BIOMEDICAL CHROMATOGRAPHY, VOL. 5 , 193-197 (1991)

Determination and Purification of Metallothioneins by High Performance Liquid Chromatography Aihua Pan,* Zhenxin Wang and Binggen Ru Protein Engineering Laboratory, Department of Biology, Peking University, Beijing 100871, China

A rapid, reproducible and sensitive high performance liquid chromatography (HPLC) method for the determination and purification of metallothionein-I (MT-I) and metallothionein-I1(MT-11) in mouse and rabbit livers has been developed. Methallothioneins (MTs) were separated by an HPLC anion exchange column, eluted through a linear gradient of Tris buffer and the peak containing MTs was determined by atomic absorption spectrophotometry. Furthermore, the content of MT-I or MT-I1 was calculated by protein peak area in a short time (about 20 min). The sample to be tested was homogenized, centrifuged and saturated by cadmium. MT-I and MT-I1 were eluted at 15.9 and 19.3 min, respectively. The following mouse liver cytosols were tested: controls, Cd-injected samples and 60Co-irradiatedsamples. A detection limit of 5 pg/g liver was established for this method. We have analysed more than 100 biological samples and obtained satisfactory results.

INTRODUCTION

Metallothioneins (MTs) are proteins with a molecular weight of about 6.500 Dalton and with a high affinity to heavy metal. They are found throughout the biological realm, and are especially plentiful in mammalian liver and kidney tissues. They contain large amounts of cystine (which accounts for about 30% of the amino residue), which allows them to bond with metals. Normally there are about seven atoms of metal in each molecule of protein, with zinc, copper and cadmium being the most common elements (Shaikh, 1979). The primary biological function of MTs is the storage, transport, and replacement of trace elements (Cousins, 198.5) as well as the detoxification of heavy metals (Cherian et al., 1983). In addition, they have an obvious relationship to the regulation of hormones and of cell metabolism (Udom et al., 1980), to the control of cell division and growth (Lebeau et al., 1985), to UV response (Schorpp et al., 1984) and to anti-radical function (Thornalley and Vasak, 198.5). The MT levels dramatically increase in human and animal tissues in heavy metal pollution (or poisoning), because MTs are also heavy metal reducing proteins (Durnam and Palmiter, 1981). In a few countries the MT levels in blood and urine are now used as an indicator of the degree of heavy metal contamination of workers. In the United States, M T levels are also used to indicate the degree of contamination of marine life (Chang et af., 1980). The purpose of this paper is to establish a high performance liquid chromatography (HPLC) method for determination of trace levels of MTs in animal tissues by use in tests of environmental contamination o f humans and animals, and in research on the biological control mechanisms of MT. Suzuki (1980) used gel filtration HPLC associated with atomic absorption spectrophotometry (AAS) to separate and purify rat liver MTs, and also separated Authoi t o whom wrrc\pondcncc \hould he ,iddrc\wd 0269-3879/91/050 193-05 $05.00

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and purified MTs from several other organisms, including tortoises, spot salamanders, water lizards and several species of frogs. However, with gel filtration it is impossible to separate the different MT subtypes. Klauser et af. (1983) established reversed phase column HPLC, and used this method to separate and purify MTs from many animal organs, such as horse kidneys, rat, rabbit, and chicken livers, and to discover the multiformity of MTs in the human liver. Later, Lehman and Klaassen (1986) established the method of HPLC associated with AAS to determine the MT content in different animal organs. The solution eluted from the HPLC negative ion exchange column directly enters the AAS to determine the atomic absorption of Cd in the sample, and then derives the amount of MT-I and MT-I1 in the sample based on the peak area of atomic absorption of Cd. The elution buffer which they used was pH 7.4,0.2 M Tris-HCI buffer. Elution with a linear Tris-HCI gradient (from 0 to 40% of 0.2 M Tris-HCI) was completed in 12 min, but the effectiveness of separation was not ideal. In addition, measurement through the peak area of atomic absorption of Cd is subject to distortion by the Cd-binding protein of nonMTs. Thus the results contain a certain degree of error.

EXPERIMENTAL Preparation of MTs. ( I ) Prepurution of mouse liver MTs. Method A: MTs were prepared using an improved version o f the method established by Tsunoo et ul. (1978). Female Swiss mice were injected with 50 pg CdCI2 per mouse on the first, second and fourth days, 150 pg per mouse on the sixth day and 250 pg per mouse on the ninth day. Three days after the last injection of CdCL, the mice were sacrificed and the livers homogenized and then centrifuged at 96,000 g and 4 "C for 60 min. The supernatants were collected and filtrated through a Sephadex G-SO column (4.8 x 95 cm) and eluted by pH 8.6, 0.01 M Tris-HCI buffer. The MT-containing fractions were combined and collected, and ion exchange chromatography

194

AIHUA PAN, ZHENXIN WANG AND BINGGEN R U

performed on a D E A E Sephadex A-SO colutnn (2.5 x 35 cm) and eluted with a linear Tris-HCI gradient (0.01-0.25 M) at p H 8.6. The peaks of MT-I and MT-I1 were collected, lyophilized separately and stored at -80 "C or -20 "C. Method B: This method is based on that established by Van der Mallie and Garvey (1978). with some improvement. It relies primarily on the heat stability of MTs. The process up to the centrifugation stage is the same as in Method A. After centrifugation, the supernatant is placed in a boiling water bath for 2 min, and then Centrifuged at 4 "C, 10,400 g for 30 min. Afterwards, separation and purification are performed as in Method A . (2) Preparation ojrahhit liver M 7 3 . Rabbit liver MTs were obtained through an improved version of the method of Kimura et al. (1979). Rabbit liver MT-I and MT-I1 were isolated from rabbits exposed to cadmium (by 21 subcutaneous injections of 1 mg/Kg body weight at intervals of 2-3 days). O n the second day after the last injection the animals were killed. To one volume of the liver homogenate in 0.01 M Tris-HCI buffer, pH 8.6, cold ethanol and chloroform ( I .00: I .05 :0.08, by volume) were added with cooling. To the supernatant obtained by centrifugation three volumes of cold ethanol were subsequently added. The solution was allowed to stand at -20 "C overnight. The produced precipitate was collected by centrifugation. It was dissolved in 0.02 M ammonium carbonate and then charged to a Sephadex G-50 column. The elution was carried out by use of 0.02 M ammonium carbonate. The MT-containing fraction on the gel filtration was further separated into MT-I and MT-I1 by ion exchange chromatography on a D E A E Sephadex A-50 column.

Preparation of samples. A mixture of MT-I and MT-I1 was dissolved in pH 8.6, 0.01 M Tris-HCI buffer, and the CdCI2 solution was added to it. The final concentration of CdCI2was 50 ppm; it was allowed to stand for 15 min at room tcmperature, placed in a boiling water bath for 2 min and then centrifuged at 4"C, 13,000rpm for 15 min. The standard sample was ready for use in HPLC analysis. The other samples were mouse livers reduced by C d and ""Coirradiation or non-exposed. An equal volume o f buffer (pH 8.6, 0.01 M Tris-HCI) was added to livers, which were then homogenized, placed in boiling water for 2 min and centrifuged for 15 min. The CdCI2 solution was added to the supernatant to a concentration of 50 ppm. It was allowed to stand at room temperature for 15 min, placed in'a boiling water bath for 2 min, and recentrifuged at 13,000rpm for 15 mn. The sample preparation was then complete.

HPLC ion exchange chromatography. The [IPLC instrument consisted of two pumps (Varian LCSO60) controlled by a computer system (Varian CDS401) and an injector equipped with a LOO pL sample loop. Chromatography was performed on an anion exchange column (DEAE-SPW 7.5 cm X 7.5 mm; Waters Associates). Tris-HCI at concentrations of 10 and 250 mM (pH 8.6 at room temperature) was used as the mobile phase. MT-I and MT-I1 were eluted with a linear gradient from 0 to 100% B in 15 min (A: 1 0 m Tris-HCI, ~ p H 8.6; B: 250 mM Tris-HCI, pH 8.6) at a flow rate of 1 mL/min. In preliminary experiments, proteins were detected at 254 nm. The fractions eluted from HPLC were separately collected and their Cd concentrations were simultaneously determined

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Figure 1. Chromatographic properties of purified MTs as determined by HPLC and AAS. MT-I and MT-II (10 and 10 pg, respectively) in a volume of 100 yL were injected onto the anion exchange column and eluted with a gradient of Tris-HCI mobile phase (0-100% B in 15 m i n where A = 0.01 M Tris-HCI, pH 8.6; B = 0.25 M Tris-HCI, pH 8.6). The concentration of Cd was determined by AAS (see Experimental Section). (a) Mouse liver MTs, (b) rabbit liver MTs.

HPLC DETERMINATION A N D PURIFICATION OF META121D'rHIONEINS

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MT-I and MT-II. Proteins were saturated with Cd (final concentration of 50 ppm) after which varying amounts of MT-I and MT-II (final volume of 100 ILL) were injected onto the anion exchange column. by an atomic absorption spectrophotonieter (Hitachi 180-50). After each gradient run, the column was purged with mobile phase B (250 mM Tris-HCI, pH 8.6) to remove any remaining contaminants. When broad peaks or low plate counts were observed. the column was regenerated by making several injections (1-2 ml,) of 0.1 N NaOH or, if necessary, 30% acetic acid. Columns were regenerated several times so that the averagc column life exceeded several hundred sample injections

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properties of heat-denatured mouse liver cytosol which was induced by Cd. The sample was saturated further with Cd in virro. MT Calibration curve. A quantity of freeze-dried MT-I and MT-I1 was dissolved in pH 8.6 1 0 m Tris-HCI ~ buffer, and the protein content deterniined with Bio-Rad protein assay reagent. The different amounts of MT-1 and MT-IT were determined by HPLC ion exchange chromatography. The peak areas of MT-I or MT-I1 reported by computer were used to construct the calibration curve. A 1OOpL aliquot of the supernatant was applied to the ion exchange column and was eluted as described above. The determination of the location of MT-I and MT-I1 in the tested sample was based on the AAS peak of Cd content and the appearance time of MT-I and MT-I1 on the calibration curve based on the peak area of MT. and then calculated according to the following equation:

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RESULTS

Time (min) Figure 3. Chromatographic properties of heat-denatured control mouse liver cytosol. The sample was treated further with Cd in vifro. A volume of 100 pL was injected onto the anion exchange column and eluted with a gradient of Tris-HCI mobile phase as described in The Experimental Section.

The ion exchange chromatograms of thc purified MT-I and MT-I1 are shown in Fig. 1. The retention time of mouse liver MT-I is 15.9 min and that for MT-1 I is 19.3 inin (the concentrations of Tris-HC1 buffer for these

AIHUA PAN. LHENXIN WANG AND BINGGEN RU

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Table 1. Concentrationsof metallothioneinsin mouse livers as determined by HPLC" MT-I

MT-II

Total

Controlb ND" ND" Cd-treatedd 231 +23 845 10 315t33 6oCo irradiation (400Rad)e 153k30 857 t 105 1010f 134 6oCoirradiation (600Rad)' 251 k44 916f118 1167 f 161 aResults are expressed as ug M T l g liver and represent the mean k SD of six mice. bMice were injected with saline and livers were removed six days later for analysis. 'Not detected. 'Mice were injected with Cd as CdCI2 (150pglmouse) and livers were removed six days later for M T analysis. "Mice were exposed t o 400 !?ad of 6oCo irradiation and livers were removed 20 h later for MT analysis. 'Mice were exposed to 600 rad of "Co irradiation and livers were removed 20 h later for MT analysis.

times are 0.17 M and 0.22 M , respectively). Thc rctention time of rabbit liver MT-I is 14.3 min and that of MT-TI is 18.3 rnin (the concentrations of Tris-HC1 buffer for these times are 0.15 M and 0.21 M , respectively). Each type of sample was repeated ten times and all gave satisfactory results. Figure 2 gives the calibration curves for MT-I and MT-IT. Each concentration sample was repeated three times and the results were averaged.

It was impossible to detect MTs in control mouse livers using HPLC ion cxchange chromatography (Fig. 3). However, the livers of mice exposed to CdCl, and '('Co irradiation, respectively, showed a clear increase in both MT-I and MT-TI (Figs 4 and 5 ) . Table 1 shows the MT-I and MT-I1 content of mouse livers treated in all three ways, as determined by HPLC ion exchange chromatography.

DISCUSSION Wc made some improvement of Klaasen, principally by changing the pH of the equilibrium and elution buffers (from 7.4 to 8.6) and also their concentrations (from 0.2 to 0.25 M). The linear gradient of the buffer was from 0 to 100% 0.25 M Tris-HC1 in 30 min. In this way, satisfactory separation results could be obtained. Also, we used the peak area of protein to calculate the MT content directly, thus avoiding intcrference of the Cd-binding protein\ of nun-MTs. This method has several special characteristics. The most important is that it makes it possible to separate MT-I and MT-I1 from a sample in a short period of time (30 min), without the Sephadex G-50 gel filtration followed by ion exchange chromatography, as required in the usual chromatographic method. Thus the experi-

HPLC UE'I ERMINATION A N D PURIFICATION OF METALLOTHIONEINS

mental procedure is simplified while still obtaining satisfactory results. Secondly, this method makes it possible to directly assess the amount of MT-I and MT-11 in a sample through the peak area of protein on ion exchange chromatography, thus avoiding the influence of other factors and increasing the accuracy of the measurement, especially in determining t h e MT-I and MT-I1 content in different organs of various animals. Another characteristic of this method is that samples can be tested after relatively simple preparation, and testing itself takes only 30 min. Furthermore the ion exchange HPLC method has excellent repeatability. In our experiment, we tested nearly 100 samples. The HPLC chromatograms for individual samples of each type were very consistent. Thus, this method is a fast, accurate and relatively sensitive way of testing MT levels. However, because its degree of sensitivity is about 5 pg/g tissue, some samples, such as blood and urine, cannot be tested for MT content by this method without preconcentration. From the point of view of methodology, the best way

I97

to test MT levels within organic tissues is the HPLC method. Cd or Ag/hemoglobin saturation assay is also a good way of testing MT levels in organic tissues (Scheuhammer and Cherian, 1986), but only for total MT content determination. As for the testing of MT levels in body fluids, such as blood and urine. RIA (Garvey et af., 1982) and Enzyme-Linked Immunosorbent Assay (ELISA) (Thamos et al., 1986) are acknowledged as methods with high sensitivity and specificity, but the experimental proccss involved is complex and time-consuming. A t present, many methods of testing for MT have been estahlished (Menra and Bremner, 1983; Dieter et al., 1986), but there is still a need for a simple, fast, sensitive method. Acknowledgements This research i s supported by the China National High Technical Grant 863-103-20-11, National Seventh Five-year Research Grant 75-71-01-10 and The Third World Academy of Sciences Rcscarch Grant No. 48-CHN 6 .

REFERENCES

Chang, C. C., Lauwerys, R., Bernard, A., Roels, H., Buchet, J. P. and Garvey, J. S . (1980). Environ. Res. 23, 422. Cherian, M. G. and Nordberg. M. (1983). Toxicol. 28, 1. Cousins, R. J. (1985). Physiol. Rev. 65(21, 238. Dieter, H. H., Muller, L., Abel, J. and Summer, K. H. (1986). Toxicol. Appl. Pharmacol. 85, 380. Durnam, D. M. and Palmiter, R. D. (1981). J. Biol. Chem. 256(11), 5712. Garvey, J. S..Van der Mallie, R. J. and Chang, C. C. (1982). EDS Met. Enzym. 84, 121. Kirnura, M.,Otaki, N. and Imano, M. (1979). In Metallothionein, ed. by Kaji, J. H. R. and Nordberg, H., pp. 163-167. Birkhauser, Basel. Klauser, S., Kaji, J. H. R. and Wilson, K. J. (1983). Biochem. J. 209, 71. Lebeau, M.,Diaz, M. O., Karin, M. and Rowley, J. D. (1985). Nature 313, 709. Lehrnan, L. D., Klaassen, C. D. (1986). Anal. Biochem. 153, 305.

Mayo, K. E. and Palmiter, R. D. (1982). J. Biol. Chem. 257(6), 3061. Menra, F. K. and Bremner, I. (1983). Biochem. J. 213, 459. Shaikh, 2. A. (1979). In Metallothionein, ed. by Kaji, J. H. R. and Nordberg, M. pp. 331-336, Birkhauser, Basel. Scheuharnmer, A. N. and Cherian, M. G. (1986). Toxicol. Appl. Pharmacol. 82, 417. Schorpp, M., Mallick, U., Dahrnsdorf, H. J. and Herrlich, P. (1984). Cell37. 861. Suzuki, K. T. (1980). Anal. Biochem. 102, 31. Tharnos, D. G., Linton, H. J. and Garvey, J. S. (1986). J. Immunol. Methods 89, 239. Thornalley, P. J. and Vasak, M. (1985). Blochim. Biophys. Acta 827, 36. Tsunoo, H., Kino, K., Nakajima, H., Hata. A.. I. Y. Huang and Yoshida, A. (1978). J. Biol. Chem. 253, 4172. Udorn, A. 0. and Brady, F. 0. (1980). Biochern. J. 187, 329. Van der Mallie, R. J. and Garvey, J. S. (1978). lmmunochemistry 15, 857.