Chem. 2010;82(16):6947–6957. 16. Artells E, Palacios Ò, Capdevila M, AtrianS. Mammalian MT1 and MT2 metallothioneins differ intheir metal binding abilities. Metallomics. 2013;5(10):1397–1410. 17. Hunziker PE, Kaur P, Wan M, KänzigA. Primary structures ofseven metallothioneins from rabbit tissue. BiochemJ. 1995;306(Pt 1): 265–270. 18. Beattie JH. Strategies for the qualitative and quantitative analysis ofmetallothionein isoforms bycapillary electrophoresis. Talanta. 1998;46(2):255–270. 19. Kojima Y, Hunziker PE. Amino acid analysis ofmetallothionein. Methods Enzymol. 1991;205:419–421. 20. Mehus AA, Muhonen WW, Garrett SH, Somji S, Sens DA, Shabb JB. Quantitation ofhuman metallothionein isoforms: Afamily ofsmall, highly conserved, cysteine-rich proteins. Mol Cell Proteomics. 2014; 13(4):1020–1033. 21. Braun W, Vasák M, Robbins AH, etal. Comparison ofthe NMR solution structure and the x-ray crystal structure ofrat metallothionein-2. Proc Natl Acad Sci USA. 1992;89(21):10124–10128. 22. Aras MA, Hara H, Hartnett KA, Kandler K, AizenmanE. Protein kinase Cregulation ofneuronal zinc signaling mediates survival during preconditioning. JNeurochem. 2009;110(1):106–117. 23. Ryvolova M, Hynek D, Skutkova H, Adam V, Provaznik I, KizekR. Structural changes inmetallothionein isoforms revealed bycapillary electrophoresis and Brdicka reaction. Electrophoresis. 2012;33(2):270–279. 24. Křížková S, Adam V, KizekR. Study ofmetallothionein oxidation by using ofchip CE. Electrophoresis. 2009;30(23):4029–4033. 25. Křížková S, Masařík M, Eckschlager T, Adam V, KizekR. Effects ofredox conditions and zinc(II) ions onmetallothionein aggregation revealed bychip capillary electrophoresis. JChromatogrA. 2010;1217(51): 7966–7971. 26. Merlos Rodrigo MA, Molina-López J, Jimenez Jimenez AM, etal. Theapplication ofcurve fitting onthe voltammograms ofvarious isoforms ofmetallothioneins-metal complexes. Int JMol Sci. 2017; 18(3):610. 27. van Vyncht G, Bordin G, Rodriguez AR. Rabbit liver metallothionein subisoform characterization using liquid chromatography hyphenated todiode array detection and electrospray ionization mass spectrometry. Chromatographia. 2000;52(11–12):745–752. 28. Chassaigne H, LobinskiR. Polymorphism and identification ofmetallothionein isoforms byreversed-phase HPLC with on-line ion-spray mass spectrometric detection. Anal Chem. 1998;70(13):2536–2543. 29. Tomalová I, Foltynová P, Kanický V, PreislerJ. MALDI MS and ICP MS detection ofasingle CE separation record: Atool for metalloproteomics. Anal Chem. 2014;86(1):647–654. 30. Irvine GW, Pinter TBJ, Stillman MJ. Defining the metal binding pathways ofhuman metallothionein 1a: Balancing zinc availability and cadmium seclusion. Metallomics. 2016;8(1):71–81. 31. Irvine GW, Heinlein L, Renaud JB, Sumarah MW, Stillman MJ. Formation ofoxidative and non-oxidative dimers inmetallothioneins: Implications for charge-state analysis for structural determination. Rapid Commun Mass Spectrom. 2017;31(24):2118–2124. 32. BrdičkaR. Polarographic studies with the dropping mercury electrode. Part IV. Theinfluence ofcircuit resistance onmaxima ofcurrent-voltage curves. Collect Czechoslov Chem Commun. 1936;8: 419–433. 33. Raspor B, Paić M, ErkM. Analysis ofmetallothioneins bythe modified Brdicka procedure. Talanta. 2001;55(1):109–115. 34. Křížková S, Blahova P, Nakielna J, etal. Comparison ofmetallothionein detection byusing Brdicka reaction and enzyme-linked immunosorbent assay employing chicken yolk antibodies. Electroanalysis. 2009;21(23):2575–2583. 35. Kruseova J, Hynek D, Adam V, etal. Serum metallothioneins inchildhood tumors: Apotential prognostic marker. Int JMol Sci. 2013;14(6): 12170–12185. 36. Adam V, Chudobova D, Tmejova K, etal. Aneffect ofcadmium and lead ions onEscherichia coli with the cloned gene for metallothionein (MT-3) revealed byelectrochemistry. Electrochimica Acta. 2014;140: 11–19. 37. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. TheCLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided byquality analysis tools. Nucleic Acids Res. 1997;25(24):4876–4882.