Evaluation of different purification procedures for the electrochemical quantification of mussel metallothioneins

Talanta 57 (2002) 1211– 1218 www.elsevier.com/locate/talanta

Evaluation of different purification procedures for the electrochemical quantification of mussel metallothioneins Marijana Erk *, Dusˇica Ivankovic´, Biserka Raspor, Jasenka Pavicˇic´ Ruder Bosˇko6ic´ Institute, Center for Marine and En6ironmental Research, BiJenicˇka c. 54, P.O. Box 180, HR-10002 Zagreb, Croatia Received 25 February 2002; received in revised form 24 April 2002; accepted 6 May 2002

Abstract Several different procedures were applied to purify metallothionein (MT) isolated from the digestive glands of the mussels (Mytilus gallopro6incialis) exposed to cadmium: heat treatment (at 70 and 85 °C), solvent precipitation, and gel-filtration. Using electrochemical method, the MT contents were determined. Based on the obtained results each procedure was statistically evaluated. Application of the post-hoc Scheffe´ test showed that there were significant differences between all the treatments applied. Heat treatment and solvent precipitation effectively remove high molecular weight (HMW) proteins from the samples. In untreated as well as in heat or solvent treated samples MT10 isoform remains unchanged. MT20 isoform is significantly reduced by heat treatment and drastically reduced by solvent precipitation. Due to the fact that MT20 is considered as ‘target’ MT isoform for metal effect and exposure it is recommended to use the purification procedure which less affects MT20 fraction. According to the presented results it is the heat treatment. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Metallothionein; Purification procedure; Electrochemical quantification; Mytilus gallopro6incialis

1. Introduction The need to detect and assess the impact of pollution on biological resources has become the primary issue in many countries. Extensive studies in this field have lead to the development of indicators of the biological effects of contaminants on organisms. These biomarkers reflect the health status of organisms at lower organization

* Corresponding author. Tel.: + 385-1-468-0216; fax: + 385-1-468-0242 E-mail address: [email protected] (M. Erk).

levels (molecular or cellular), respond rapidly to stress, have high toxicological relevance, and can be used as early-warning indicators of damage before the irreversible changes in the ecosystems occur [1]. One of the biomarkers of metal exposure and effect are metallothioneins (MTs), which are inducible, low molecular mass proteins, of characteristic metal-binding properties because of high cystein content [2,3]. One of their putative roles in the organisms is detoxification of toxic metals (cadmium, mercury, silver). Studies on marine and freshwater bivalves performed by different research groups agreed, in general, on the detox-

0039-9140/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 2 3 9 - 4

1212

M. Erk et al. / Talanta 57 (2002) 1211–1218

ification role of MTs for non-essential metal ions [4,5]. Cadmium induced metallothioneins from the common mussel (Mytilus edulis) were shown to comprise of two groups of isoforms, MT10 and MT20 [6]. Amino acid sequencing of mussel MTs clearly showed that the most significant difference between MT10 and MT20 is the presence of two additional cysteine residues in the MT20 isoforms. The exact role of each specific MT isoform or isoform group has not been elucidated yet, but recent studies on MT gene expression [7] indicate that MT20 could be important for detoxification in cadmium exposed mussels. Therefore, it is necessary to know the basal level of this biomarker in the organism, and its changes in the polluted environment. One of the commonly used methods for the determination of the total MT level in target tissue of indicator organism is electrochemical method introduced by Brdicˇ ka [8], modified by Palecˇ ek and Pechan [9], Olafson and Sim [10], and Thompson and Cosson [11]. This method has been used in a number of environmental studies on bivalves as indicator organisms [12– 15]. It has been used as a routine, although the results depend on selected experimental conditions [16]. Clarifying the mechanism of Brdicˇ ka reaction Raspor [17] proposed the set-up of physical and chemical conditions which should be strictly followed in a protocol to achieve comparable results in different laboratories. Still, the number of non-intercalibrated protocols used to quantify MT content is huge, and this makes it difficult or even impossible to compare the results of different research teams [18]. Accordingly, Isani et al. [19] recently concluded that any comparison of MT amounts in bivalve tissues with data from the literature should take into account the isolation and quantification procedure. Starting point of a satisfactory MT quantification method is reliable and reproducible isolation and purification procedure of MTs from the biological material. It is necessary to define the isolation, purification and quantification method in detail and statistically validate each step. We have chosen S50 supernatant as the common

starting point, and applied three different purification procedures, determining the MT content in each subsample using common, Brdicˇ ka modified reaction. The aim was to assess if significant differences in purification procedures exist and to select the most appropriate one for quantification of mussel MTs. S50 supernatant contains total cytosolic proteins which were isolated from the digestive gland tissue of mussels exposed to Cd. The quantification of MTs was performed with the common method which was applicable to all subsamples, both untreated or treated by different purification procedures.

2. Experimental

2.1. Chemicals and reagents All solutions were prepared from the analytical-reagent grade chemicals and twice distilled water (Milli-Q system). Commercially available rabbit liver MT I+ II, from Sigma (M 7641, Lot 125H9512), was used as the calibrant.

2.2. Instruments Homogenization was performed in a glass homogenizer (Potter-Elverhjem) with PTFE piston attached to rotating axis (GLAS-COL, model K41, USA). Centrifugation was performed with the Sorval RC28S centrifuge by Du Pont (Wilmington, DE). To separate MT from low- (LMW) and highmolecular weight (HMW) proteins gel-filtration on Sephadex G-75 column (0.9 cm× 60 cm) with automatic fraction collector (Pharmacia) was used. Voltammetric measurements were carried out with a mAutolab instrument (Eco Chemie, The Netherlands). Double-jacket Metrohm type of polarographic cell was thermostated at (7.09 0.1) °C with the laboratory water circulator Haake D8. The pH was measured with a Metrohm (Germany) Model E603 pH meter and Metrohm combined pH electrode.

M. Erk et al. / Talanta 57 (2002) 1211–1218

2.3. Isolation procedure Adult specimens of Mytilus gallopro6incialis (4– 6 cm, from the Krka river estuary, S& ibenik, Middle Adriatic) were exposed for 20 days to 147 mg Cd l − 1 in aquarium pools (50 l) which were filled with fresh seawater every day (S =12 9 2, 18 °C). Digestive glands of 20 individuals were pooled, stored in a liquid nitrogen and transferred to the deep freeze (− 80 °C) until the analysis. The composite sample of digestive glands of Cd-exposed mussels was homogenized (Tris 0.02 mol l − 1, pH 8.6, sucrose 0.5 mol l − 1, leupeptine 3.0 ml ml − 1, phenylmethyl-sulphonylflouride, PMSF 1.5 ml ml − 1, mercaptoethanol 0.01%) and centrifuged at 50 000×g (1 h, 4 °C).

2.4. Purification procedures S50 supernatant, which contains total cytosolic proteins, was carefully separated from the pellets using glass Pasteur-pipettes. Different purification procedures applied to S50 aliquots are indicated in Scheme 1 and are the following: (a) 10 min of heat treatment at 70 and 85 °C, including an additional 10-fold dilution with 0.9% NaCl prior to heat treatment; (b) solvent precipitation using ethanol/chloroform mixtures according to Viarengo et al. [20], which is carried out in two steps with two different ethanol concentrations.

1213

(c) gel-filtration on Sephadex G-75 column, pooling the fractions to HMW, LMW, MT dimer (MT20) and MT monomer (MT10). The purification procedure (a) by heat treatment was performed in a water bath, with a subsequent separation of the precipitated proteins by centrifugation at 30 000× g, 20 min, 4 °C. Based on the purification procedure (c) S50 supernatant was eluted on Sephadex G-75 column with 0.02 mol l − 1 Tris/HCl buffer to which 0.001 mol l − 1 DTT was added as the reducing agent.

2.5. Quantification of MTs MT content was determined in each of the differently treated S50 subsamples by modified Brdicˇ ka procedure [16,17]. During analysis the critical physical and chemical parameters were strictly kept as described in the previous publication [17]. The analysis was performed on a 10 ml aliquot of the supporting electrolyte at the Metrohm 290E hanging mercury drop electrode (HMDE) of the drop surface area (2.209 0.05) mm2, in the solution of 6× 10 − 4 mol l − 1 Co(NH3)6Cl3 and ammonia buffer (1 mol l − 1 NH4Cl + 1 mol l − 1 NH4OH) of pH 9.5. The reference electrode was an Ag/AgCl/saturated KCl, and the counter electrode a platinum wire. Measurement parameters were set up for the differential pulse voltammetric (DPV) mode with the negative potential scan from − 0.9 to − 1.7 V. The instrumental measuring conditions were the following: voltage pulse amplitude 0.025 V, duration of the voltage pulse application 0.057 s, a scan rate 0.005 V s − 1 and a clock time 0.5 s. All measurements were performed at constant temperature (7.09 0.1) °C [16]. Before each measurement, the solutions with MT samples were thoroughly deaerated with extra pure nitrogen, which passed during the measurement over the surface of the solution.

2.6. Statistical analysis

Scheme 1. Purification of S50 fraction which was isolated from the digestive glands of mussels exposed to Cd.

For differently purified sub-samples significant differences in the MT concentrations were evaluated using analysis of variance (ANOVA) and post hoc comparisons of means assessed by the

1214

M. Erk et al. / Talanta 57 (2002) 1211–1218

Fig. 1. MT quantification by modified Brdicˇ ka reaction on heat treated (85 °C, 10 min) S50 supernatant. Before the analysis, heat treated fraction was diluted 11 times (A) and 26 times (B). Added purified aliquots: 1 – 20 ml; 2 – 40 ml.

Scheffe´ test. These tests were performed using a standard statistical package (STATISTICA® for Windows).

3. Results and discussion The determination of proteins which contain cystein residues (including metallothioneins) is based on the linear relationship between catalytic hydrogen wave (Cat in Figs. 1– 3) and the protein concentration. Due to the fact that the response is not specific to certain types of protein, but to certain types of functional groups which are common to different proteins (SH-groups) efficient purification is essential for getting reliable data on MT content. For the electrochemical method of MT analysis, the HMW proteins are interfering and have to be removed before the analysis [10]. The concentration of MT in the unknown sample is evaluated from the height of the Cat signal and the slope of the calibration straight line. The appropriate mussel MT reference material is still not available. As a substitute the commercially available rabbit liver MT is used as the calibrant for MT quantification, but its properties can differ from lot to lot [18,21]. Modified Brdicˇ ka reaction is suitable for detecting low concentrations of MT. The linear response range is narrow (from 39.2 to 313.6 mg l − 1 MT I + II used as calibrant, and the correspond-

ing current range from − 0.083 to − 0.335 mA) and special care has to be taken that the Cat signal height of the unknown sample is within the linear calibration range [16]. The unfavorable case of MT analysis is illustrated in Fig. 1A and B. Undiluted S50 supernatant was heat-treated at 85 °C, 10 min (Scheme 1). Due to the fact that the supernatant is isolated from the tissue of mussels which were exposed to Cd the MT content is high. Before the analysis the aliquot was diluted 11 times (Fig. 1A) and 26 times (Fig. 1B) in order to fulfill the condition that the signal height is within the linear calibration range. Even at 26-fold dilution the proportional increase in Cat signal height has not been achieved (compare voltammograms 1 and 2 in Fig. 1B). From an analytical point of view, high dilution factors are not recommended, because the dilution itself might cause the bias. The unfavorable approach illustrated in Fig. 1A and B was improved by 10-fold dilution of S50 supernatant with 0.9% NaCl prior to heat treatment at 70 and 85 °C for 10 min. This modification brings several benefits for analytical determination of MT: reduces MT coprecipitation with HMW proteins and makes almost unnecessary further sample dilution before the analysis. The Cat signal height is recorded within the linear calibration range and it proportionally increases after the subsequent aliquot addition (Fig. 2, curves 1 and 2). The proportional increase in the

M. Erk et al. / Talanta 57 (2002) 1211–1218

1215

Fig. 2. MT quantification by modified Brdicˇ ka reaction of S50 supernatant diluted 10 times with 0.9% NaCl before heat treatment at 85 °C, 10 min. Before the analysis, heat treated fraction was diluted 3 times. Added purified aliquots: 1 – 20 ml; 2 – 40 ml.

peak height after the addition of the second aliquot of MT serves as the internal control of the reliability of the measured quantity. To analyze the sample of S50 supernatant purified by solvent precipitation according to Viarengo et al. [20], we applied also the electrochemical procedure and obtained voltammograms (Fig. 3) with the same characteristics (same shape and same peak potentials of Co2 + , RS2Co and Cat signals) as voltammograms of heat-treated sample (Fig. 1B). As already explained for the case of the samples undiluted prior to heat treatment, a similar problem occurred here. Due to the high MT content, 21-fold dilution of the sample prior to analysis was necessary to record the signal height within the calibration range. In spite of very large dilution it was not possible to achieve the proportional increase in Cat signal height. Statistical evaluation of the significant differences between the average results on MT content in purified S50 supernatant after heat treatment at 70 and 85 °C and solvent precipitation (Scheme 1) are shown in Table 1. The results obtained exhibit the statistical differences between the specific sample treatments applied, as evaluated by one-way ANOVA (PB 0.01). Further application of the post hoc Scheffe´ test showed that there are significant differences in MT concentrations between the three applied treatments. Opposite to our results, Geret et al. [22] found no significant differences between MT amounts measured in the

digestive gland sample prepared using solvent treatment and heat treatment. In order to determine which molecular weight component was affected the most by the different purification procedures, gel-filtration of previously prepared samples was performed (Scheme 1). In Fig. 4, the summary of the determined MT values after separation by gel-filtration chromatography is shown. Since the subsamples of S50 purified by gel filtration were diluted during the process of elution, they did not have to be additionally diluted prior to electrochemical measurement of MT. As is obvious from Fig. 4A in untreated S50 supernatant after separation on Sephadex G-75

Fig. 3. MT quantification by modified Brdicˇ ka reaction of S50 supernatant purified by solvent precipitation according to Viarengo et al. [20]. Before the analysis, the sample was diluted 21 times. Added purified aliquots: 1 – 20 ml; 2 – 40 ml.

M. Erk et al. / Talanta 57 (2002) 1211–1218

1216

Table 1 Statistical analysis of the results on quantification of MT in differently purified samples (two subsamples and two independent measurements) Treatment

C (MT)/ mg ml−1

P

Mean HT70 °C HT85 °C SP value 9S.D. HT70 °C HT85 °C SP

1013 9107 873 9 78 733 9 28

0.0053* 0.0053* 0.0000*

0.0000* 0.0060*

0.0060*

* Differences are significant at PB0.01.

column a relatively large amount of HMW proteins is present. If S50 supernatant is purified by heat treatment at two temperatures (Fig. 4B and C) the following occurs. Heat treatment is efficient in removing HMW proteins from the S50 supernatant, but at the same time MT20 content is reduced, too. Geret et al. [22] reported that a coprecipitation of MTs during the heat denaturation could lead to an underestimation of the MT content. Comparing untreated sample (Fig. 4A) with heat treated at 70 °C (Fig. 4B), the average

amount of HMW proteins decreases from 45.8 to 9.3 mg ml − 1 (decrease of 80% relative to untreated sample), while MT20 decreases from 70.7 to 37.3 mg ml − 1 (decrease of 47%). At the same time the average content of MT10 does not change and LMW decreases from 4.3 to 1.9 mg ml − 1 (decrease of 56% relative to untreated sample). Comparing untreated sample (Fig. 4A) with heat treated at 85 °C (Fig. 4C) the average amount of HMW proteins decreases from 45.8 to 8.9 mg ml − 1 (decrease of 81%), while MT20 decreases from 70.7 to 29.5 mg ml − 1 (decrease of 58%). At the same time the average content of MT10 does not change and LMW decreases from 4.3 to 3.5 mg ml − 1. From the presentation in Fig. 4A–C it could be concluded that heat treatment at both temperatures, 70 and 85 °C, is equally efficient in removing the HMW proteins which could interfere with the subsequent MT analysis by modified Brdicka procedure (S50 was not diluted with 0.9% NaCl before heat treatment). It is also clear that at 85 °C an additional amount of MT20 is removed if compared with the heat treatment at the lower temperature (70 °C). MT20 form is not fully saturated with Cd and therefore, possible formation

Fig. 4. MT quantification by modified Brdicka reaction in gel-filtration pooled fractions eluted on untreated S50 supernatant (A), heat treated S50 supernatant, undiluted before heat treatment (B and C) and solvent treated S50 supernatant (D).

M. Erk et al. / Talanta 57 (2002) 1211–1218

of intra and inter molecular disulfide bonds might occur since the free SH-groups are highly susceptible to oxidation [18]. The breakage of the disulfide bonds could be caused by more severe purification conditions (85 °C) leading to the fragmentation of MT dimers and to the redistribution of the amounts of MT20 and MT10 (Fig. 4B and C). Interesting information follows that MT10 fraction is not affected by any purification procedure, and is the same in untreated as well as in heat or solvent treated samples. MT10 isoform remain nearly unchanged in untreated as well as in differently purified S50 fractions. The explanation could be that due to the fact that S50 supernatant was isolated from the digestive gland tissue of mussels which were exposed to elevated Cd concentrations, the MT10 isoform is saturated with Cd and therefore resistant to oxidation and polymerization. That form of MT is chemically stable and resists to any physical or chemical treatment. In comparison with untreated S50 supernatant (Fig. 4A), the purification procedure using solvent precipitation results (Fig. 4D) in the drastic reduction of MT20 fraction (from 70.7 to 10.4 mg ml − 1, which represents the decrease of 85% relative to untreated sample), while as explained before the MT10 fraction remains unchanged. Solvent precipitation procedure is more efficient in removing HMW proteins as indicated if Fig. 4A is compared with Fig. 4D. It is obvious that solvent purification procedure of S50 supernatant causes a substantial lost of MT20 (Fig. 4D). Using cloning and characterization of metallothionein cDNA in M. edulis, Barsˇyte et al. [7] found that the MT20 isoform represents a Cd-inducible form of MT, whereas MT10 is a basally-expressed form. Applying the same techniques Lemoine et al. [23] concluded that MT20 was induced by CdCl2, while an induction of MT10 was detected with ZnCl2. Therefore, the content of MT20 isoform could serve as an indicator of mussel exposure to cadmium, and it could be considered as ‘target’ MT isoform in biomonitoring. Consequently, it is recommended to use the purification procedure which less affects MT20 fraction. According to the presented results it would be the heat treatment.

1217

4. Conclusions Reliable data on MT content in mussel tissue rely on the consistent and reproducible isolation, purification and quantification procedures. It is necessary to define the isolation, purification and quantification steps in detail and statistically validate each step. In our study the common S50 supernatant, isolated from the digestive gland tissue of mussels which were exposed to elevated Cd concentrations, was subjected to three different purification procedures, determining the MT content with the common, Brdicka modified reaction. Statistically significant differences in all three purification procedures were determined and the following is concluded: “ Both heat treatments (at 70 and 85 °C) and solvent treatment efficiently remove HMW proteins from S50 supernatant, which would otherwise interfere with the electrochemical measurement. “ In untreated as well as in heat or solvent treated samples MT10 isoform remains unchanged. That could be explained by the fact that S50 supernatant was isolated from the digestive gland tissue of mussels which were exposed to elevated Cd concentrations. MT10 isoform is saturated with Cd and therefore resistant to oxidation and polymerization. “ MT20 isoform is significantly reduced by heat treatment and drastically reduced by solvent precipitation. Due to the fact that MT20 is considered as ‘target’ MT isoform for metal effect and exposure it is recommended to use the purification procedure which less affects MT20 fraction and that is heat treatment. From a quantification point of view, it is necessary to strictly follow the experimental conditions which influence the catalytic signal height like buffer, depolarizer concentration, pH value, temperature, type of calibrant, linear calibration range. Dilution of the supernatant prior to the heat treatment is beneficial, because the coprecipitation of MTs with HMW proteins is reduced and it is not necessary to dilute the sample prior to electrochemical determination.

1218

M. Erk et al. / Talanta 57 (2002) 1211–1218

Acknowledgements The financial support of the Ministry of Science and Technology of the Republic of Croatia, project No. 00981511, is gratefully acknowledged.

References [1] J. Lo´ pez-Barea, In: G.H. Degen, J.P. Seitler, P. Bentley (Eds.), Toxicology in Transition, Proceedings of the 1994 EUROTOX Congress, 1994, p. 57. [2] M.J. Stillman, Coord. Chem. Rev. 144 (1995) 461. [3] P.-A. Binz, J.H.R. Ka¨ gi, in: C.D. Klaassen (Ed.), Metallothionein IV, Birkha¨ user Verlag, Basel, 1999, p. 7. [4] G. Bordin, (Ed.) Cell. Mol. Biol., 46(2) (2000), special issue. [5] W.J. Langston, M.J. Bebianno, G. Burt, in: W.J. Langston, M.J. Bebianno (Eds.), Metal Metabolism in Aquatic Environments, Chapmann and Hill, London, 1998, p. 219. [6] E.A. MacKay, J. Overnell, B. Dunbar, I. Davidson, P.E. Hunziker, J.H.R. Ka¨ gi, J.E. Fothergill, Eur. J. Biochem. 218 (1993) 183. [7] D. Barsˇyte, K.N. White, D.A. Lovejoy, Comp. Biochem. Physiol. C 122 (1999) 287.

[8] R. Brdicˇ ka, Collect. Czech. Chem. Commun. 5 (1933) 112. [9] E. Palecˇ ek, Z. Pechan, Anal. Biochem. 42 (1971) 59. [10] R.W. Olafson, R.G. Sim, Anal. Biochem. 100 (1979) 343. [11] J.A.J. Thompson, R.P. Cosson, Mar. Environ. Res. 11 (1984) 137. [12] J. Pavicˇ ic, B. Raspor, D. Martincˇ icˇ , Mar. Biol. 115 (1993) 435. [13] M.J. Bebianno, L.M. Machado, Mar. Poll. Bull. 34 (1997) 666. [14] G. Bordin, J. McCourt, F. Cordeiro Raposo, A.R. Rodriguez, Mar. Biol. 129 (1997) 453. [15] C. Amiard-Triquet, F. Rainglet, C. Larroux, F. Regoli, H. Hummel, Ecotoxicol. Environ. Safety 41 (1998) 96. [16] B. Raspor, M. Paicˇ , M. Erk, Talanta 55 (2001) 109. [17] B. Raspor, J. Electroanal. Chem. 503 (2001) 159. [18] M. Dabrio, A.R. Rodrı´guez, G. Bordin, M.J. Bebianno, M. De Ley, I. S& esta´ kova, M. Vasˇa´ k, M. Nordberg, J. Inorg. Biochem. 88 (2002) 123. [19] G. Isani, G. Andreani, M. Kindt, E. Carpene`, Cell. Moll. Biol. 46 (2000) 311. [20] A. Viarengo, E. Ponzano, F. Dondero, R. Fabbri, Mar. Environ. Res. 44 (1997) 69. [21] R.P. Cosson, Cell. Mol. Biol. 46 (2000) 295. [22] F. Geret, F. Rainglet, R.P. Cosson, Mar. Environ. Res. 46 (1998) 545. [23] S. Lemoine, Y. Bifot, D. Sellos, R.P. Cosson, M. Laulier, Mar. Biotechnol. 2 (2000) 195.