Determination of Metallothionein at the Femtomole Level by Constant

Chem. 73, 20, 4801-4807 ... Analytical Chemistry 2004 76 (19), 5930-5936 ... Electrochemical analysis of Os(VI)-modified glycoproteins and label-free ...
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Anal. Chem. 2001, 73, 4801-4807

Determination of Metallothionein at the Femtomole Level by Constant Current Stripping Chronopotentiometry Rene´ Kizek,†,‡ Libusˇe Trnkova´,‡ and Emil Palecˇek*,†

Institute of Biophysics, Academy of Sciences of the Czech Republic, Kra´ lovopolska´ 135, CZ-612 65 Brno, Czech Republic, and Department of Theoretical and Physical Chemistry, Faculty of Science, Masaryk University, Kotla´ rˇska´ 2, CZ-611 37 Brno, Czech Republic

Metallothionein (MT) from rabbit liver was analyzed by differential pulse polarography, cyclic voltammetry, square wave voltammetry, and chronopotentiometric stripping analysis (CPSA) with a hanging mercury drop electrode under various conditions. The highest sensitivity of the MT determination was obtained with CPSA which produced a well-developed peak H due to catalytic hydrogen evolution at highly negative potentials. The highest peak H was obtained in borate buffer close to pH 8.0. In this medium, subnanomolar concentrations of MT were detectable. In the adsorptive transfer stripping (medium exchange) experiments, determination of few femtomoles of MT in 5-µL aliquots of the analyte was possible. CPSA determination of MT in various tissues of carp (Cyprinus carpio) yielded values in agreement with the published data. Metallothioneins (MTs) are low molecular weight proteins (6000-10 000), the first of which was isolated from equine renal cortex.1 Later, similar proteins from kidney, liver, and intestine tissues of other mammals, as well as from birds, fish, crustacean mussels, fungi,2 and plants,3 and from metal-resistant microorganisms4-6 were characterized. By the end of 1997, more than 170 amino acid sequences of MT from 50 species were known.7 Each of them consists of a chain of 61 or 62 amino acids. The chains have remarkably similar composition containing no aromatic amino acids.8 All contain 20 cysteine residues whose positions are invariant along the chain: 10 cysteines are found in five Cys-X-Cys units, where X is a variable residue, seven additional cysteines are present in the Cys-Cys-X-Cys-Cys (positions 33* Corresponding author: (tel) +4205 746241; (fax) +420541211293; (e-mail) [email protected]. † Academy of Sciences of the Czech Republic. ‡ Masaryk University. (1) Marfoshes, M.; Vallee, B. L. J. Am. Chem. Soc. 1957, 79, 4813-4814. (2) Lerch, K. Nature 1980, 284, 368-372. (3) Rauser, W. E.; Curvetto, N. R. Nature 1980, 287, 563-565. (4) Higham, D. P.; Sadler, P. J.; Scawen, M. D. Science 1984, 225, 1043-1045. (5) Kojima, Y.; Kagi, J. H. R. Trends Biochem. Sci. 1978, 3, 90-93. (6) Boulanger, I.; Goodman, M. C.; Forte, P. C.; Fesik, W. S.; Armitage, M. Biochemistry 1983, 80, 1501-1505. (7) Rodriguez, A. R. In Methallothionein IV; Klaassen, C., Ed.; Birkhauser Verlag Basel: Basel, Switzerland, 1999; pp 85-91. (8) Kojima, Y.; Berger, C.; Vallee, B. L.; Kagi, J. H. R. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3413-3418. 10.1021/ac010126u CCC: $20.00 Published on Web 09/18/2001

© 2001 American Chemical Society

37) and Cys-X-Cys-Cys (positions 57-60) groups.6 The metal content of MTs varies with the species, the tissues, the stage of development, and the exposure of the organism to metals. Through thiolate bonds, the protein binds various metallic cations such as Cd, Zn, Cu, and Hg. The MT biosynthesis is closely controlled by the level of exposure of an organism to salts of these metals.9 Cys residues can coordinate seven divalent metal ions in two distinct metal clusters.10-13 Due to the lack of any measurable biological activity, the quantitation of MT has proved to be a difficult task, and investigators have considered unique structural aspects of the molecule in order to develop analytical procedures. Therefore, efforts to detect MTs rely predominantly either on determination of metal binding or sulfhydryl contents or on immunological assays. Since 1933, cystine/cysteine-containing proteins have been known to produce characteristic dc polarographic waves in cobaltcontaining solutions due to catalytic hydrogen evolution. About 30 years ago, we showed that by using differential pulse polarography (DPP) instead of the dc polarography and by changing the composition of the background electrolyte a great increase of the sensitivity of the protein determination could be achieved.14 The DPP technique (using DME as a working electrode) has successfully been applied to the analysis of metallothioneins and has become a routine method for their determination in various tissues.15 DPP takes advantage of the high cysteine content in a MT and overcomes many of the shortcomings of other analytical methods.15 Being capable of picomole-level sensitivity, very rapid, and highly reproducible, the technique provides a useful procedure for monitoring MT concentrations in natural samples, e.g., during MT isolation from tissues or other physiological experiments.15-17 Pulse polarography and square wave and linear sweep voltammetry with mercury and carbon electrodes were used to (9) Vallee, B. L. In Metallothionein; Kagi, J. H. R., Nordberg, M., Eds.; Birkhauser: Basel, Switzerland, 1979. (10) Vasak, M.; Kagi, R. H. Biochemistry 1981, 78, 6709-6713. (11) Talanta 1998, 46 (2) (special issue). (12) Otvos, D. J.; Armitage, I. M. Biochemistry 1980, 77, 7094-7098. (13) Oz, G., Pountney, D. L., Armitage, I. M. Metallothionein structure update; Birkhauser Verlag: Basel, Switzerland, 1999. (14) Palecek, E.; Pechan, Z. Anal. Biochem. 1971, 42, 59-71. (15) Olafson, R. W.; Olsson, P. E. Methods Enzymol. 1991; 205, 205-213. (16) Piotrowski, J. R.; Bolanowska, W.; Sapota, A. Acta Biochem. Pol. 1973, 20, 207-215. (17) Chen, R. W.; Ganther, H. E. Environ. Physiol. Biochem. 1975, 5, 378-388.

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Figure 1. Comparison of the peak H of 100 ng/mL MT detected by DPP and constant current CPSA in 0.75 M (NH4OH + NH4Cl) and 1 mM [Co(NH3)6]Cl3: (A) DPP peak of MT, modulation time 0.05 s, modulation amplitude 50 mV, and drop time 1 s; (B) CPSA peak of MT (s), solution without MT (- -), tA 60 s, EA -0.10 V, and Istr -5 µA.

study binding of metals to MT.7,18-25 Immunochemical determination26,27 of MTs has been applied, providing higher sensitivity than the DPP method.28 Involvement of these and other methods in the analysis of MT was summarized in 1998 in a special issue of Talanta.11 Recently we have shown that some peptides, as well as MT, produce a well-developed catalytic signal in constant current stripping analysis (CPSA) at a hanging mercury drop electrode (HMDE) (peak H). This signal is produced by peptides and proteins at potentials around -1.7 V.29,30 CPSA has been shown to be more sensitive for the determination of tested peptides, in comparison with the voltammetric analysis in cobalt solutions.29,30 In this study, we have used CPSA and HMDE to study a rabbit liver MT under different conditions. The highest signal has been observed in borate buffers. We have shown that the peak H gives (18) Nieto, O.; Hellemans, G.; Bordin, G.; De Ley, M.; Rodrı´guez, A. R. Talanta 1998, 46, 315-324. (19) Nieto, O.; Rodrı´guez, A. R. Electroanalysis 1999, 11, 175-182. (20) Munoz, A.; Rodrı´guez, A. R. Analyst 1995, 120, 529-532. (21) Sestakova, I.; Miholova, D.; Vodickova, H.; Mader, P. Electroanalysis 1995, 7, 237-245. (22) Sestakova, I.; Vodickova, H.; Mader, P. Electrochemistry 1998, 10, 764769. (23) Sestakova, I.; Kopanica, M.; Havran, L.; Palecek, E. Electroanalysis 2000, 12, 100-104. (24) Sestakova, I.; Mader, P. Cell. Mol. Biol. 2000, 46, 257-267. (25) Studnickova, M.; Turanek, J.; Zabrsova, H.; Krejci, M.; Kysel, M. J. Electroanal. Chem. 1997, 421, 25-32. (26) Eaton, D. L.; Stacey, N. H.; Wong, K. L.; Klaassen, D. C. Toxicol. Appl. Pharmacol. 1980, 55, 393-402. (27) Brady, F. D.; Webb, M. J. Biol. Chem. 1981, 256, 3931-3935. (28) Zelazowski, A. J.; Piotrowski, J. K. Acta Biochem. Pol. 1977, 24, 97-103. (29) Tomschik, M.; Havran, L.; Fojta, M.; Palecek, E. Electroanalysis 1998, 10, 403-407. (30) Tomschik, M.; Havran, L.; Palecek, E.; Heyrovsky´, M. Electroanalysis 2000, 12, 274-279.

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higher sensitivity than DPP or dc voltammetry, and is comparable to that of immunoassays. MATERIALS AND METHODS Materials. The metallothionein from rabbit liver (containing 5.9% Cd and 0.5% Zn) was purchased from Sigma Chemical Co. as the product No. M 7641 (MW 7143). The components of the supporting electrolytes (ammonium phosphate, sodium phosphate, sodium borate) were analytical purity grade products of Lachema (Brno, Czech Republic). All solutions were prepared using deionized water (Millipore, Milli-Q). Apparatus and Methods. Electrochemical measurements were performed with an Autolab analyzer (EcoChemie, Utrecht, The Netherlands) in connection with VA-Stand 663 (Metrohm, Zurich, Switzerland). The standard cell with three electrodes was used. The working electrode was either the HDME or a dropping mercury electrode (DME) with a drop area of 0.4 mm2. The reference electrode was the Ag/AgCl/3 M KCl electrode, and a graphite electrode was used as the auxiliary electrode. All experiments were carried out at 25 °C under air. The supporting electrolyte 0.2 M NH4OH + NH4Cl containing 1 mM [Co(NH3)6]Cl3 described by Brdicka32-34 was prepared according to ref 14. The software GPES 4.4 supplied by EcoChemie was employed for smoothing and baseline correction. Standard solutions were prepared from a purified rabbit metallothionein (1.0 mg/ L) in deionized water. Analysis of MT in Tissues. Metallothionein was prepared according to the modified methods.31 Samples of 0.5-1.0 g of (31) Olafson, R. W.; Sim, R. G. Anal. Biochem. 1979, 100, 343-351. (32) Brdicka, R. Collect. Czech. Chem. Commun. 1933, 5, 112-128. (33) Brdicka, R. Collect. Czech. Chem. Commun. 1933, 5, 148-164. (34) Brdicka, R. Collect. Czech. Chem. Commun. 1936, 8, 366-376.

Figure 2. CV, DPP, SWV, and CPSA of MT (100 ng/mL) in 0.1 M H3BO3 + 0.05 M Na2B4O7, pH 7.6: (A) CV, tA 60 s, initial potential -0.10 V, and scan rate 0.08 V/s; (B) DPP, modulation time 0.05 s, modulation amplitude 50 mV, initial potential -0.10 V, step potential 0.0045 V, and drop time 1 s; (C) SWV, tA 60 s and frequency 350 Hz; (D) CPSA, tA 60 s, EA -0.10 V, and Istr -5 µA.

different tissues from fish were used. Samples were made up to 5 mL, following homogenization in saline (0.9% NaCl solution) at 4 °C. One-milliliter aliquots of each sample was placed in tubes and kept at 90 °C in Techne Dri-Block DB-2D for 6 min with occasional stirring and then cooled to 4 °C. Denaturated homogenates were centrifuged at 4 °C and 10000g for 20 min (Centrifuge MPW-365). Homogenates were cooled on ice and placed into tubes, and aliquots of the supernatant fraction were mixed with supporting electrolyte. RESULTS AND DISCUSSION We attempted to detect MT in a cobalt-containing background electrolyte at a concentration of 100 ng/mL (13.1 nM), i.e., at a concentration close to the detection limit of the DPP analysis, up to now considered to be the most sensitive electrochemical technique for MT determination.15 At this MT concentration, we obtained a DPP peak of MT which differed only slightly from the noise (Figure 1A). In contrast, the CPSA produced a welldeveloped peak of MT at relatively short accumulation time (tA) of 60 s at HMDE (Figure 1B). We have termed the signal observed at very negative potentials as peak H (hydrogen evolution).29 To obtain this peak, the presence of cobalt or other multivalent ions was not necessary. These results suggested that peak H in combination with the stripping technique is better suited for the determination of low MT concentrations than the usual DPP analysis in cobalt-containing solutions (Figure 1). Among several buffers used, the borate buffer (pH 7.6) was the most suitable as a background electrolyte for CPSA of MT.

Using 0.1 M borate buffer (pH 7.6) we have applied different electrochemical techniques, i.e., cyclic voltammetry (CV), DPP, square wave voltammetry (SWV), and CPSA to the determination of 13.1 nM MT (Figure 2A-D, respectively). Under these conditions, only CPSA produced a well-developed peak H. We studied the effect of composition of the background electrolyte. Earlier29 we showed that MT at nanomolar concentrations produced a well-developed CPSA peak H in 0.2 M ammonium phosphate (pH 8.0). In the present paper, we studied the effect of buffer composition on the CPSA signal of 13.1 nM MT (Figure 3A). In sodium phosphate buffer (pH 7.6) the peak was obtained at -1.70 V; in ammonium phosphate buffer (pH 7.6) peak H was shifted by ∼60 mV to negative potentials, the peak being ∼60% higher (not shown). An even higher peak H was observed in 0.2 M NH4OH + NH4Cl at pH 8.5; in the presence of 1 mM [Co(NH3)6]Cl3 this peak increased almost 10-fold (Figure 3A1). Use of borate as a background electrolyte resulted in a dramatic increase of peak H (Figure 3B). The strong increase in peak height in the borate buffer was surprising. Boric acid can act as an effective proton donor by utilizing a water molecule for the proton-transfer process.35 It was thus tempting to suggest that the large increase of the MT signal in the borate buffer might be due to this property of the borate. Earlier we showed that analysis of proteins29 and nucleic acids36-38 can easily be performed in very small volumes (usually 3-10 µL) (35) Jencks, W. P. Chem. Rev. 1972, 72, 705-719. (36) Palecek, E. Bioelectrochem. Bioenerg. 1986, 15, 275-295. (37) Palecek, E.; Postbieglova, I. J. Electroanal. Chem. 1986, 214, 359-371.

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Figure 3. (A, B) CPS curves of MT at concentrations of (A) 100 ng/mL, (A1) (9) 0.2 M NH4OH + NH4Cl + 1 mM [Co(NH3)6]Cl3; (s) 0.2 M NH4OH + NH4Cl, pH 8.5; (A2) sodium borate (0.1 M H3BO3 + 0.05 M Na2B4O7, pH 7.6, and tA 60 s; (B) 5 ng/mL in sodium borate, pH 7.6, tA 240 s, and Istr -1 µA; other details in Figure 2. (D) Adsorptive transfer (medium exchange) CPSA of MT (100 ng /mL). Accumulation of MT at HMDE was performed from a 5-µL MT solution drop, without stirring, tA 120 s, EA -0.1 V, and Istr -1 µA. The HMDE was washed and transferred to the background electrolyte in the usual electrolytic cell where the CPSA was performed; (bp) MT adsorption from borate, (b) (0.1 M H3BO3 + 0.05 M Na2B4O7, pH 7.6) followed by transfer to phosphate, (p) (0.2 M NaH2PO4 + Na2HPO4, pH 7.6), (bb) MT adsorption from borate followed by transfer to borate, and (pb) MT adsorption from phosphate followed by transfer to borate; (D) Comparison of AdT CPSA responses of MT using different buffers for MT adsorption and for CPSA (two examples are shown in C). The peak height obtained in bb experiment was taken as 100%.

by the so-called adsorptive transfer stripping voltammetry (AdTSV) method. The procedure is based on the immobilization of the analyte from a small drop of a solution on HMDE followed by washing and electrochemical measurement in a cell containing blank background electrolyte. In AdTSV (medium exchange), the adsorption of the biomacromolecule is effectively separated from the electrode process, which can proceed in a medium different from that used for the adsorption. We took advantage of AdT CPSA to separately test the effect of borate on adsorption and on the electrode process. We obtained the highest peak when adsorption was performed from sodium borate followed by a transfer to sodium phosphate where peak H was measured (Figure 3C,D). Adsorption from phosphate followed by borate resulted in a much smaller peak, differing only little from that obtained after adsorption from phosphate followed by measurement in the same medium. These results clearly show that the borate buffer has little effect on the electrode process but influences strongly MT adsorption. The increase of peak H observed in conventional CPSA in borate might be thus due to the orientation of the MT molecules (38) Palecek, E.; Jelen, F.; Teijeiro, C.; Fucik, V.; Jovin, T. M. Anal. Chim. Acta 1993, 273, 175-186.

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at the electrode surface. It should be noted that in contrast to conventional CPSA, the AdT measurements were performed without stirring the MT solution and the peak heights did not correspond to those of conventional CPSA with stirring of the MT sample. Furthermore, we have studied the dependence of the potential and height of peak H on buffer concentration, pH, stripping current (Istr), MT concentration, and accumulation time tA using conventional CPSA and borate buffer as a background electrolyte. The height of peak H decreased with increasing stripping current in the usual way30 showing an optimum at -1 µA for 10-50 nM MT (not shown). At accumulation time tA of 300 s and Istr of -1 µA, the dependence of peak H on MT concentration, ranging from 1 to 600 nM (Figure 4A), showed a shape resembling the Langmuir adsorption isotherm. Using AdTS CPSA at tA 120 s, we obtained a linear calibration between 1.6 and 50 ng/mL (Figure 4B). At concentrations below 1.0 ng/mL a nonlinearity was observed under these conditions. Peak H of 13.1 nM MT increased with accumulation time almost linearly up to ∼3 min and then declined from linearity, until it became independent of tA between 15 and 50 min (Figure 5).

Figure 4. Dependence of the height of peak H on MT concentration: (A) conventional CPSA (1-600 ng/mL) 0.1 M H3BO3 + 0.05 M Na2B4O7, pH 7.6, and tA 300 s; (B) AdT CPSA (1-50 ng/mL) accumulation of MT from 0.1 M H3BO3 + 0.05 M Na2B4O7, pH 7.6, followed by transfer and CPSA in 0.2 M NaH2PO4 + Na2HPO4, pH 8.0. Other conditions as in Figure 3.

Peak H increased steeply when the pH was raised, showing a maximum height close to pH 7.6, and decreased less steeply with further increase in pH (not shown). Between pH 6.5 and 7.5, the peak potential Ep shifted with increasing pH to less negative values by 10 mV per pH unit. Between 7.6 and 9.0, Ep shifted to more negative values (6 mV per pH unit between pH 7.6 and 8.0, 2 mV per pH unit between pH 8.0 and 9.0). Considering the results we obtained by AdT CPSA showing that the nature of the buffer is more important for MT adsorption than for the electrode process (Figure 3), we did the same type of the medium exchange experiment to study the effect of pH on MT adsorption and the MT electrode process connected with catalytic hydrogen evolution. In this experiment, we either adsorbed MT at constant pH and measured at different pH’s or vice versa (Figure 3). Our results showed that the height of peak H was little influenced by the pH of adsorption (pHads) (Figure 6A1) if the measurements were performed at pH 7.2 (pHmeas) (Figure 6B1). On the other

hand, the peak height at pHmeas 8.0 (Figure 6B2) was strongly influenced by pHads (Figure 6A2). The highest signals were obtained only if the both pHads and pHmeas were 8.0, i.e., close the isoelectric point of MT (pI 8.2) in solution.39 Our results suggest that the charge of MT might play an important role in the electrode process yielding peak H, but because of the complexity of the processes to which MT seems to be subjected at the electrode, we prefer to continue our experimental work before making conclusions about the role of MT ionization at the electrode. More details will be published elsewhere. In agreement with the behavior of the catalytic hydrogen evolution signal,30 the peak H increased with buffer concentration (at pH 7.6), and Ep shifted to negative values (Figure 7). Above 0.25 M, precipitation of the buffer components took place. We tested also the effect of concentration of a more soluble sodium phosphate (pH 7.6). We observed a steep increase of the peak height between 0.2 and 0.3 M, but raising the concentration of the phosphate to 0.5 M did not induce any increase in the height of peak H (Figure 7). Despite the great increase in the height of peak H between 0.2 and 0.3 M phosphate, the peak height obtained in 0.1 M borate was still much higher than the peak in 0.3 M phosphate in agreement with the critical role of borate in the MT adsorption (Figure 3). We attempted to find the lowest concentration of MT that would yield a well-developed and reproducible peak H. Such a peak was obtained with 0.5 nM MT (at tA 5 min (Figure 3B); this concentration of MT was determined under the given conditions with a standard deviation of 9.5%. MT at 50 pM concentration produced an equally well-developed peak H. However, the standard deviation increased to 20-30%, which was probably due to adsorption of MT on the glass wall of the electrochemical cell. Using AdTS CPSA, in which MT adsorption on the cell wall cannot take place, we obtained a linear calibration between 1.6 and 50 ng/mL, tA 120 s (Figure 4B). At concentrations below 1.0 ng/mL (131 pM), a nonlinearity was observed under these conditions. An MT concentration of 1.6 ng/mL (210 pM) corresponds to ∼1 fmol in 5 µL of MT solution, still far above the expected detection

Figure 5. Dependence of the height of peak H of MT on accumulation time: (A) time interval 0-600 s; (B) time interval 0-3 000 s, Istr -1 µA. Other conditions as in Figure 3.

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Figure 6. Dependence of the height of MT peak H on pH: (A) relative peak heights (A1) conventional CPSA in 0.1 M H3BO3 + 0.05 M Na2B4O7 (2); (A1) AdTS CPSA MT adsorption from borate, pH 7.2 (pHads 7.2) followed by transfer into blank borate buffer, pH 7.2 (b); (A2) AdT CPSA, MT adsorption from borate, pH 8.0; (pHads 8.0) followed by transfer into blank borate buffers, pH 8.0. (A1) AdTS CPSA, MT adsorption from different pH’s followed by transfer into borate buffer, pH 7.2 (pHmeas 7.2) (9); (A2) AdTS CPSA, MT adsorption from different pH’s followed by transfer into blank borate buffer, pH 8.0 (pHmeas 8.0) (9). Peak heights obtained at pH 8.0 were taken as 100%. Other conditions as in Figure 3. (B) Column graph showing absolute peak height values obtained in AdTS CPSA at different pH’s. MT adsorption was performed at different pH’s: (1, 7.2; 2, 7.6; 3, 8.0; 4, 8.6 while CPSA was carried out at constant pH: (B1) pHmeas 7.2; (B2) pHmeas 8.0, tA 120 s, and Istr - 1 µA. Other conditions of AdT CPSA as in (A).

We have used the CPSA peak H for the determination of MT in fish organs. Other proteins that may interfere with the determination were removed by heating the sample at 90 °C and protein precipitation (see Materials and Methods) with MT remaining in solution, as described by Olafson.15 The MT peak obtained from carp (Cyprinus carpio L.) spleen, liver, and testes is typically as shown in Figure 8A. The MT content determined by CPSA in these organs, using the calibration curve (Table 1), was in a agreement with the results of other authors40-43 obtained by means of ELISA, RIA, and DPP.

Figure 7. Dependence of the height of peak H on borate and phosphate buffers concentrations: (b) sodium borate; (O) sodium phosphate. The peak height obtained with 0.1 M buffer was taken as 100%; tA 120 s, Istr - 1 µA, and other conditions as in Figure 3.

limit of the AdT CPSA. Unless a special cell is used in which adsorption of MT on the wall is prevented, AdT CPSA is perhaps best suited for determination of low concentrations of MT (Figure 8B). 4806 Analytical Chemistry, Vol. 73, No. 20, October 15, 2001

CONCLUSIONS Use of CPSA, so far the most sensitive electrochemical method for MT determination, is proposed in this paper. CPSA peak H29,30,44 is related to the dc polarographic “pre-sodium wave” discovered about 70 years ago by Herles and Vancura45 and Heyrovsky and Babicka.46 This wave, which is due to hydrogen (39) Kaegii, J. H. R., Koima, Y. Experientia Suppl. 1987, 52, 25-61. (40) Thompson, J. A. J.; Cosson, R. P. Mar. Environ. Chem. 1984, 11, 137152. (41) Eaton, D.; Toal, B. F. Pharmacology 1982, 66, 134-142. (42) Duquesne, S.; Janquin, M. A.; Hogstrand, C. Fresenius J. Anal.Chem. 1995, 352, 589-595. (43) Wong, K. L.; Klaassen, C. D. J. Biol. Chem. 1979, 254, 12399-12403. (44) Heyrovsky, M. Electroanalysis 2000, 12, 490-495.

Figure 8. (A) CPSA determination of the MT content in different tissues: (9) testes, (b) spleen, and (2) liver (µg of MT/g of tissue); other conditions as in Figure 4. (B) Adsorptive transfer (medium exchange) CPSA of MT. Accumulation of MT (5 ng/mL) at the electrode was performed from a 5-µL drop of MT solution in water without stirring, other conditions as in Figure 3.

Table 1. Tissue Levels of Metallothionein (Cyprinus carpio) tissue

µg of MT/g of tissue, CPSA

liver spleen testes

1.5 ( 0.5 2.0 ( 0.7 11 ( 2

evolution catalyzed by free NH2 and SH groups in the protein molecule,47 was considered not to be suitable for analytical purposes because it was usually not sufficiently well shaped as it was too close to the discharge of the supporting electrolyte.44 Several factors contribute to the higher sensitivity of the CPSA determination (using peak H) as compared to dc polarography or DPP with DME in cobalt-containing solutions15 or in the absence of cobalt (Figure 2): (A) compared to polarography or voltammetry, CPSA allows us to reach more negative potentials48 necessary to obtain a well-developed peak H which in voltammetric measurements is too close to the background discharge; (B) (45) Herles, F.; Vancura, A. Czech. Acad. Sci. 1932, 42, 4-14. (46) Heyrovsky, J.; Babicka, J. Collect. Czech. Chem. Commun. 1930, 370-378. (47) Kuta, J.; Palecek, E. Modern polarographic (voltammetric) techniques in biochemistry and molecular biology; John Wiley and Sons: London, 1983. (48) Kalvoda, R. Chem. Listy 1960, 54, 1265-1268. Heyrovsky, J. Forejt J Oscillographic polarography; SNTL: Prague, 1953 (in Czech). (49) Palecek, E.; unpublished results, 2001. (50) Brazdova, M., Kizek, R., Havran, L., Palecek, E. Bioelectrochemistry, in press. (51) Palecek, E., Kizek, R.; unpublished results, 2001.

efficient baseline correction improves the peak shape at low MT concentrations; (C) MT is strongly adsorbed at HMDE and can be accumulated at its surface during the waiting time. A welldeveloped peak H is obtained not only with MT and peptides29,30 but also with core domain and C-terminal domain of the tumor suppressor protein p53.49 As distinct from the well-known protein double-wave produced in cobalt-containing solutions,33 peak H is yielded also by proteins not containing SH or S-S groups,14,50 making it possible to analyze a wider spectrum of proteins, including histones and protamines.51 In the medium exchange (AdTS) experiments using 3 µL of the analyte, subfemtomole amounts of MT can be determined by CPSA. These amounts of the analyte are comparable or even lower than those necessary for immunoassays. More work will be necessary to better understand the nature and potentialities of the peak H in peptide and protein analysis. ACKNOWLEDGMENT We are indebted to Dr. Jiri Jarkovsky for the isolation of fish tissues and Prof. Jiri Hala, Dr. Ludek Havran, Dr. Zdenek Pechan, and Dr. Michael Heyrovsky for discussions. The authors thank Jan Vacek for technical assistance. This work was supported by Grants Z 5004920, 204/97/K084, and A4004110 from the Academy of Sciences of the Czech Republic. Received for review January 26, 2001. Accepted July 26, 2001. AC010126U

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