Proc. Nati. Acad. Sci.

USA
Vol. 88, pp. 3057-3059, April 1991
Biochemistry

Metalloselenonein, the selenium analogue of metallothionein:

Synthesis and characterization of its complex with copper ions
(synthetic peptide/selenocysteine)
TADAO OIKAWA, NOBUYOSHI ESAKI, HIDEHIKO TANAKA*, AND KENJI SODAt
Institute for Chemical Research, Kyoto University, Uji Kyoto-Fu 611, Japan
Communicated by Thressa C. Stadtman, January 16, 1991

ABSTRACT We used an automated peptide synthesizer to Table 1. Solid-phase synthesis protocol

produce a peptide, metalloselenonein, that contains selenocys- Mixing
teine residues substituted for all cysteine residues in Neurospora Step Reagents and operation time, min
crassa copper metallothionein. Metalloselenonein binds 3 mol
of Cu(I) per mol. This adduct shows a broad absorption band 1. Deprotection 60%o CF3COOH in DMF 1
between 230 and 400 nm and a fluorescence band at 395 nm, 2. Wash CH2Cl2 (three times) 1
which can be attributed to copper-selenolate coordination. The 3. Neutralization 10%1o DIEA in DMF (twice) 1
circular dichroism spectrum of the copper-metalloselenonein 4. Wash DMF (six times) 1
complex shows a positive band around 245 nm attributable to 5. Coupling Symmetric anhydride 20
asymmetry in metal coordination. method in DMF
6. Wash DMF (twice), 1
CH2CI2 (four times) 1
A selenocysteine residue occurs as an integral moiety in the 7. Ninhydrin test
active center of the selenium-containing enzymes such as
glycine reductase, formate dehydrogenase, and glutathione DMF, dimethylformamide; DIEA, N,N-diisopropylethylamine.
peroxidase (1). The catalytic role of the selenocysteine res- OCH2-Pam resin (0.5 mmol/g of resin; CIZ, p-chlorobenzyl-
idue is attributed to the high reactivity of the selenol group: oxycarbonyl) (10). The tert-butyloxycarbonyl (Boc) group
selenols have much lower redox potentials than their sulfur was used for the NO terminus, and the side-chain protecting
counterparts (2). Thus, attention has been paid to synthesis groups were as follows: Asp(OBzl), Sec(MeBzl), and
of selenocysteine-containing polypeptides and proteins, Ser(OBzl) (Bzl, benzyl; MeBzl, methylbenzyl; Sec, seleno-
which may have properties that the sulfur counterparts do not cysteine). The coupling reactions were carried out with 2.0
Downloaded from https://www.pnas.org by 1.38.109.41 on August 8, 2024 from IP address 1.38.109.41.

have (3). mmol of protected amino acid. The yield in the coupling
Metallothioneins are low molecular weight, cysteine-rich reaction was determined with ninhydrin (11).
proteins that bind various kinds of metal ions (4). The The peptidyl resin produced (about 1.0 g) was treated with
biological functions of metallothioneins have been proposed anhydrous hydrogen fluoride containing 15% anisole and
to be involved in provision of physiological metals for met- 2.5% ethyl methyl sulfide at 0°C for 1 hr. After evaporation
alloenzymes and in storage and detoxification of heavy of hydrogen fluoride, the residue was washed with diethyl
metals. These functions depend on high affinity for metal ions ether and extracted with 2 M acetic acid, and the extract was
and the reactivity of cysteine residues in a metal-thiolate Iyophilized.
cluster. Here we describe the synthesis of metalloselenonein, The crude peptide was purified by reverse-phase high-
in which all the cysteine residues in the Neurospora crassa performance liquid chromatography (HPLC). Special care
copper metallothionein are replaced by selenocysteines, and was taken to avoid air oxidation during the purification
its interaction with copper ions. procedure. All buffers were kept under a constant stream of
nitrogen. Preparative separations were done with an Ultron
MATERIALS AND METHODS C18 column (25 x 1 cm; Shinwakako, Kyoto) at a flow rate of
2.4 ml/min, whereas analytical separations were carried out
13-Chloro-L-alanine was synthesized from L-serine (5); diso- on an Ultron N-C18 column (15 x 0.46 cm) at a flow rate of
dium diselenide was prepared by the method of Klayman and 0.8 ml/min. Both columns were programmed with a 30-min
Griffin (6); L-selenocysteine was prepared from disodium linear gradient from 5% to 50% acetonitrile in water. Triflu-
diselenide and /-chloro-L-alanine (7); Se-(p-methylbenzyl)- oroacetic acid was added at a concentration of 0. 1% (vol/vol)
L-selenocysteine was obtained from L-selenocysteine and to water and acetonitrile. Peptides were detected at 210 nm.
a-bromo-p-xylene by a modification of the procedure of Amino acid analysis was performed with a Beckman 7300
Erickson and Memfield (8); N-(tert-butyloxycarbonyl)-Se- amino acid analyzer. Peptides were hydrolyzed in 6 M HCI
(p-methylbenzyl)-L-selenocysteine was prepared from Se-(p- (Pierce) at 108°C in a sealed vessel under reduced pressure for
methylbenzyl)-L-selenocysteine and S-(tert-butyloxycar- 9 hr with a Waters Pico-Tag automatic acid hydrolysis
bonyl)-4,6-dimethyl-2-thiopyrimidine by a modification of system. Selenocysteine was determined after conversion into
the procedure of Nagasawa et al. (9). Other reagents, the best Se-carboxymethylselenocysteine with iodoacetic acid. The
grade commercially available, were used without further numbers of amino acid residues were calculated on the basis
purification. of a molecular weight of 2548 for metalloselenonein.
Metalloselenonein was synthesized in an Applied Biosys- All procedures were performed in a nitrogen glove box in
tems 430A peptide synthesizer programmed as shown in which oxygen concentration was kept below 10 ppm. All
Table 1. The synthesis was started on 1 g of Boc-Lys(CIZ)- solutions were degassed prior to use. Metalloselenonein (390
The publication costs of this article were defrayed in part by page charge *Present address: Faculty of Agriculture, Okayama University,
payment. This article must therefore be hereby marked "advertisement" Okayama 700, Japan.
in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.
3057
 3058 Biochemistry: Oikawa et al. Proc. Natl. Acad. Sci. USA 88 (1991)
nmol) was incubated in 20 mM Tris HCI buffer (pH 8.6) Table 2. Amino acid analysis of metalloselenonein
containing 5.85 ,umol of CuCl2 and 1.2 mmol of 2-mercapto- Number of residues
ethanol at 30°C for 30 min. The solution was applied to an
Asahipak GS-220 HPLC column (500 x 7.6 mm; Asahi- Amino acid Calculated Found
Kasei, Tokyo) equilibrated with 20 mM Tris HCI buffer (pH Ala 1 1.0
8.6). Absorption spectra were recorded using a Shimadzu Asx 3 2.4
MPS-2000 spectrophotometer in a sealed cuvette under re- Gly 6 6.0
duced pressure. Circular dichroism spectra were recorded in Lys 1 0.7
a Jasco J-600 spectropolarimeter using a 1-mm cuvette at Sec 7 7.1*
room temperature (about 20°C). Fluorescence spectra were Ser 7 6.6
measured with a Hitachi MPF-4 fluorescence spectropho- *Selenocysteine (Sec) was determined as Se-carboxymethylseleno-
tometer at 25°C. The copper content was determined by the cysteine after alkylation with iodoacetic acid.
method of standard additions with a Shimadzu AA-670G
atomic absorption spectrophotometer equipped with a graph- preparative HPLC was about 2%. The low yield is probably
ite furnace atomizer. Absorption was monitored at 324.8 nm attributable to the irreversible adsorption of metalloselenon-
with deuterium-arc background correction. We took an av- ein to the HPLC column. Selenocysteine is oxidized to give
erage of three independent determinations. All solutions used selenocysteic acid by performic acid oxidation in the same
for copper analysis were passed through a Chelex (Bio-Rad) manner as cysteine. However, selenocysteic acid decom-
column (1 x 10 cm) to remove free copper ions and stored in poses during acid hydrolysis of proteins. Therefore, we
polyethylene bottles. analyzed selenocysteine in the form of Se-carboxymethyl-
selenocysteine after alkylation with iodoacetic acid (15).
RESULTS Selenium compounds are generally highly susceptible to
We synthesized metalloselenonein by the solid-phase method
oxidative degradation. However, we have found that the
selenol of metalloselenonein is oxidized much more slowly
(12) with an automated peptide synthesizer. Its sequence is than that of selenocysteine. When metalloselenonein (0.4
H-Gly-Asp-Sec-Gly-Sec-Ser-Gly-Ala-Ser-Ser-Sec-Asn-Sec- mM) was incubated in 0.1 M Tris*HCI buffer (pH 8.0) at 370C
Gly-Ser-Gly-Sec-Ser-Sec-Ser-Asn-Sec-Gly-Ser-Lys-OH, for 2 hr, all of the initial selenocysteine residues remained
where Sec means selenocysteine. The amino terminus and intact, whereas 52% of free selenocysteine was oxidized to
the side chains of amino acids were protected with acid-labile selenocysteine under the same conditions.
groups: amino terminus, tert-butyloxycarbonyl group; aspar- The metalloselenonein complex with Cu(I) was isolated by
tic acid and serine, benzyl group. However, no masking HPLC with an Asahipak GS-220 gel filtration column. The
group for the selenol of selenocysteine has been developed. first major fraction (Mr 2700) contained 3 mol of copper per
We used a p-methylbenzyl group to protect the thiol of mol. The absorption spectrum of the copper complex was
cysteine. Several techniques have been developed to reduce characterized by a broad absorption band between 230 and
side reactions during the peptide synthesis and were used in 400 nm with a shoulder around 260 nm (Fig. 2). However,
Downloaded from https://www.pnas.org by 1.38.109.41 on August 8, 2024 from IP address 1.38.109.41.

the present synthesis. For example, Pam resin, which has metalloselenonein itself showed no absorption above 260 nm.
acid-stable linkage to the resin support, was used to reduce Therefore, the broad absorption band around 260 nm ob-
loss of premature peptides during acidolytic deprotection served for the copper complex is most probably attributable
cycles (13, 14). We used symmetrical anhydride coupling in to a copper-selenolate complex. The circular dichroism of
dimethylformamide for 20 min to obtain satisfactory coupling the copper-metalloselenonein complex revealed a negative
yields of >99% for all coupling steps except for the step of band at 220 nm and a positive band at 245 nm (Fig. 2).
Ala8-Ser9 synthesis: only 96.7% coupling yields were Metalloselenonein itself showed only a negative band at 220
achieved.
Metalloselenonein synthesized as described above was
purified by preparative HPLC after reduction with NaBH4.
The final preparation of metalloselenonein was eluted as a
single symmetrical peak upon analytical HPLC (Fig. 1), and
was found to be homogeneous by amino acid analysis of the 0
acid hydrolysates (Table 2). The overall recovery in the o

-0to
S-
0
1
(A
.0
0

0
5 2
.0'4 l L,1
CY)
0

U)
g-
C) 0
>I
0 ~)
.01 n r 0)

-2 L
Time (min) 220 300 400
FIG. 1. Reverse-phase HPLC profile of metalloselenonein. The Wavelength (nm)
crude peptide was applied to a column equilibrated with 0.1%
trifluoroacetic acid in 5% acetonitrile. The column was washed with FIG. 2. Absorption spectra and circular dichroism spectra of
the same buffer at a flow rate of 0.8 ml/min followed by elution with metalloselenonein. The solid lines show the spectra of the copper
a linear gradient between 0.1% trifluoroacetic acid in water and 50%1 complex of metalloselenonein in 20 mM Tris HCl buffer (pH 8.6), and
acetonitrile containing 0.1% trifluoroacetic acid (solvent B). the dotted lines show the spectra of metalloselenonein in 50 mM HCI.
 Biochemistry: Oikawa et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3059

complex is also attributable to transitions of a charge-transfer

type of the Cu(I)-selenolate complex. Metalloselenonein
binds three copper ions per molecule, in contrast to the native
ation and reconstituted metallothionein of N. crassa, which binds
six copper ions per molecule (23, 24). In the copper complex
u) of metallothionein, copper atoms are contained in a compact
polynuclear cluster with the thiolate of the cysteine residues
(25). The difference in ionic radius between sulfur and
selenium (ref. 26 and references therein) most likely accounts
for the observed difference in the coordination mode between
the two copper complexes.
We thank Dr. Hiromu Sakurai (Department of Pharmaceutical
Sciences, Tokushima University) for helpful suggestions. This work
was supported in part by special coordination funds from the Science
and Technology Agency of the Japanese Government.
200 300 400 500 600
1. Stadtman, T. C. (1980) Annu. Rev. Biochem. 49, 93-110.
Wavelength (nm) 2. Williams, R. J. P. (1978) in New Trends in Bioinorganic Chem-
istry (Academic, New York), pp. 253-260.
FIG. 3. Fluorescence spectrum of copper-metalloselenonein 3. Wu, Z. P. & Hilvert, D. (1989) J. Am. Chem. Soc. 111,
complex. 4513-4514.
4. Nordberg, M. & Kojima, Y. (1979) in Metallothionein, eds.
nm, which is attributable to an amide transition. Therefore, Kagi, J. H. R. & Nordberg, M. (Birkhaeuser, Basel), pp.
the positive band at 245 nm shows asymmetry in the copper- 41-117.
selenolate coordination. The copper-metalloselenonein com- 5. Walsh, C., Schonbrumm, A. & Abels, R. P. (1971) J. Biol.
plex showed an emission spectrum with a maximum at 395 Chem. 246, 6855-6866.
6. Klayman, D. J. & Griffin, S. T. (1973) J. Am. Chem. Soc. 95,
nm when it was excited at 245 nm (Fig. 3). Addition of 1 M 197-199.
HCl to the solution containing the complex led to complete 7. Chocat, P., Esaki, N., Tanaka, H. & Soda, K. (1985) Anal.
disappearance of not only the absorption band around 260 nm Biochem. 148, 485-489.
but also the fluorescence band at 395 nm. This indicates 8. Erickson, B. W. & Merrifield, R. B. (1973) J. Am. Chem. Soc.
displacement of copper with protons in the complex upon 95, 3757-3763.
addition of HCI. Furthermore, both electron-spectral bands 9. Nagasawa, T., Kuroiwa, K., Narita, K. & Isowa, Y. (1973)
Bull. Chem. Soc. Jpn. 46, 1269-1272.
were lost when the complex was oxidized in air. 10. Mitchell, A. R., Kent, S. B. H., Engelhard, M. & Merrifield,
R. B. (1978) J. Org. Chem. 43, 284-285.
11. Sarin, V. K., Kent, S. B. H., Tam, J. P. & Merrifield, R. B.
Downloaded from https://www.pnas.org by 1.38.109.41 on August 8, 2024 from IP address 1.38.109.41.

DISCUSSION (1981) Anal. Biochem. 117, 147-157.

The selenium analogues of various sulfur-containing peptides 12. Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154.
and proteins have been prepared previously. The acid-labile 13. Merrifield, R. B. (1986) Science 232, 341-347.
14. Mitchell, A. R., Erickson, B. W., Ryabtsev, M. N., Hodges,
sulfur in several iron-sulfur proteins was replaced by sele- R. S. & Merrifield, R. B. (1976) J. Am. Chem. Soc. 98, 7357-
nium: Fe2S2 putidaredoxin (16) and parsley ferredoxin (17); 7362.
Fe4S4 ferredoxin from Clostridium pasteurianum (18). A 15. Cone, J. E., Martin del Rio, R., Davis, J. N. & Stadtman, T. C.
derivative of 8-galactosidase whose methionine residues (1976) Proc. Natl. Acad. Sci. USA 73, 2659-2663.
were extensively replaced by selenomethionine residues was 16. Fee, J. A. & Palmer, G. (1971) Biochim. Biophys. Acta 245,
obtained from a selenium-resistant mutant of Escherichia coli 175-195.
grown on sodium selenate (27). Selenium analogues of gluta-
17. Wilson, G. S., Tsibris, J. C. M. & Gunsalus, I. C. (1973) J.
Biol. Chem. 248, 6059-6061.
thione (19), oxytocin (20), and somatostatin (21) were syn- 18. Meyer, J. & Moulis, M. M. (1981) Biochem. Biophys. Res.
thesized by the manual liquid-phase method (22), which is Commun. 103, 667-673.
much less efficient than the automatic solid-phase method 19. Theodoropoulos, D., Schwartz, I. L. & Waltoder, R. (1967)
based on symmetric anhydride coupling chemistry. By using Biochemistry 6, 3927-3932.
an automated peptide synthesizer according to the standard 20. Walter, R. & Chan, W. Y. (1967) J. Am. Chem. Soc. 89,
protocol, we have achieved the synthesis of a peptide (met- 3892-3898.
alloselenonein) that contains selenocysteine residues in place 21. Hartrodt, B., Neubert, K., Bierwolf, B., Blech, W. & Jakubke,
of all cysteine residues in copper metallothionein of N. H.-P. (1980) Tetrahedron Lett. 21, 2393-23%.
22. Popenoe, E. A. & du Vigneaud, V. (1953) J. Am. Chem. Soc.
crassa. 75, 4879-4880.
The copper-metalloselenonein complex showed a broad 23. Lerch, K. (1980) Nature (London) 284, 368-370.
absorption band between 230 and 400 nm with a shoulder 24. Kull, F. J., Reed, M. F., Elgren, T. E., Ciardelli, T. L. &
around 260 nm. This is probably attributable to a Se*-Cu(I) Wilcox, D. E. (1990) J. Am. Chem. Soc. 112, 2291-2298.
transition in the copper-selenolate complex. The circular 25. Smith, T. A., Lerch, K. & Hodgson, K. 0. (1986) Inorg. Chem.
dichroism spectrum of the Cu-metalloselenonein complex 25, 4677-4680.
showed a positive band around 245 nm. This indicates 26. Klayman, D. L. & Gunther, W. H. H. (1973) Organic Sele-
nium Compounds: Their Chemistry and Biology (Wiley, New
asymmetry in the copper-selenolate complex because the York).
band is absent from the spectrum of free metalloselenonein. 27. Coch, E. H. & Green, R. C. (1971) Biochim. Biophys. Acta 230,
The fluorescence spectrum of the copper-metalloselenonein 223-236.