Jens
Hogeback
a,
Miriam
Schwarzer
a,
Christoph A.
Wehe
a,
Michael
Sperling
ab and
Uwe
Karst
*a
aInstitute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 28/30, 48149 Münster, Germany. E-mail: uk@uni-muenster.de; Fax: +49 251 83 36013; Tel: +49 251 83 33141
bEuropean Virtual Institute for Speciation Analysis (EVISA), Mendelstr. 11, 48149 Münster, Germany
First published on 29th September 2015
The interaction of mercury species with human erythrocytes is studied to investigate possible high molecular binding partners for mercury species. Human blood hemolysate was spiked with methylmercury and investigated by means of liquid chromatography (LC) coupled to electrospray ionization time of flight mass spectrometry (ESI-ToF-MS) and inductively coupled plasma mass spectrometry (ICP-MS). Beside adduct formation of mercury species with hemoglobin, the main compound of the erythrocytes, mercury binding to the enzyme carbonic anhydrase was revealed. Due to an enzymatic digest of the protein-mercury adduct, the binding site at the free thiol group of the protein was identified. These results indicate that carbonic anhydrase might play a role in mercury toxicity.
MeHg+, which is taken up with contaminated food is absorbed almost entirely by the blood stream, whereas THI, in cases where it was used as multidose vaccine preservative, is injected directly into the body leading to increased mercury levels.5,6 However due to the intramuscular administration of thiomersal containing vaccines, relatively high local concentrations at time and point of injection are likely, before it is later diluted and distributed via the blood stream.
In biological systems, a large number of biomolecules with free thiol groups are available. Due to the high affinity of mercury towards sulfur, reactions with free thiol groups of peptides and proteins with different mercury species may occur.7–10 It is assumed that the interaction of mercury species with thiols plays an important role for mercury transport and for toxic mechanisms of action.11
Studies on the fractionation of whole blood from mammals following total mercury determination showed that the bulk of blood alkyl mercury is bound to the erythrocytes.12 Moreover, half of the erythrocyte-mercury was found to be coordinated to high molecular compounds.13,14 Since hemoglobin is by far the most abundant protein in erythrocytes and contains free thiol groups, it is a potential target molecule for mercury binding. The interaction of mercury species with hemoglobin was investigated in the 1970s. Rabenstein et al. showed in their NMR studies that mercury species are coordinated to the free thiol groups of hemoglobin.15,16 Janzen et al. compared the adduct formation of human and rat hemoglobin with THI and MeHg+.17 Recently, Rayas et al. identified hemoglobin as major binding site for MeHg+ in dolphin liver.18
The second most abundant protein of the erythrocytes is carbonic anhydrase (CA), which is found in all animals and photosynthesizing organisms. It catalyzes the reversible hydration of carbon dioxide. Human CA from erythrocytes contains one cysteine residue and with that one free thiol group, which may interact with mercury species.19 In the context of crystal structure investigations of human CA isoforms, mercury species were used as phasing agents in X-ray diffraction experiments and interaction with thiol groups was detected.20,21 However, these experiments were not conducted under physiological conditions and not brought into relation to mercury toxicity.
Ekinci et al. showed that Hg2+ is a competitive inhibitor of CA.22 Hence, it is not known, which effect organic mercury species may have with respect to the enzymatic activity of CA.
In this study, the interaction of different mercury species with human CA was investigated by carrying out incubation experiments at physiological conditions. It is expected that adduct formation at the free thiol group takes place. In addition, an enzymatic digest of the protein adducts was carried out to prove the proposed binding site of mercury in the protein.
Incubation solution with a physiological pH of 7.4 was prepared by dissolution of PBS tablets according to the manufacturer's instructions and pH value was adjusted with 1% aqueous ammonia to the intended pH. Fresh stock solutions of CA1, THI, chymotrypsin, ammonium bicarbonate, sodium chloride and calcium chloride were prepared daily by dissolution of the solids in deionized water. MeHg+ solutions were prepared by dissolving MeHgCl in a mixture of methanol and water 3:1 (v/v) and diluted to the intended concentration with deionized water.
For ESI-ToF-MS investigations, the LC outlet was directly coupled to an ESI source of a micrOTOF from Bruker Daltonics (Bremen, Germany). The following parameters were used for analyses of the incubated CA1 and hemolysate in the positive ion mode: mass range: m/z 500–2200, end plate offset: −500 V, capillary: 4000 V, nebulizer gas: 1.4 bar, dry gas: 8 L min−1, dry temperature: 190 °C, capillary exit: 150.0 V, skimmer 1: 50.0 V, skimmer 2: 22.0 V, hexapole 1: 23.0 V, hexapole 2: 21.0 V, hexapole RF: 400.0 V, transfer time: 60.0 μs, pre-pulse storage: 30.0 μs, lens 1 storage: 40 V, lens 1 extraction: 21.3 V.
For analyses of the enzymatic digests, the following ESI-ToF-MS parameters were used: mass range: m/z 100–2000: end plate offset: −500 V, capillary: 4000 V, nebulizer gas: 1.5 bar, dry gas: 8 L min−1, dry temperature: 200 °C, capillary exit: 150.0 V, skimmer 1: 50.0 V, skimmer 2: 26.5 V, hexapole 1: 23.0 V, hexapole 2: 21.4 V, hexapole RF: 300.0 V, transfer time: 70.0 μs, pre-pulse storage: 19.0 μs, lens 1 storage: 40 V, lens 1 extraction: 20.9 V.
The instrument was routinely calibrated using ammonium formate clusters as external standards. Furthermore, at the end of each data acquisition, internal calibration was performed using ammonium formate clusters as well. The software Data Analysis (Bruker Daltonics) was used for spectra evaluation. Deconvolution of protein mass spectra was performed using MagTran 1.02, which is based on a charge state deconvolution algorithm developed by Zhang and Marshall.23
For ICP-MS investigations, a quadrupole-based ICP-MS (iCap Qc, Thermo Fisher Scientific, Bremen, Germany) was used. The LC outlet was directly coupled to a PFA MicroFlow nebulizer (Elemental Scientific, Omaha, NE, USA) and a Peltier-cooled (−5 °C) cyclonic spray chamber. The instrument was controlled by the Qtegra ISDS software (Thermo Fisher Scientific). The following ICP-MS parameters were used: Plasma power: 1550 W, nebulizer gas: 0.6 L min−1, cool gas: 14 L min−1, auxiliary gas: 0.8 L min−1, oxygen as additional gas: 45 mL min−1, recorded isotopes 199Hg, 200Hg and 202Hg with a dwell time of 0.1 s, each.
Separations of the hemolysate were performed on a BioBasic C18 column (2.1 × 150 mm, 5 μm, 4 × 4 mm guard column, 300 Å) from Thermo Fisher Scientific (Dreieich, Germany). In both cases, the injection volume was 5 μL and the column oven had a temperature of 40 °C. Separation was performed using a binary gradient with aqueous formic acid (0.1%) as eluent A and acetonitrile as eluent B at a flow rate of 300 μL min−1. However, another gradient was used for peptide separation. Both gradient profiles are given in Table 1.
Protein separations | |||||||
t/min | 0 | 2 | 20 | 24 | 27 | 29 | 35 |
B/% | 35 | 35 | 42 | 70 | 70 | 35 | 35 |
Enzymatic digest | |||||||
t/min | 0 | 2 | 19 | 23 | 28 | 30 | |
B/% | 10 | 10 | 80 | 80 | 10 | 10 |
Fig. 1 Charge distributions and deconvoluted mass spectra of native CA1 (a and b) and after incubation with MeHg+ (c and d) and THI (e and f). |
The mass spectra were deconvoluted in order to obtain neutral mass spectra (Fig. 1b, d and f). An experimental mass of 28781.0 Da was found for the unmodified CA1, which is in a good agreement with values described in literature or calculated from its primary structure.24,25
In the deconvoluted mass spectra of the sample after incubation with MeHg+ (Fig. 1d), a second signal arises, which can be assigned to a mass of 28995.7 Da. The mass difference of 214.7 Da in relation to the unmodified CA1 can be explained by a substitution of one proton (1.0079 Da) with a MeHg+ unit (215.6337 Da). The obtained mass difference is in good agreement with the calculated (214.6258 Da).
A similar reaction can be observed in the deconvoluted mass spectrum after incubation with THI (Fig. 1f). Here, also a second signal arises with a mass of 29009.7 Da. The mass difference of 228.7 Da indicates adduct formation with an EtHg+ unit. The found mass difference correlates well to a theoretical difference of 228.6524 Da. THI has hydrolyzed to thiosalicylic acid and EtHg+. These results indicate that MeHg+ and EtHg+ from THI are bound to CA1. It can be assumed that the mercury species are coordinated to the single free thiol group in the protein. To specify the expected binding site, an enzymatic digest was performed.
Trypsin and chymotrypsin were used in parallel for the digestion to obtain different peptide patterns. The tryptic digest of CA1 results in 24 specific peptides, in which the peptide T19 (#174–213) is the peptide featuring the only cysteine residue and consists of 40 amino acids. The digestion with chymotrypsin produced 45 fragments from CA1. The peptide C40, which contains the cysteine, consists of 14 amino acids (#210–223).
Fig. 2 shows the mass spectra of the T19 peptide after tryptic digest in the +4 charge state and the C40 peptide after chymotryptic digest in the +2 charge state, respectively. The isotopic patterns of the unmodified peptide are displayed in Fig. 2a and d. The resulting peptides after incubation with MeHg+ and following digestion show a characteristic isotopic pattern. Seven stable mercury isotopes cause a typical isotopic pattern of mercury containing peptides (Fig. 2b and e). The obtained isotopic pattern of T19 and C40 after incubation with THI show that adduct formation with EtHg+ has taken place at the cysteine containing peptide (Fig. 2c and f). In all cases, the obtained masses are in good agreement with calculated values, which are listed in Table 2.
Sum formula | Exp. m/z | Calc. m/z | Dev. [ppm] | |
---|---|---|---|---|
T19 | [C216H314N46O60S]4+ | 1136.5672 | 1136.5657 | 1.3 |
T19 + MeHg+ | [C217H316N46O60SHg]4+ | 1190.0633 | 1190.0603 | 2.6 |
T19 + EtHg+ | [C218H318N46O60SHg]4+ | 1194.0682 | 1194.0647 | 2.9 |
C40 | [C65H116N16O24S]2+ | 768.4029 | 768.4006 | 3.0 |
C40 + MeHg+ | [C66H118N16O24SHg]2+ | 876.3946 | 876.3966 | −2.3 |
C40 + EtHg+ | [C67H120N16O24SHg]2+ | 883.4044 | 883.4060 | −1.8 |
Since mercury binding was shown in both digestion experiments at the only cysteine containing peptide it can be considered as proof that mercury species are bound to the thiol function.
For analysis of the hemolysate, a method was developed to separate the α- and β-subunits of hemoglobin, which are the main constituents of erythrocytes. In Fig. 3a, the extracted ion chromatograms (XIC) of charge state +13 of the α- and β-chain are shown. After deconvolution of the charge distribution spectra, neutral masses of 15126.2 Da for the α-chain and 15867.0 Da for the β-chain were determined. The obtained deconvoluted masses are in good accordance with calculated values from amino acid sequence and literature.26,27
Fig. 3 LC/ESI-ToF-MS chromatogram of human blood hemolysate with the XICs of the most abundant compounds (a). Deconvoluted mass spectra of the proteins CA2 (b), CA1 (c), αHb (d) and βHb (e). |
The complex of the hemoglobin subunits and the heme group is released through organic eluents during chromatography. The heme group appears as dimer with a mass of 1231.4 Da. The monomer and trimer were observed as well in lower intensities. The isotopic pattern of the recorded and calculated dimer matches well. The formation of heme dimer and other oligomers has been described by Pashynska et al.28
Besides hemoglobin, charge distributions of two other proteins were observed. Deconvoluted masses of 28780.9 Da and 29156.5 Da were determined, which can be assigned to human CA1 and human carbonic anhydrase 2 (CA2), respectively. The found protein masses agree well with values described in literature.24,25,29
Fig. 4a displays a LC/ICP-MS chromatogram of MeHg+ spiked hemolysate. The mercury trace shows four main peaks. The first can be assigned to unbound MeHg+, which elutes with the void volume. The other three signals can be assigned comparing retention times of the LC/ESI-ToF-MS measurements (Fig. 3b). The XIC of the MeHg+ protein adducts are in good agreement with the mercury signals obtained by LC/ICP-MS. The +13 charge state of the α-Hb + MeHg+ adduct (m/z 1181.5) was detected at 9.4 min with ICP- and ESI-ToF-MS. A peak of the MeHg+ adduct of the CA1 was detected at 8.3 min in LC/ICP-MS and LC/ESI-MS chromatograms as well. A signal for the MeHg+ adduct with CA2, which is expected at 6.2 min, could not be detected, as it is overlapped by the tailing peak of the free MeHg+ species. Moreover, the mercury background in this region is caused by slight species decomposition due to the low pH during separation.
Fig. 4 LC/ICP-MS (a) and LC/ESI-ToF-MS chromatogram of hemolysate, which was incubated with MeHg+. Deconvoluted mass spectrum (c) of the 9–12 min fraction of (b). |
To show that adduct formation of MeHg+ has taken place, a deconvoluted mass spectrum of the 9–12 min fraction was generated (Fig. 4c). There are five main signals. The first two signals at 15126.3 Da and 15340.9 Da can be assigned to the hemoglobin α-chain and its corresponding MeHg+ adduct. The mass difference of 214.6 Da can be explained by a substitution of the proton (1.0079 Da) of the free thiol group with one MeHg+ (215.6337 Da) unit, as described earlier. Furthermore, signals for the β-chain and its corresponding single and double MeHg+ adduct can be expected, because the hemoglobin β-subunit contains two free cysteine residues. The detected mass of the free β-chain of 15867.2 Da correlates well to values from literature. The single and double MeHg+ adducts with masses of 16082.3 Da and 16296.0 Da create mass differences of 215.1 Da and 428.8 Da relating to the free β-chain. The obtained mass differences are in good agreement with the calculated due to an addition of one and two MeHg+.
In addition to the detected mass increase of the protein after adduct formation, an effect on the retention time was observed (Fig. 3a and 4b). The free α-chain has a retention time of 8.6 min, whereas the MeHg+ adduct elutes at 9.3 min. Due to the additional MeHg+ group, the protein becomes less polar, and because of the used reversed phase conditions, a separation of the α-chain and the α-chain MeHg+ adduct was achieved. A similar effect was observed in case of the β-chain, whereas the shift in retention time was not as distinctive as for the α-chain. The unmodified β-chain elutes at 10.5 min, the single MeHg+ adduct at 10.7 min and the double MeHg+ adduct at 10.9 min. The shift in retention time was also observed after incubation experiments of the hemolysate with EtHg+. In this case, the retention times were shifted even more than for MeHg+.
The amino acid sequence of human CA1 and CA2 shows that the protein contains one free cysteine residue each and it is thus assumed that similar reactions with mercury species take place at the free thiol group. Fig. 5a shows an enlargement of the mass spectra with the charge distributions of the free hemoglobin α chain and the coeluting CA1. The signal doublets of the CA1 indicate adduct formation, which can be confirmed due to deconvolution of this spectrum, which is shown in Fig. 5b. Two signals with masses of 28781.3 Da for the free CA1 and 28995.8 Da for the corresponding MeHg+ adduct can be assigned. The mass difference of 214.5 Da corresponds well to expected values due to an addition of MeHg+ as described above. In case of the CA2, similar reactions were observed (Fig. 5c and d). After deconvolution, masses of 29156.9 Da and 29371.9 Da can be assigned to the free protein and its adduct. The mass difference of 215.0 Da is still in good correlation to the expected value, whereas the lower abundance of the CA2 in the samples may lead to slight mass inaccuracies.
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