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Metallothionein isoforms from horse, rabbit and rat separated by capillary zone electrophoresis at low pH
Talanta 46 (1998) 291 – 300
Metallothionein isoforms from horse, rabbit and rat separated by capillary zone electrophoresis at low pH Tore W. Wilhelmsen a, Pa˚l A. Olsvik b, Sverre W. Teigen a, Rolf A. Andersen b,* a
Pharmaceutical Department, The Norwegian Medicines Control Authority, S6en Oftedals6ei 6, N-0950 Oslo, Norway b Department of Zoology, Norwegian Uni6ersity of Science and Technology, N-7055 Trondheim, Norway Received 4 November 1996; received in revised form 6 January 1997; accepted 20 February 1997
Abstract A comparative study of MT isoforms in rat liver and in commercial Sigma MT preparations from rabbit liver and horse kidney was performed using capillary zone electrophoresis (CZE). Electropherograms revealed the co-migration of MT forms from these species. A special form, the a-form (not binding Cd), occurred in various MT samples in different amounts, depending on the method used for MT purification. In the rabbit liver electropherogram a main form appeared (the b-form), which might be a modified MT form. A band of unknown composition, running ahead of the rat liver MT-I and -II forms on polyacrylamide gels, not having Cd binding affinity, probably had its counterpart in a yet unidentified CZE peak. CZE electropherograms of purified MT samples may contain main peaks that do not represent genuine and functional MT isoforms. Results are also presented which indicate that at low pH the MT-II form is more unstable than MT-I. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Metallothionein isoforms; Anion exchange chromatography; Polyacrylamide gel electrophoresis; Capillary zone electrophoresis
1. Introduction Recent interest in metallothionein (MT) research, including ecotoxicological aspects has increased tremendously, this protein is used as a biomarker for heavy metal pollution and various types of stress. The purification of MT from heat treated liver cytosols of cadmium (Cd) treated animals generally involves gel permeation followed by anion exchange chromatography. Our method differs * Corresponding author.
from other previous procedures [1,2] in that the gel permeation step was run in Tris buffer of the same low ionic strength as the starting buffer used in the gradient system for anion exchange to obtain the final separation [3]. In this way dialysis and lyophilization were omitted in order to prevent oligomerizations and oxidation of the reactive MT protein [4]. A typical elution profile for rat CdMT obtained by anion exchange chromatography monitored at 254 nm includes three peaks [3]. The fractions corresponding to those eluted as the second and third contain high amounts of added 109Cd, while
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those that correspond to the first peak does not contain the isotope, even though they had a relatively high absorbance at 254 nm (see Fig. 1). It is speculated that the second and third peak contain MT-I and -II, respectively. The first eluting peak was designated the a-form. Samples of material collected from these three peaks were run on capillary zone electrophoresis (CZE). This was done in a sodium phosphate buffer at low pH. The fused silica capillary was linearly coated with acrylamide [5]. One important advantage of using this neutral hydrophilic surface compared with uncoated or positively coated capillaries (e.g. polyamine polymers) is that appropriate buffers can be designed to produce separations either toward the cathode (normal polarity) or to the anode (reversed polarity). This offers additional selectivity without significant peak broadening caused by electroendosmotic flow. An important factor in using acidic buffer conditions is metal dissociation (below MTs pI 3.8– 4.4) causing apothioneins, thereby adding more resolving power to the system than at high pH [6–10]. At neutral or alkaline pH metal containing isoforms may co-migrate because of little differences in the net charge of the two main MT-I and MT-II types [11]. In our MT measurements using the CZE technique the a-form was also found in commercial rabbit and horse MT preparations. In rabbit liver a major form occurred, designated the b-form, most probably a spontaneous modified MT form. The stability of the various MT forms was also tested. The CZE technique, which separates compounds based on their charge to mass ratio, has proven successful for fast, sensitive and reproducible separation of MT isoforms [6 – 10,12–15].
55% relative humidity. They were fed ad libitum with an EWOS commercial pelleted diet (EWOS, So¨derta¨lje, Sweden). The animals had free access to water.
2.2. Chemicals The isotope 109CdCl2 (sp.act. 760 mCi mg − 1 Cd, carrier free) was obtained from DuPont de Nemours, Wilmington, DE. The column materials Sephadex G-75 and DEAE-Sephadex A-50 were obtained from Pharmacia, Uppsala, Sweden. All chemicals were of analytical grade. 3-Amino-9ethylcarbazole, bovine serum albumin, cadmium sulphate (3CdSO4 · 8H2O), Coomassie Brilliant Blue R, N,N%-dimethylformamide, sodium acetate, Trizma base, Trizma hydrochloride, Trizma pre-set crystals, horse kidney MT (lot 73H9544) and rabbit liver MT (lot 93H9559; new lot 44H9568) were from Sigma, St. Louis, MO. Bromophenol blue, glycerol, glycine buffer substance, methanol, 4-chloro-1-naphthol, perhydrol (30% H2O2), ortho-phosphoric acid (99% pure cryst.), potassium chloride, sodium hydroxide pellets and zinc acetate were all from E. Merck, Darmstadt, Germany. Sodium dodecyl sulfate (SDS), N,N%methylene-bis-acrylamide (BIS), acrylamide (99.9% pure), ammonium persulfate, N,N,N%,N%tetra-methylethylenediamine (TEMED) and 2mercaptoethanol were all from Bio-Rad Laboratories, Life Science Group, Richmond, CA. Fluorinert™ liquid (FC-77) came from 3M, Haven, Belgium. The nitrocellulose membrane filter (2 mm) was obtained from Schleicher & Schuell, Dassal, Germany. Peroxidase conjugated IgG fraction of goat anti-rabbit IgG was prepared by Cooper Biomedical, Malvern, PA.
2.3. Metallothionein induction and preparation 2. Experimental
2.1. Animal housing and maintenance Two male rats (Rattus nor6egicus, Mol: WIST, 200–300 g) were used in the experiments. The animals were kept in macrolon cages and maintained in 12 h light/12 h darkness at 20°C and
Because MT isoforms are labile it was thought necessary to give a rather comprehensive description of the procedure for MT preparation. Cold 3CdSO4 · 8H2O was injected intraperitoneally to rats each day for a period of 4 days, 0.35 mg kg − 1 on day 1, then 1.4 mg kg − 1 on day 2, 3 and 4. After killing the animals on day 6 the livers were quickly removed and chilled on ice.
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Fig. 1. (A) The major Cd binding fractions from the chromatography of rat liver cytosol on the Sephadex G-75 column pooled and rechromatographed on a DEAE Sephadex A-50 column with a linear Tris buffer gradient from 5 to 500 mM, pH 8.5. Absorbance 254 nm ——. Refractive index · · $ · · $ · · . See text for further details. (B) Individual fractions assayed for added 109Cd marker —— — . MT-I is shown to elute at a refractive index of 1.3345, MT-II at 1.3360.
The livers were then homogenized in 5 mM Tris buffer, pH 8.5 (1:4 w/v) using a glass-Teflon homogenizer (Potter-Elvehjem) rotating at 1400 rpm. After centrifugation at 20 300 g for 30 min at 4°C the supernatant was heat treated by equilibrating it on a water bath during temperature elevation from 20 to 80°C. Then the supernatant was kept at this temperature for 2 min, after which it was quickly chilled. The heat treated mixture was centrifuged at 20 300 g for 30 min at 4°C and the supernatant saved. An aliquot of the heat treated supernatant (6– 10 ml) was applied to a Sephadex G-75 column (2.5× 72.5 cm) and eluted with 5 mM Tris buffer pH 8.5 at a flow rate of 1 ml min − 1. Supernatant samples were equilibrated for 5 min with 10 ml of Cd isotope solution, containing 100 000 cpm ml − 1 prior to gel permeation. Fractions (each 4.25 ml) were collected and analysed for 109Cd by a Packard Minaxy Auto-gamma counter, model 5550 (Packard Instruments, IL).
The peak radioactivity fractions eluted from the Sephadex G-75 column were pooled and applied directly on a DEAE-Sephadex A-50 anion exchange column (1.5× 25 cm) equilibrated with 5 mM Tris buffer, pH 8.5. After washing in (100 ml buffer) elution was started with a linear Tris buffer gradient (5–500 mM, pH 8.5, 600 ml). Eluate fractions of 4.25 ml were collected and analysed for UV absorption at 254 nm and for 109 Cd. The peak fractions were then pooled and lyophilized to get the final purified MT preparations. Tris salt was not removed.
2.4. Polyacrylamide gel electrophoresis and staining The Bio-Rad Protean II Slab Cell was used for SDS polyacrylamide electrophoresis using the Laemmli reducing buffer system [16] as well as the native gel system. The stacking gel for the SDS system contained 4% acrylamide (0.35% bis), separation gel 12% (1% bis). The sample buffer con-
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sisted of 4.0 ml aq. dest., 1.0 ml 0.5 M Tris– HCl (pH 6.8), 0.8 ml glycerol, 1.6 ml 10% (w/v) SDS, 0.4 ml 2-mercaptoethanol and 0.05% (w/v) bromophenol blue. Protein samples were diluted 1:4 with this buffer and heated to 80°C for 2 min to minimize negative charge differences and disulphide linkages prior to each run. For native polyacrylamide gel electrophoresis the method by Jovin et al. [17] was used, except for riboflavin being replaced by TEMED and ammonium persulfate for upper gel polymerization. The stacking gel contained 2.5% acrylamide, separation gel 7.5%. The Protean II slab cell apparatus was run at 40 V (16 h) overnight using the Bio-Rad Power Supply, model 250/2.5. The acrylamide gels were stained with Coomassie Brilliant Blue.
2.5. Electroblotting of MT, identification of Cd binding proteins, Western blotting The electrotransfer of MT from polyacrylamide gels to nitrocellulose filters was per-
Fig. 2. Polyacrylamide SDS gel electrophoresis, Western blotting, autoradiography and native gel electrophoresis of our purified MT isoforms. In lanes (1), (2) the individual MT-II and MT-I preparations, respectively, Coomassie stained; (3), (4) Western blotting of MT-I and MT-II, respectively; (5), (6) autoradiography. In lane (7) MT-I and MT-II isoforms mixed, subjected to native gel electrophoresis. Arrowheads indicate positions of the SDS molecular weight markers (from top to bottom: ovalbumin, 45 kDa; carbonic anhydrase, 31; soybean trypsin inhibitor, 21.5; lysozyme, 14.4).
formed using the Bio-Rad Trans-Blot Cell according to the method employed by Towbin et al. [18]. The polyacrylamide gels (0.75 mm in thickness) were run as previously described [16]. After electrophoresis the gels were equilibrated in 500 ml Tris–glycine electrotransfer buffer (3.03 g Tris base, 14.4 g glycine, 200 ml methanol, aq. dest. to 1l) for 30 min. Then the electrotransfer was performed for 2 h at 60 V (Bio-Rad Power Supply, model 250/2.5). To identify Cd binding proteins using autoradiography [19] the filter was equilibrated for 2 h in 400 ml of 10 mM Tris buffer, pH 7.4 at 4°C. The filter was then submerged for 10 min at room temperature in 25 ml of 10 mM Tris buffer, pH 7.4 containing 1 mCi 109CdCl2 ml − 1, 0.1 mM zinc acetate and 0.1 M KCl. The filter was then washed twice in aq. dest. at room temperature and completely dried. Autoradiography was performed at −70°C for 38–48 h using Kodak X-Omat AR (XAR-5) film and DuPont Cronex Quanta II intensifying screens. The films were manually processed with Kodak LX 24 developer and AL 4 fixative. Using nitrocellulose filters prepared as described above, our anti rat MT antibodies (developed in rabbits) were subjected to Western blotting [3,20]. The filters were washed in phosphate buffered saline for 15 min and then kept for 30 min in a solution consisting of 3 g bovine serum albumin to 100 ml phosphate buffered saline. The filter was then treated with antibody (rabbit serum without further purification) at a concentration of 40 ml 10 ml − 1 blocking solution by gentle shaking overnight. The antibody solution was removed by washing with phosphate buffered saline. The filter was then treated with peroxidase conjugated IgG fraction goat anti-rabbit IgG in the concentration of 10 ml (undiluted) 10 ml − 1 blocking solution. After shaking for 2 h the filter was developed in a mixture of 2 ml aminoethylcarbazole (AEC, fresh stock), 50 ml sodium acetate buffer, pH 5 and 25 ml hydrogen peroxide. The stock solution consisted of 0.25 g AEC to 25 ml dimethylformamide.
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Fig. 3. Mixtures of our rat MT isoforms subjected to capillary zone electrophoresis (CZE) at 200 mM sodium phosphate, pH 2.0. The individual forms were diluted 10 times from stock solutions (see text) and mixed in equal volumes. (A) The a-form (17 mg ml − 1)+ MT-I (16 mg ml − 1)+ MT-II (29 mg ml − 1), run at 10 kV. (B) MT-I (25 mg ml − 1) +MT-II (44 mg ml − 1). (C) MT-I (37 mg ml − 1)+ MT-II (22 mg ml − 1), volume 3:1. (B) and (C) run at 12 kV. (D – F) the individual a-form (50 mg ml − 1), MT-I (49 mg ml − 1), MT-II (87 mg ml − 1), run at 10 kV. Given concentrations represent end values after mixing.
2.6. Capillary zone electrophoresis of MT samples The BioFocus™ 3000 Capillary Electrophoresis System with Spectra Software version 3.00, Integration Software version 3.01 and BioFocus Capillary Cartridge, 17 cm× 25 mm I.D., 375 mm O.D., polyacrylamide coated (12.5 cm to the detector window), from Bio-Rad Laboratories, Life Science Group, Richmond, CA., were used for MT analysis.
The lyophilized a-peak sample prepared in our laboratory was diluted in 200 ml aq. dest., the MT-I sample in 500 ml aq. dest and MT-II in 1 ml (stock solutions). These samples were again diluted 1:9 in aq. dest. before CZE. The commercial Sigma reference samples of rabbit (lot 93H9559, new lot 44H9568) and horse MT (lot 73H9544), claimed to be essentially salt free, were dissolved in 10 mM Trizma pre-set crystal buffer at pH 9.1 to a final concentration of 180 mg ml − 1. Further
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Fig. 4. Separation of mixed samples of horse and rat MT isoforms by CZE in 200 mM sodium phosphate, pH 2.0 at 10 kV. (A) Commercial horse MT preparation alone (180 mg ml − 1) (rat isoform mixture alone shown in Fig. 3), (B) horse MT (90 mg ml − 1)+ rat MT (93 mg ml − 1), (C) horse MT (90 mg ml − 1) +the individual rat a-form (25 mg ml − 1). Given concentrations represent end values after mixing.
Fig. 5. Separation of mixed samples of rabbit, rat and horse MT isoforms by CZE in 200 mM sodium phosphate, pH 2.0 at 10 kV. (A) Commercial rabbit MT preparation alone (old lot 93H9559) (180 mg ml − 1), (B) rabbit MT (90 mg ml − 1) +rat MT (93 mg ml − 1), (C) rabbit MT (90 mg ml − 1)+ horse MT (90 mg ml − 1). Given concentrations represent end values after mixing.
dilutions of the samples were done in aq. dest. The MT samples were kept as stock solutions at − 70°C. Samples were mixed by volume ratio 1:1, except for rat MT-I and MT-II which were also mixed 3:1 (Fig. 3C). All CZE measurements were performed in 200 mM sodium phosphate at pH 2.0 (19.6 g H3PO4 diluted
in aq. dest., titrated by 1 M NaOH to pH 2.0 and then aq. dest. to 11). The experiments were done at constant voltage (10 or 12 kV) giving a maximum current of 55 mA in the capillary. Detection was performed at 200 nm. The cartridge and the carusel were thermostated at 20°C by the Peltier thermoelectric cooling system with Fluorinert™ liquid.
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Fig. 6. Stability of the commercial rabbit MT preparations kept under atmospheric conditions, pH 9.1 at room temperature and measured by CZE in 200 mM sodium phosphate, pH 2.0. (A) Old lot (93H9559), 65 h (old lot, 0 h shown in Fig. 5A), (B) old lot, 60 days, (C) new lot (44H9568), 0 h. The b-form is clearly distinguished from the old lot, not in the new lot. In (B) peaks are merged. (A) and (B) run at 12 kV, (C) run at 10 kV.
All samples were applied hydrodynamically under pressure (20 psi× s) and analysed using polarity from the positive to the negative electrode. The important washing procedure between each single run was as follows: 15 s with 50 mM H3PO4 at pH 1.85; 10 s with electrophoresis buffer (see above); 10 s with 0.25 mM NaOH and finally electrophoresis buffer for 15 s.
3. Results and discussion Fig. 1A shows that freshly purified MT after anion exchange chromatography on the DEAESephadex A-50 column, monitored at 254 nm, elutes as three peaks. When peak material was tested for 109Cd binding affinity (Fig. 1B), it turned out that the second and third eluting peak contained MT-I and MT-II, respectively. The non metal binding material (Fig. 1) eluting at refractive index of 1.3333 in the Tris buffer gradient, thereby appearing in front of the two MT isoforms, was designated the a-form. Our rat MT preparations gave three individual bands on Coomassie stained SDS polyacrylamide and native gels (Fig. 2). Both gel systems separated the two common MT isoforms with the I
form running ahead of the II form. The faint band appearing in front of these two isoforms may correspond to an unknown protein discovered previously in the MT-II fraction by Beattie et al. [12] and assumed to be hemoglobin. This band may, however, represent a precursor of active metal binding MT [3]. The functional studies represented by Western blotting, the autoradiography data (Fig. 2, lanes 3–6) as well as the 109Cd binding study shown in Fig. 1B, show that MT-I and MT-II isoforms are only functionally capable of metal binding, not the material in the faint band. The metal binding in this band in lane 6 is probably unspecific caused by presence of the Cd isotope in excess. When MT samples isolated in our experiments on the DEAE-Sephadex A-50 column were pooled and run through the CZE system, three peaks were seen (Fig. 3A). Runs of mixtures of MT forms in different proportions clearly demonstrates that the first eluting peak in the CZE electropherogram at low pH corresponds to the a-form, the second to MT-II and the third peak to the MT-I isoform (Fig. 3A–C). In addition to the running voltage, the positions of the various MT forms in the electropherogram are largely dependent on ionic strength and pH, at acidic pH the
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MT-II form is more positively charged than the MT-I form. In separate runs of individual MT forms, as shown in Fig. 3E, F, MT-I migrates faster than MT-II in spite of the higher charge of the latter. This is explained by the higher ionic strength of the MT-II sample, being eluted at a higher salt than MT-I in the anionic exchange gradient (Fig. 1A). When the MT samples are mixed and thereby run under equal conditions, as in Fig. 3A– C, MT-II migrates faster than the MT-I form. Further experiments to determine the content of the a-peak were not carried out. It is, however, unlikely that it contains low molecular weight sulphydryls due to improper heat treatment, as is suggested from the work of Olafson and Olsson [21]. Such species are removed when selecting the MT containing fractions from the Sephadex G-75 column prior to further processing on the ion exchange column. In the commercial rabbit MT
Fig. 7. Stability of the rat MT isoforms kept under atmospheric conditions, pH 9.1 and measured by CZE in 200 mM sodium phosphate, pH 2.0 at 12 kV. (A) Room temperature at 0, 4, 20 and 65 h, (B) refrigerated at 5°C at 0, 20, 44 and 68 h. Each point gives the mean of two or three individual runs.
Fig. 8. Regression analysis of individual isoforms for the rabbit MT preparation (old lot 93H9559) under atmospheric conditions, pH 9.1 and room temperature measured by CZE in 200 mM sodium phosphate, pH 2.0 at 10 kV covering the concentration range of 1.4 – 180 mg ml − 1. Migration time and area reproducibility of 92% was observed. Achieved regression of normalized peak areas (mV× s/migration time, see text) was r 2 =0.99896 for the b-form, r 2 =0.99931 for MT-II, r 2 =0.99805 for MT-I and r 2 =0.99903 for total MT. Individual points represent the mean of three separate runs.
preparation used in the present work the a-form could only be seen in the old lot (lot 93H9559) at concentrations above 500 mg ml − 1 MT. In a recently purchased preparation (new lot 44H9568) this form could be seen at 200 mg ml − 1. The a-form was also present in MT preparations studied by others [9,10]. We believe that the presence of this form in MT samples depends on the ability of picking only the MT-I and -II containing fractions in the purification procedure. The isoelectric focusing used as the final step in the isolation of the rabbit liver Sigma MT preparation [22] seems to be more effective in achieving this than the anion exchange procedure used for the Sigma horse kidney MT separation [23,24]. A proper interpretation of the CZE electropherograms of MT samples should include the functionality of separated forms. When the commercial horse MT preparation and our rat MT preparations were pooled prior to CZE, marked co-migration between peaks was observed (Fig. 4A–C), with only minor differences confined to the a-peak and the MT-II peak. Corresponding results for rabbit commercial and rat MTs as well as for commercial rabbit and horse MT preparations, are shown in Fig. 5A–C with minor differences related only to the MT-I
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peak. Co-migration at this level therefore seems to be related to mutual factors between isoforms, no further studies were carried out. It can also be seen that the rabbit MT preparation had one dominant form, marked as the b-form in Fig. 5A, which did not concur with any of those present in horse and rat MTs. In Fig. 6A,B it is shown that this form developed gradually with time at pH 9.1 under atmospheric conditions at room temperature. The electropherogram of the new lot of rabbit MT, however, did not show this peak (Fig. 6C), indicating the rabbit b-form develops spontaneously during storage. In recent work on rabbit MT isoforms by Virtanen et al. [6] their major g-peak was characterized as a modified isoform, which probably corresponds to the b-form described in the present work. A corresponding peak is also seen in electropherograms from other workers [8– 10] having run rabbit MT preparations using the CZE technique at acidic conditions. When Fig. 3A is compared with Fig. 5B, it is interesting to note that a small indication of the b-form presence was also seen in our rat liver MT preparation as the part ahead of the MT-II peak in Fig. 3A. By using our CZE technique, stability of the freshly prepared rat MT preparations were tested at pH 8.5 under atmospheric conditions at room temperature and refrigerated at 5°C over a period of 65 and 68 h, respectively. Analysis of aliquots showed that the a-form did not degrade in contrast to MT-I and -II (Fig. 7). Under these conditions the I form was more stable than the II form. Corresponding results (not shown) were found for the rabbit (pH 9.1) and horse MT (pH 9.1) preparations. At tested levels the a-form was not found in rabbit. The observation that the MT-I form is more stable than the MT-II form is surprising [25]. At pH 2.0, however, at which the stability measurements were performed, metal is stripped from the protein resulting in apoprotein (apothionein) formation. Under these conditions the MT-II form may be more vulnerable than the MT-I form. During degradation experiments, new peaks were not observed in our rat MT forms. The regression analysis of individual MT forms in rabbit covering the range 1.4 – 180.0 mg ml − 1 is shown in Fig. 8. The observed detection limit for
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MT preparations below 5 mg ml − 1 corresponds to the sensitivity level found by others [8,12,13,15]. Other MT preparations studied in the present work gave similar results (not shown). The concentration of the individual isoforms in the rat liver MT preparations was determined by measuring the total and relative percentage levels of the individual forms. These values were then related to the measured total level of forms in the horse Sigma preparation, for which the MT concentration was given by the supplier. This preparation contained similar isoform types as found in rat liver (Fig. 4). In the commercial rabbit preparation the a-form was seen only at higher concentrations, and it contained considerable amounts of the b-form. It is important to keep in mind that when the sample is applied hydrodynamically (pressure) to the CZE capillary as in this work, the separated forms move through the detector window at different speeds. Peak area is a better measure for actual amounts than peak height when using different running conditions, i.e. voltage and unequal salt concentration in the samples. To compare peak areas in stability testing and regression, however, they must be divided by the corresponding migration times to give normalized values (Fig. 8) [26,27].
Acknowledgements The authors would like to thank Prof. Stellan Hjerte´n, Department of Biochemistry, BMC, S751 23 Uppsala, Sweden, for his critical review of the manuscript. We are especially grateful for the gift of coated capillaries from the Bio-Rad Laboratories, Life Science Group, Richmond, CA., and to Director Lars Th. Wold, Bio-Test A/S, N-1580 Rygge, Norway, for the courtesy of free disposal of the BioFocus™ 3000 system.
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Metallothionein isoforms from horse, rabbit and rat separated by capillary zone electrophoresis at low pH Tore W. Wilhelmsen a, Pa˚l A. Olsvik b, Sverre W. Teigen a, Rolf A. Andersen b,* a
Pharmaceutical Department, The Norwegian Medicines Control Authority, S6en Oftedals6ei 6, N-0950 Oslo, Norway b Department of Zoology, Norwegian Uni6ersity of Science and Technology, N-7055 Trondheim, Norway Received 4 November 1996; received in revised form 6 January 1997; accepted 20 February 1997
Abstract A comparative study of MT isoforms in rat liver and in commercial Sigma MT preparations from rabbit liver and horse kidney was performed using capillary zone electrophoresis (CZE). Electropherograms revealed the co-migration of MT forms from these species. A special form, the a-form (not binding Cd), occurred in various MT samples in different amounts, depending on the method used for MT purification. In the rabbit liver electropherogram a main form appeared (the b-form), which might be a modified MT form. A band of unknown composition, running ahead of the rat liver MT-I and -II forms on polyacrylamide gels, not having Cd binding affinity, probably had its counterpart in a yet unidentified CZE peak. CZE electropherograms of purified MT samples may contain main peaks that do not represent genuine and functional MT isoforms. Results are also presented which indicate that at low pH the MT-II form is more unstable than MT-I. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Metallothionein isoforms; Anion exchange chromatography; Polyacrylamide gel electrophoresis; Capillary zone electrophoresis
1. Introduction Recent interest in metallothionein (MT) research, including ecotoxicological aspects has increased tremendously, this protein is used as a biomarker for heavy metal pollution and various types of stress. The purification of MT from heat treated liver cytosols of cadmium (Cd) treated animals generally involves gel permeation followed by anion exchange chromatography. Our method differs * Corresponding author.
from other previous procedures [1,2] in that the gel permeation step was run in Tris buffer of the same low ionic strength as the starting buffer used in the gradient system for anion exchange to obtain the final separation [3]. In this way dialysis and lyophilization were omitted in order to prevent oligomerizations and oxidation of the reactive MT protein [4]. A typical elution profile for rat CdMT obtained by anion exchange chromatography monitored at 254 nm includes three peaks [3]. The fractions corresponding to those eluted as the second and third contain high amounts of added 109Cd, while
0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 3 9 - 9 1 4 0 ( 9 7 ) 0 0 3 4 0 - 8
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those that correspond to the first peak does not contain the isotope, even though they had a relatively high absorbance at 254 nm (see Fig. 1). It is speculated that the second and third peak contain MT-I and -II, respectively. The first eluting peak was designated the a-form. Samples of material collected from these three peaks were run on capillary zone electrophoresis (CZE). This was done in a sodium phosphate buffer at low pH. The fused silica capillary was linearly coated with acrylamide [5]. One important advantage of using this neutral hydrophilic surface compared with uncoated or positively coated capillaries (e.g. polyamine polymers) is that appropriate buffers can be designed to produce separations either toward the cathode (normal polarity) or to the anode (reversed polarity). This offers additional selectivity without significant peak broadening caused by electroendosmotic flow. An important factor in using acidic buffer conditions is metal dissociation (below MTs pI 3.8– 4.4) causing apothioneins, thereby adding more resolving power to the system than at high pH [6–10]. At neutral or alkaline pH metal containing isoforms may co-migrate because of little differences in the net charge of the two main MT-I and MT-II types [11]. In our MT measurements using the CZE technique the a-form was also found in commercial rabbit and horse MT preparations. In rabbit liver a major form occurred, designated the b-form, most probably a spontaneous modified MT form. The stability of the various MT forms was also tested. The CZE technique, which separates compounds based on their charge to mass ratio, has proven successful for fast, sensitive and reproducible separation of MT isoforms [6 – 10,12–15].
55% relative humidity. They were fed ad libitum with an EWOS commercial pelleted diet (EWOS, So¨derta¨lje, Sweden). The animals had free access to water.
2.2. Chemicals The isotope 109CdCl2 (sp.act. 760 mCi mg − 1 Cd, carrier free) was obtained from DuPont de Nemours, Wilmington, DE. The column materials Sephadex G-75 and DEAE-Sephadex A-50 were obtained from Pharmacia, Uppsala, Sweden. All chemicals were of analytical grade. 3-Amino-9ethylcarbazole, bovine serum albumin, cadmium sulphate (3CdSO4 · 8H2O), Coomassie Brilliant Blue R, N,N%-dimethylformamide, sodium acetate, Trizma base, Trizma hydrochloride, Trizma pre-set crystals, horse kidney MT (lot 73H9544) and rabbit liver MT (lot 93H9559; new lot 44H9568) were from Sigma, St. Louis, MO. Bromophenol blue, glycerol, glycine buffer substance, methanol, 4-chloro-1-naphthol, perhydrol (30% H2O2), ortho-phosphoric acid (99% pure cryst.), potassium chloride, sodium hydroxide pellets and zinc acetate were all from E. Merck, Darmstadt, Germany. Sodium dodecyl sulfate (SDS), N,N%methylene-bis-acrylamide (BIS), acrylamide (99.9% pure), ammonium persulfate, N,N,N%,N%tetra-methylethylenediamine (TEMED) and 2mercaptoethanol were all from Bio-Rad Laboratories, Life Science Group, Richmond, CA. Fluorinert™ liquid (FC-77) came from 3M, Haven, Belgium. The nitrocellulose membrane filter (2 mm) was obtained from Schleicher & Schuell, Dassal, Germany. Peroxidase conjugated IgG fraction of goat anti-rabbit IgG was prepared by Cooper Biomedical, Malvern, PA.
2.3. Metallothionein induction and preparation 2. Experimental
2.1. Animal housing and maintenance Two male rats (Rattus nor6egicus, Mol: WIST, 200–300 g) were used in the experiments. The animals were kept in macrolon cages and maintained in 12 h light/12 h darkness at 20°C and
Because MT isoforms are labile it was thought necessary to give a rather comprehensive description of the procedure for MT preparation. Cold 3CdSO4 · 8H2O was injected intraperitoneally to rats each day for a period of 4 days, 0.35 mg kg − 1 on day 1, then 1.4 mg kg − 1 on day 2, 3 and 4. After killing the animals on day 6 the livers were quickly removed and chilled on ice.
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Fig. 1. (A) The major Cd binding fractions from the chromatography of rat liver cytosol on the Sephadex G-75 column pooled and rechromatographed on a DEAE Sephadex A-50 column with a linear Tris buffer gradient from 5 to 500 mM, pH 8.5. Absorbance 254 nm ——. Refractive index · · $ · · $ · · . See text for further details. (B) Individual fractions assayed for added 109Cd marker —— — . MT-I is shown to elute at a refractive index of 1.3345, MT-II at 1.3360.
The livers were then homogenized in 5 mM Tris buffer, pH 8.5 (1:4 w/v) using a glass-Teflon homogenizer (Potter-Elvehjem) rotating at 1400 rpm. After centrifugation at 20 300 g for 30 min at 4°C the supernatant was heat treated by equilibrating it on a water bath during temperature elevation from 20 to 80°C. Then the supernatant was kept at this temperature for 2 min, after which it was quickly chilled. The heat treated mixture was centrifuged at 20 300 g for 30 min at 4°C and the supernatant saved. An aliquot of the heat treated supernatant (6– 10 ml) was applied to a Sephadex G-75 column (2.5× 72.5 cm) and eluted with 5 mM Tris buffer pH 8.5 at a flow rate of 1 ml min − 1. Supernatant samples were equilibrated for 5 min with 10 ml of Cd isotope solution, containing 100 000 cpm ml − 1 prior to gel permeation. Fractions (each 4.25 ml) were collected and analysed for 109Cd by a Packard Minaxy Auto-gamma counter, model 5550 (Packard Instruments, IL).
The peak radioactivity fractions eluted from the Sephadex G-75 column were pooled and applied directly on a DEAE-Sephadex A-50 anion exchange column (1.5× 25 cm) equilibrated with 5 mM Tris buffer, pH 8.5. After washing in (100 ml buffer) elution was started with a linear Tris buffer gradient (5–500 mM, pH 8.5, 600 ml). Eluate fractions of 4.25 ml were collected and analysed for UV absorption at 254 nm and for 109 Cd. The peak fractions were then pooled and lyophilized to get the final purified MT preparations. Tris salt was not removed.
2.4. Polyacrylamide gel electrophoresis and staining The Bio-Rad Protean II Slab Cell was used for SDS polyacrylamide electrophoresis using the Laemmli reducing buffer system [16] as well as the native gel system. The stacking gel for the SDS system contained 4% acrylamide (0.35% bis), separation gel 12% (1% bis). The sample buffer con-
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sisted of 4.0 ml aq. dest., 1.0 ml 0.5 M Tris– HCl (pH 6.8), 0.8 ml glycerol, 1.6 ml 10% (w/v) SDS, 0.4 ml 2-mercaptoethanol and 0.05% (w/v) bromophenol blue. Protein samples were diluted 1:4 with this buffer and heated to 80°C for 2 min to minimize negative charge differences and disulphide linkages prior to each run. For native polyacrylamide gel electrophoresis the method by Jovin et al. [17] was used, except for riboflavin being replaced by TEMED and ammonium persulfate for upper gel polymerization. The stacking gel contained 2.5% acrylamide, separation gel 7.5%. The Protean II slab cell apparatus was run at 40 V (16 h) overnight using the Bio-Rad Power Supply, model 250/2.5. The acrylamide gels were stained with Coomassie Brilliant Blue.
2.5. Electroblotting of MT, identification of Cd binding proteins, Western blotting The electrotransfer of MT from polyacrylamide gels to nitrocellulose filters was per-
Fig. 2. Polyacrylamide SDS gel electrophoresis, Western blotting, autoradiography and native gel electrophoresis of our purified MT isoforms. In lanes (1), (2) the individual MT-II and MT-I preparations, respectively, Coomassie stained; (3), (4) Western blotting of MT-I and MT-II, respectively; (5), (6) autoradiography. In lane (7) MT-I and MT-II isoforms mixed, subjected to native gel electrophoresis. Arrowheads indicate positions of the SDS molecular weight markers (from top to bottom: ovalbumin, 45 kDa; carbonic anhydrase, 31; soybean trypsin inhibitor, 21.5; lysozyme, 14.4).
formed using the Bio-Rad Trans-Blot Cell according to the method employed by Towbin et al. [18]. The polyacrylamide gels (0.75 mm in thickness) were run as previously described [16]. After electrophoresis the gels were equilibrated in 500 ml Tris–glycine electrotransfer buffer (3.03 g Tris base, 14.4 g glycine, 200 ml methanol, aq. dest. to 1l) for 30 min. Then the electrotransfer was performed for 2 h at 60 V (Bio-Rad Power Supply, model 250/2.5). To identify Cd binding proteins using autoradiography [19] the filter was equilibrated for 2 h in 400 ml of 10 mM Tris buffer, pH 7.4 at 4°C. The filter was then submerged for 10 min at room temperature in 25 ml of 10 mM Tris buffer, pH 7.4 containing 1 mCi 109CdCl2 ml − 1, 0.1 mM zinc acetate and 0.1 M KCl. The filter was then washed twice in aq. dest. at room temperature and completely dried. Autoradiography was performed at −70°C for 38–48 h using Kodak X-Omat AR (XAR-5) film and DuPont Cronex Quanta II intensifying screens. The films were manually processed with Kodak LX 24 developer and AL 4 fixative. Using nitrocellulose filters prepared as described above, our anti rat MT antibodies (developed in rabbits) were subjected to Western blotting [3,20]. The filters were washed in phosphate buffered saline for 15 min and then kept for 30 min in a solution consisting of 3 g bovine serum albumin to 100 ml phosphate buffered saline. The filter was then treated with antibody (rabbit serum without further purification) at a concentration of 40 ml 10 ml − 1 blocking solution by gentle shaking overnight. The antibody solution was removed by washing with phosphate buffered saline. The filter was then treated with peroxidase conjugated IgG fraction goat anti-rabbit IgG in the concentration of 10 ml (undiluted) 10 ml − 1 blocking solution. After shaking for 2 h the filter was developed in a mixture of 2 ml aminoethylcarbazole (AEC, fresh stock), 50 ml sodium acetate buffer, pH 5 and 25 ml hydrogen peroxide. The stock solution consisted of 0.25 g AEC to 25 ml dimethylformamide.
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Fig. 3. Mixtures of our rat MT isoforms subjected to capillary zone electrophoresis (CZE) at 200 mM sodium phosphate, pH 2.0. The individual forms were diluted 10 times from stock solutions (see text) and mixed in equal volumes. (A) The a-form (17 mg ml − 1)+ MT-I (16 mg ml − 1)+ MT-II (29 mg ml − 1), run at 10 kV. (B) MT-I (25 mg ml − 1) +MT-II (44 mg ml − 1). (C) MT-I (37 mg ml − 1)+ MT-II (22 mg ml − 1), volume 3:1. (B) and (C) run at 12 kV. (D – F) the individual a-form (50 mg ml − 1), MT-I (49 mg ml − 1), MT-II (87 mg ml − 1), run at 10 kV. Given concentrations represent end values after mixing.
2.6. Capillary zone electrophoresis of MT samples The BioFocus™ 3000 Capillary Electrophoresis System with Spectra Software version 3.00, Integration Software version 3.01 and BioFocus Capillary Cartridge, 17 cm× 25 mm I.D., 375 mm O.D., polyacrylamide coated (12.5 cm to the detector window), from Bio-Rad Laboratories, Life Science Group, Richmond, CA., were used for MT analysis.
The lyophilized a-peak sample prepared in our laboratory was diluted in 200 ml aq. dest., the MT-I sample in 500 ml aq. dest and MT-II in 1 ml (stock solutions). These samples were again diluted 1:9 in aq. dest. before CZE. The commercial Sigma reference samples of rabbit (lot 93H9559, new lot 44H9568) and horse MT (lot 73H9544), claimed to be essentially salt free, were dissolved in 10 mM Trizma pre-set crystal buffer at pH 9.1 to a final concentration of 180 mg ml − 1. Further
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Fig. 4. Separation of mixed samples of horse and rat MT isoforms by CZE in 200 mM sodium phosphate, pH 2.0 at 10 kV. (A) Commercial horse MT preparation alone (180 mg ml − 1) (rat isoform mixture alone shown in Fig. 3), (B) horse MT (90 mg ml − 1)+ rat MT (93 mg ml − 1), (C) horse MT (90 mg ml − 1) +the individual rat a-form (25 mg ml − 1). Given concentrations represent end values after mixing.
Fig. 5. Separation of mixed samples of rabbit, rat and horse MT isoforms by CZE in 200 mM sodium phosphate, pH 2.0 at 10 kV. (A) Commercial rabbit MT preparation alone (old lot 93H9559) (180 mg ml − 1), (B) rabbit MT (90 mg ml − 1) +rat MT (93 mg ml − 1), (C) rabbit MT (90 mg ml − 1)+ horse MT (90 mg ml − 1). Given concentrations represent end values after mixing.
dilutions of the samples were done in aq. dest. The MT samples were kept as stock solutions at − 70°C. Samples were mixed by volume ratio 1:1, except for rat MT-I and MT-II which were also mixed 3:1 (Fig. 3C). All CZE measurements were performed in 200 mM sodium phosphate at pH 2.0 (19.6 g H3PO4 diluted
in aq. dest., titrated by 1 M NaOH to pH 2.0 and then aq. dest. to 11). The experiments were done at constant voltage (10 or 12 kV) giving a maximum current of 55 mA in the capillary. Detection was performed at 200 nm. The cartridge and the carusel were thermostated at 20°C by the Peltier thermoelectric cooling system with Fluorinert™ liquid.
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Fig. 6. Stability of the commercial rabbit MT preparations kept under atmospheric conditions, pH 9.1 at room temperature and measured by CZE in 200 mM sodium phosphate, pH 2.0. (A) Old lot (93H9559), 65 h (old lot, 0 h shown in Fig. 5A), (B) old lot, 60 days, (C) new lot (44H9568), 0 h. The b-form is clearly distinguished from the old lot, not in the new lot. In (B) peaks are merged. (A) and (B) run at 12 kV, (C) run at 10 kV.
All samples were applied hydrodynamically under pressure (20 psi× s) and analysed using polarity from the positive to the negative electrode. The important washing procedure between each single run was as follows: 15 s with 50 mM H3PO4 at pH 1.85; 10 s with electrophoresis buffer (see above); 10 s with 0.25 mM NaOH and finally electrophoresis buffer for 15 s.
3. Results and discussion Fig. 1A shows that freshly purified MT after anion exchange chromatography on the DEAESephadex A-50 column, monitored at 254 nm, elutes as three peaks. When peak material was tested for 109Cd binding affinity (Fig. 1B), it turned out that the second and third eluting peak contained MT-I and MT-II, respectively. The non metal binding material (Fig. 1) eluting at refractive index of 1.3333 in the Tris buffer gradient, thereby appearing in front of the two MT isoforms, was designated the a-form. Our rat MT preparations gave three individual bands on Coomassie stained SDS polyacrylamide and native gels (Fig. 2). Both gel systems separated the two common MT isoforms with the I
form running ahead of the II form. The faint band appearing in front of these two isoforms may correspond to an unknown protein discovered previously in the MT-II fraction by Beattie et al. [12] and assumed to be hemoglobin. This band may, however, represent a precursor of active metal binding MT [3]. The functional studies represented by Western blotting, the autoradiography data (Fig. 2, lanes 3–6) as well as the 109Cd binding study shown in Fig. 1B, show that MT-I and MT-II isoforms are only functionally capable of metal binding, not the material in the faint band. The metal binding in this band in lane 6 is probably unspecific caused by presence of the Cd isotope in excess. When MT samples isolated in our experiments on the DEAE-Sephadex A-50 column were pooled and run through the CZE system, three peaks were seen (Fig. 3A). Runs of mixtures of MT forms in different proportions clearly demonstrates that the first eluting peak in the CZE electropherogram at low pH corresponds to the a-form, the second to MT-II and the third peak to the MT-I isoform (Fig. 3A–C). In addition to the running voltage, the positions of the various MT forms in the electropherogram are largely dependent on ionic strength and pH, at acidic pH the
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MT-II form is more positively charged than the MT-I form. In separate runs of individual MT forms, as shown in Fig. 3E, F, MT-I migrates faster than MT-II in spite of the higher charge of the latter. This is explained by the higher ionic strength of the MT-II sample, being eluted at a higher salt than MT-I in the anionic exchange gradient (Fig. 1A). When the MT samples are mixed and thereby run under equal conditions, as in Fig. 3A– C, MT-II migrates faster than the MT-I form. Further experiments to determine the content of the a-peak were not carried out. It is, however, unlikely that it contains low molecular weight sulphydryls due to improper heat treatment, as is suggested from the work of Olafson and Olsson [21]. Such species are removed when selecting the MT containing fractions from the Sephadex G-75 column prior to further processing on the ion exchange column. In the commercial rabbit MT
Fig. 7. Stability of the rat MT isoforms kept under atmospheric conditions, pH 9.1 and measured by CZE in 200 mM sodium phosphate, pH 2.0 at 12 kV. (A) Room temperature at 0, 4, 20 and 65 h, (B) refrigerated at 5°C at 0, 20, 44 and 68 h. Each point gives the mean of two or three individual runs.
Fig. 8. Regression analysis of individual isoforms for the rabbit MT preparation (old lot 93H9559) under atmospheric conditions, pH 9.1 and room temperature measured by CZE in 200 mM sodium phosphate, pH 2.0 at 10 kV covering the concentration range of 1.4 – 180 mg ml − 1. Migration time and area reproducibility of 92% was observed. Achieved regression of normalized peak areas (mV× s/migration time, see text) was r 2 =0.99896 for the b-form, r 2 =0.99931 for MT-II, r 2 =0.99805 for MT-I and r 2 =0.99903 for total MT. Individual points represent the mean of three separate runs.
preparation used in the present work the a-form could only be seen in the old lot (lot 93H9559) at concentrations above 500 mg ml − 1 MT. In a recently purchased preparation (new lot 44H9568) this form could be seen at 200 mg ml − 1. The a-form was also present in MT preparations studied by others [9,10]. We believe that the presence of this form in MT samples depends on the ability of picking only the MT-I and -II containing fractions in the purification procedure. The isoelectric focusing used as the final step in the isolation of the rabbit liver Sigma MT preparation [22] seems to be more effective in achieving this than the anion exchange procedure used for the Sigma horse kidney MT separation [23,24]. A proper interpretation of the CZE electropherograms of MT samples should include the functionality of separated forms. When the commercial horse MT preparation and our rat MT preparations were pooled prior to CZE, marked co-migration between peaks was observed (Fig. 4A–C), with only minor differences confined to the a-peak and the MT-II peak. Corresponding results for rabbit commercial and rat MTs as well as for commercial rabbit and horse MT preparations, are shown in Fig. 5A–C with minor differences related only to the MT-I
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peak. Co-migration at this level therefore seems to be related to mutual factors between isoforms, no further studies were carried out. It can also be seen that the rabbit MT preparation had one dominant form, marked as the b-form in Fig. 5A, which did not concur with any of those present in horse and rat MTs. In Fig. 6A,B it is shown that this form developed gradually with time at pH 9.1 under atmospheric conditions at room temperature. The electropherogram of the new lot of rabbit MT, however, did not show this peak (Fig. 6C), indicating the rabbit b-form develops spontaneously during storage. In recent work on rabbit MT isoforms by Virtanen et al. [6] their major g-peak was characterized as a modified isoform, which probably corresponds to the b-form described in the present work. A corresponding peak is also seen in electropherograms from other workers [8– 10] having run rabbit MT preparations using the CZE technique at acidic conditions. When Fig. 3A is compared with Fig. 5B, it is interesting to note that a small indication of the b-form presence was also seen in our rat liver MT preparation as the part ahead of the MT-II peak in Fig. 3A. By using our CZE technique, stability of the freshly prepared rat MT preparations were tested at pH 8.5 under atmospheric conditions at room temperature and refrigerated at 5°C over a period of 65 and 68 h, respectively. Analysis of aliquots showed that the a-form did not degrade in contrast to MT-I and -II (Fig. 7). Under these conditions the I form was more stable than the II form. Corresponding results (not shown) were found for the rabbit (pH 9.1) and horse MT (pH 9.1) preparations. At tested levels the a-form was not found in rabbit. The observation that the MT-I form is more stable than the MT-II form is surprising [25]. At pH 2.0, however, at which the stability measurements were performed, metal is stripped from the protein resulting in apoprotein (apothionein) formation. Under these conditions the MT-II form may be more vulnerable than the MT-I form. During degradation experiments, new peaks were not observed in our rat MT forms. The regression analysis of individual MT forms in rabbit covering the range 1.4 – 180.0 mg ml − 1 is shown in Fig. 8. The observed detection limit for
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MT preparations below 5 mg ml − 1 corresponds to the sensitivity level found by others [8,12,13,15]. Other MT preparations studied in the present work gave similar results (not shown). The concentration of the individual isoforms in the rat liver MT preparations was determined by measuring the total and relative percentage levels of the individual forms. These values were then related to the measured total level of forms in the horse Sigma preparation, for which the MT concentration was given by the supplier. This preparation contained similar isoform types as found in rat liver (Fig. 4). In the commercial rabbit preparation the a-form was seen only at higher concentrations, and it contained considerable amounts of the b-form. It is important to keep in mind that when the sample is applied hydrodynamically (pressure) to the CZE capillary as in this work, the separated forms move through the detector window at different speeds. Peak area is a better measure for actual amounts than peak height when using different running conditions, i.e. voltage and unequal salt concentration in the samples. To compare peak areas in stability testing and regression, however, they must be divided by the corresponding migration times to give normalized values (Fig. 8) [26,27].
Acknowledgements The authors would like to thank Prof. Stellan Hjerte´n, Department of Biochemistry, BMC, S751 23 Uppsala, Sweden, for his critical review of the manuscript. We are especially grateful for the gift of coated capillaries from the Bio-Rad Laboratories, Life Science Group, Richmond, CA., and to Director Lars Th. Wold, Bio-Test A/S, N-1580 Rygge, Norway, for the courtesy of free disposal of the BioFocus™ 3000 system.
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