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Synthesis, characterization, and radical scavenging effects of polynuclear thiolate complexes
Journal of Inorganic Biochemistry 70 (1998) 7±10
Synthesis, characterization, and radical scavenging eects of polynuclear thiolate complexes ShengLi Guo, Errun Ding, ShengMing Liu, YuanQi Yin
*
Lanzhou Institute of Chemical Physics, Academia Sinica, Lanzhou 730000, People's Republic of China Received 28 February 1997; received in revised form 2 December 1997; accepted 15 December 1997
Abstract Three thiolate complexes (Me4 N)2 [M4 (o-SC6 H4 CH3 )10 ], [M Zn (1); Cd (2)] and [c-l4 -S)(SCH2 Ph)12 S4 Zn8 ] (3) were synthesized and characterized by elemental analyses, IR, and 1 H NMR spectroscopic techniques. The suppression abilities of complexes for the hydroxyl radical (OH) and TEMP (HO-N-O) were determined by EPR. The former were also measured by its reaction with 3-methylthiobutyraldehyde (MBA) to give propylene and methane analyzed by GC. The results show that the complexes possess selectively scavenging eects: on the hydroxyl radicals, abilities decreased across the series 3 > NMT > 2 1 (NMT native metallothionein), whereas, on TEMP radicals, the order was 1 > 3 > NMT > 2. Ó 1998 Elsevier Science Inc. All rights reserved.
1. Introduction
2. Experimental
Metallothioneins (MTs) constitute a family of low molecular weight (Mr 6100 Dalton), cysteine-rich metal binding proteins that are found ubiquitously distributed in nature [1±4]. Crystal structure determination showed [5], the structure of MT isoform II from rat liver was folded into two domains: the amino terminal domain (b) of residues 1±29 enfolds a three-metal cluster of CdZn2 (Cys)9 , while the carboxyl terminal domain (a) of residues 30±61 enfolds a four-metal cluster of Cd4 (Cys)11 . All seven metal sites have tetrahedral coordination geometry. One of important physiological roles of MTs is providing cells with radioresistance ± a property shown by MTs probably because of their high cysteinyl thiolate content [6]. Dance and coworkers have reported a lot of complexes contained E4 S10 , E4 M10 S16 (E S, Se; M Zn, Cd) cores [7±9]. Since hydroxyl radical is a primary free radical generated during radiolysis of aerobic aqueous solution, the investigation of the reaction of the radical with MT and its model complexes will be of great interest. In this paper, we report the synthesis, characterization, and inhibitory action for free radicals of the thiolate polynuclear metal complexes as a contrast to native MT.
Chemical: o-methyl benzenethiol, 5,5-dimethyl-1pyrroline-N-oxide (DMPO), 2,2,6,6-tetramethyl-4-hydroxyl-1-piperidine-N-oxide (TEMP) radical were purchased from Aldrich Chemical. 3-Methylthiobutyralde hyde (MBA) was prepared similarly as in [10]. Rat liver metallothionein (NMT) was supplied by Beijing Institute of Science and Technology. All other reagents were A.R. grade. All of the reactions were performed in Schlenk-type ¯ask under dry nitrogen. Physical measurements: C, H, N, and S were determined by a Vario EL elemental analyzer. The IR spectra were recorded on a Nicolet-10DX FT-IR instrument in KBr disk in the range of 4000±400 cmÿ1 . 1 H NMR spectra were obtained on a Varian FT-80A spectrometer using CD3 CN as solvent and TMS as internal reference. EPR spectra were performed on a Varian E-115 electron paramagnetic resonance spectrometer. Gas chromatography was carried out on a Shimadzu GC-9A instrument. Preparation of the complexes: (a) Tetranuclear complexes 1,2 [9,11]
*
Corresponding author. Fax: +86 931 8417088; e-mail: hcom@ns. lzb.ac.cn. 0162-0134/98/$19.00 Ó 1998 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 2 - 0 1 3 4 ( 9 8 ) 0 0 0 0 6 - 3
A solution of 4.42 g (32.4 mmol) of ZnCl2 in 30 ml of methanol was added to a solution of 81 mmol of sodium
8
S.L. Guo et al. / J. Inorg. Biochem. 70 (1998) 7±10
o-methyl benzenethiolate (from 1.85 g of sodium and 10.0 g of o-methyl benzenethiol) in 90 ml of methanol. After the solution was stirred for 40 min, sodium chloride was removed by ®ltration and tetramethylammonium bromide (2.2 g, 14.3 mmol) was added. The resultant precipitate became microcrystalline upon further stirring for 14 h. The product was collected by ®ltration, washed with methanol and recrystallized from acetonitrile, aording 8.14 g (61.3%) of white solid. IR data (cmÿ1 ): 2968, 2945, 1375(CH3 ), 3048, 1548, 1464, 1128, 1061, 1030, 744, 442(Ph), 675(vc±s ). 1 H NMR: 6.94 (m, 10H, H of position 6 of Ph), 6.73 (m, 30H, H of position 3,4,5 of Ph), 3.12 (s, 24H, NMe4 ), 2.26 (s, 30H, SC6 H4 CH3 ). 2. IR data (cmÿ1 ): 2967, 2942, 1375(CH3 ), 3054, 1586, 1464, 1061, 1044, 743, 440(Ph), 673(vc±s ). 1 H NMR: 7.30 (m, 10H, H of position 6 of Ph), 6.85 (m, 30H, H of position 3,4,5 of Ph), 3.01 (s, 24H, NMe4 ), 2.25 (s, 30H, SC6 H4 CH3 ). (b) Octanuclear complex 3:
The zinc chloride anhydrous (9.1 g, 0.067 mol) was added to a ¯ask containing 150 ml of acetonitrile, and stirred for 0.5 h. Then, a solution of sodium benzylthiolate (14.6 g, 0.1 mol) in 50 ml of methanol was added dropwise, stirred for 2 h. Subsequently, tetramethylammonium chloride (4.6 g, 0.042 mol) was added agitated for further 5 h and ®ltered. The ®ltration was diused by diethyl ether for ca. one month, the colorless crystals were produced, which were isolated by ®ltration, washed with cooled diethyl ether and dried in vacuum. Yield: 7.63 g, 56.14%. IR data (cmÿ1 ): 3010, 1378(NMe4 ), 3057, 1601, 1491, 1240, 949, 774, 702(Ph), 675(vc±s ). 1 H NMR: 7.32-7.11 (m, 60H, Ph), 3.67 (s, 24H, CH2 ), 3.06 (s, 24H, NMe4 ). Experiment of suppression of radicals: The hydroxyl radicals(OH) were produced by the system of H2 O2 / (Fe2 -EDTA)/L -ascorbic acid [12] and determined both by EPR spectra and its reaction with MBA to give propylene and methane. EPR technique: mixed each 0.02 ml of Fe2 -EDTA (4 mM), L -ascorbic acid (8 mM), DMPO(50 mM), H2 O2 (4 mM), tested complexes (0.3 mM in 20% acetonitrile for 1±3, and PBS buer for NMT) and PBS buer (0.8 M, pH 7.4), in an EPR thin tube and incubation for 0.5 h at a constant 37 C, then the spin adduct DMPO-OH was detected on EPR
instrument. For GC method mixed 1 ml of all above reactant except replacing DMPO with MBA (4 mM), the produced amount of propylene and methane was measured and the suppression ratio for OH was calculated from the following expression [13]: M0 ÿ M Suppression ratio 100 ; M0 where M; M0 stands for content of propylene and methane in the presence and absence of complexes to test the quenching action of TEMP radical (20 mM) by EPR method using thiolate compounds as inhibitor was similarly carried out only by replacing (Fe2 -EDTA)/H2 O2 / L -ascorbic acid and DMPO with TEMP. 3. Results and discussion Chemical characterization: Three thiolate complexes were prepared and characterized by elemental analyses, IR, and 1 H NMR spectra, the structure of complex 3 was solved by X-ray crystal structure analysis in our previous work [14]. The elemental analyses data (Table 1) are in good agreement with the calculated values. IR spectra of the complexes are similar. Some characteristic bands of phenyl ring (dCH 743; UC±C 440 of ortho-disubstituted phenyl ring of 1,2 and d@CH 774, 702 of monosubsititued phenyl ring of 3), and C±S bond (vC±S 675 cmÿ1 ) appeared in their IR spectra, reveals that thiolate ligand are all coordinated to metal center. 1 H NMR spectra of the complexes are simple, as there are only three kinds of hydrogen in the complexes: action (MMe4 ), phenyl ring and substituents on it (CH3 or CH2 S). Scavenging radical reaction: Upon incubation of hydrogen peroxide with ferrous salt and ascorbic acid, hydroxyl radicals are generated in Fenton-type reaction [12]. Their production can be easily monitored by the hydroxyl radical spin adduct DMPO-OH. The maximum concentration of DMPO-OH in the absence and presence of thiolate complexes or NMT was detected. The results (Fig. 1) showed that they have the inhibiting action for OH radicals. The hydroxyl radicals spin adduct DMPO-OH was characterized by g 2.0054 and hyper®ne splitting constants aN arH 1:49 mT, while for TEMP g 2.0060 and aN 1.58 mT. In contrast to hydroxyl radical, the scavenging eect on the same molar concentration of TEMP, had been smaller, and quenching order: 1 > 3 > NMT > 2, which diers from
Table 1 Elemental analyses data and melting point of the complexes Compound no. Found
1 2 3
Melting point ( C)
Calc.
C%
H%
N%
S%
C%
H%
N%
S%
56.82 51.18 47.69
5.52 5.28 4.48
1.75 1.53 1.04
19.35 17.50 23.84
57.06 50.96 47.80
5.73 5.14 4.67
1.71 1.53 1.21
19.55 17.53 23.60
196±197 132±133 >320(dec.)
S.L. Guo et al. / J. Inorg. Biochem. 70 (1998) 7±10
Fig. 1. The EPR spectra of the (Fe2 -EDTA)/H2 O2 / L -ascorbic acid reaction in the presence of the spin trapping agent DMPO and scavenger with a (control-no further additions), b (+0.3 mM complex 2), c (+0.3 mM complex 1), d (+0.3 mM of NMT), e (+0.3 mM complex 3). Spectra a±e are plotted in the same x-axis scale, but they are arbitrarily shifted from each other.
that for OH radicals: 3 > NMT > 1 2, such suppression selectivity may be attributed to TEMP radicals being more stable than DMPO-OH analogue. The complex 3 has best scavenging eect on hydroxyl radicals, the native MT has second one, however, the suppression action of tetranuclear complexes 1 and 2 are the weakest of all tested scavenger (see Fig. 2). Hydroxyl radicals had ever been detected by their reaction with methional to give ethylene [15]. Dierent from this, we developed a new method which employed 3-methylthiobutyraldehyde as a probe, the OH radicals concentration was measured indirectly from content of propylene and methane (very little amount of ethylene being neglected) given by the reaction of OH radicals with 3-methylthiobutyraldehyde (Eq. (1)). C H3 SHCH2 CHO OH
CH3
! CH3 CH@CH2 CH4 CH2 @CH2 90%
9%
1%
1
The result obtained from this method is identical with that of the result from EPR technique, see Table 2. Thus, it is seemed that the scavenging eects of complexes on hydroxyl radicals had mainly relevance to the mole number of metal atom of the complexes, since there are seven mole Zn atom per mole of native MT, e.g., the scavenging eect decrease across the series [Zn8 ] (3) > [Zn7 ](NMT) > Zn4 (1) [Cd4 ](2). P.J. Thornalley et al. [16] had reported their studying result that the orders of quenching of DMPO-OH adduct production was: Cd±MT Zn±Mt bovine
9
Fig. 2. The EPR spectra of TEMP radical in the absence and presence of scavenger, the same denotation of a±e as Fig. 1. Spectra a±e are plotted in the same x-axis scale, but they are arbitrarily shifted from each other.
Table 2 Data of scavenging eect on OH of the complexes and NMT Compound no.
Complex conc. (mM)
Average suppression ratio(%) (Based on propylene only
(Propylene methane)
1 2 3 NMT
0.05 0.05 0.05 0.05
38.5 35.1 84.0 64.0
37.4 34.6 83.8 62.1
serum albumin. Our observance that complex 3 had a scavenging capacity towards hydroxyl radicals even greater than Zn±MT, may implicate some interrelation between the unusual structure of complex 3 and its reactivity of scavenging OH free radicals. References [1] J.H.R. K agi, M. Nordberg (Eds.), Metallothionein; Birkh auser, Basel, Switzerland, 1979. [2] D.C. Dalgarno, I.M. Armitage, in: G.L. Eichorn, L. Marzilli (Eds.), Advances in Inorganic Biochemistry, Elsevier, New York 6 (1984) 113. [3] K.B. Nielson, C.L. Atkin, D.R. Winge, J. Biol. Chem. 260 (1985) 5342.
10
S.L. Guo et al. / J. Inorg. Biochem. 70 (1998) 7±10
[4] J.H.R. Kagi, M. Vas ak, K. Lerch, D.E.O. Gilg, P. Hunziker, W.R. Bernhard, M. Good, Environ. Health Perspect. 54 (1984) 93. [5] W.F. Furey, A.H. Robbins, L.L. Clancy, D.R. Winge, B.C. Wang, C.D. Stout, Science 231 (1986) 704. [6] A. Bakka, A.S. Johnson, L. Endressen, H.E. Rugstad, Experientia 38 (1982) 381. [7] I.G. Dance, J. Amer. Chem. Soc. 101 (1979) 6264. [8] A. Choy, D. Craig, I.G. Dance, M.L. Scudder, J. Chem. Soc. Chem. Commun. 1246 (1982). [9] I.G. Dance, A. Choy, M.L. Scudder, J. Amer. Chem. Soc. 106 (1984) 6285.
[10] Sumitomo Chemical Co., Ltd. Fr.1, 520, 328(Cl. C07 C), 05, Apr. 1968, Japan Appl. 25, Apr. 1966. [11] K.S. Hagen, D.W. Stephan, R.H. Holml, Inorg. Chem. 21 (1982) 3928. [12] C.C. Winterbourn, Biochem. J. 198 (1981) 125. [13] K.W. Yang, L.F. Wang, J.G. Wu, F. Dong, J. Inorg. Biochem. 52 (1993) 145. [14] S.L. Guo, X.M. Liu, Y.Q. Yin, Chinese J. Struct. Chem. 16 (1997) 349. [15] C. Beauchamp, I. Fridovich, J. Biol. Chem. 245 (1970) 4641. [16] P.J. Thornalley, M. Vas ak, Biochem. Biophys. Acta 827 (1985) 36.
Synthesis, characterization, and radical scavenging eects of polynuclear thiolate complexes ShengLi Guo, Errun Ding, ShengMing Liu, YuanQi Yin
*
Lanzhou Institute of Chemical Physics, Academia Sinica, Lanzhou 730000, People's Republic of China Received 28 February 1997; received in revised form 2 December 1997; accepted 15 December 1997
Abstract Three thiolate complexes (Me4 N)2 [M4 (o-SC6 H4 CH3 )10 ], [M Zn (1); Cd (2)] and [c-l4 -S)(SCH2 Ph)12 S4 Zn8 ] (3) were synthesized and characterized by elemental analyses, IR, and 1 H NMR spectroscopic techniques. The suppression abilities of complexes for the hydroxyl radical (OH) and TEMP (HO-N-O) were determined by EPR. The former were also measured by its reaction with 3-methylthiobutyraldehyde (MBA) to give propylene and methane analyzed by GC. The results show that the complexes possess selectively scavenging eects: on the hydroxyl radicals, abilities decreased across the series 3 > NMT > 2 1 (NMT native metallothionein), whereas, on TEMP radicals, the order was 1 > 3 > NMT > 2. Ó 1998 Elsevier Science Inc. All rights reserved.
1. Introduction
2. Experimental
Metallothioneins (MTs) constitute a family of low molecular weight (Mr 6100 Dalton), cysteine-rich metal binding proteins that are found ubiquitously distributed in nature [1±4]. Crystal structure determination showed [5], the structure of MT isoform II from rat liver was folded into two domains: the amino terminal domain (b) of residues 1±29 enfolds a three-metal cluster of CdZn2 (Cys)9 , while the carboxyl terminal domain (a) of residues 30±61 enfolds a four-metal cluster of Cd4 (Cys)11 . All seven metal sites have tetrahedral coordination geometry. One of important physiological roles of MTs is providing cells with radioresistance ± a property shown by MTs probably because of their high cysteinyl thiolate content [6]. Dance and coworkers have reported a lot of complexes contained E4 S10 , E4 M10 S16 (E S, Se; M Zn, Cd) cores [7±9]. Since hydroxyl radical is a primary free radical generated during radiolysis of aerobic aqueous solution, the investigation of the reaction of the radical with MT and its model complexes will be of great interest. In this paper, we report the synthesis, characterization, and inhibitory action for free radicals of the thiolate polynuclear metal complexes as a contrast to native MT.
Chemical: o-methyl benzenethiol, 5,5-dimethyl-1pyrroline-N-oxide (DMPO), 2,2,6,6-tetramethyl-4-hydroxyl-1-piperidine-N-oxide (TEMP) radical were purchased from Aldrich Chemical. 3-Methylthiobutyralde hyde (MBA) was prepared similarly as in [10]. Rat liver metallothionein (NMT) was supplied by Beijing Institute of Science and Technology. All other reagents were A.R. grade. All of the reactions were performed in Schlenk-type ¯ask under dry nitrogen. Physical measurements: C, H, N, and S were determined by a Vario EL elemental analyzer. The IR spectra were recorded on a Nicolet-10DX FT-IR instrument in KBr disk in the range of 4000±400 cmÿ1 . 1 H NMR spectra were obtained on a Varian FT-80A spectrometer using CD3 CN as solvent and TMS as internal reference. EPR spectra were performed on a Varian E-115 electron paramagnetic resonance spectrometer. Gas chromatography was carried out on a Shimadzu GC-9A instrument. Preparation of the complexes: (a) Tetranuclear complexes 1,2 [9,11]
*
Corresponding author. Fax: +86 931 8417088; e-mail: hcom@ns. lzb.ac.cn. 0162-0134/98/$19.00 Ó 1998 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 2 - 0 1 3 4 ( 9 8 ) 0 0 0 0 6 - 3
A solution of 4.42 g (32.4 mmol) of ZnCl2 in 30 ml of methanol was added to a solution of 81 mmol of sodium
8
S.L. Guo et al. / J. Inorg. Biochem. 70 (1998) 7±10
o-methyl benzenethiolate (from 1.85 g of sodium and 10.0 g of o-methyl benzenethiol) in 90 ml of methanol. After the solution was stirred for 40 min, sodium chloride was removed by ®ltration and tetramethylammonium bromide (2.2 g, 14.3 mmol) was added. The resultant precipitate became microcrystalline upon further stirring for 14 h. The product was collected by ®ltration, washed with methanol and recrystallized from acetonitrile, aording 8.14 g (61.3%) of white solid. IR data (cmÿ1 ): 2968, 2945, 1375(CH3 ), 3048, 1548, 1464, 1128, 1061, 1030, 744, 442(Ph), 675(vc±s ). 1 H NMR: 6.94 (m, 10H, H of position 6 of Ph), 6.73 (m, 30H, H of position 3,4,5 of Ph), 3.12 (s, 24H, NMe4 ), 2.26 (s, 30H, SC6 H4 CH3 ). 2. IR data (cmÿ1 ): 2967, 2942, 1375(CH3 ), 3054, 1586, 1464, 1061, 1044, 743, 440(Ph), 673(vc±s ). 1 H NMR: 7.30 (m, 10H, H of position 6 of Ph), 6.85 (m, 30H, H of position 3,4,5 of Ph), 3.01 (s, 24H, NMe4 ), 2.25 (s, 30H, SC6 H4 CH3 ). (b) Octanuclear complex 3:
The zinc chloride anhydrous (9.1 g, 0.067 mol) was added to a ¯ask containing 150 ml of acetonitrile, and stirred for 0.5 h. Then, a solution of sodium benzylthiolate (14.6 g, 0.1 mol) in 50 ml of methanol was added dropwise, stirred for 2 h. Subsequently, tetramethylammonium chloride (4.6 g, 0.042 mol) was added agitated for further 5 h and ®ltered. The ®ltration was diused by diethyl ether for ca. one month, the colorless crystals were produced, which were isolated by ®ltration, washed with cooled diethyl ether and dried in vacuum. Yield: 7.63 g, 56.14%. IR data (cmÿ1 ): 3010, 1378(NMe4 ), 3057, 1601, 1491, 1240, 949, 774, 702(Ph), 675(vc±s ). 1 H NMR: 7.32-7.11 (m, 60H, Ph), 3.67 (s, 24H, CH2 ), 3.06 (s, 24H, NMe4 ). Experiment of suppression of radicals: The hydroxyl radicals(OH) were produced by the system of H2 O2 / (Fe2 -EDTA)/L -ascorbic acid [12] and determined both by EPR spectra and its reaction with MBA to give propylene and methane. EPR technique: mixed each 0.02 ml of Fe2 -EDTA (4 mM), L -ascorbic acid (8 mM), DMPO(50 mM), H2 O2 (4 mM), tested complexes (0.3 mM in 20% acetonitrile for 1±3, and PBS buer for NMT) and PBS buer (0.8 M, pH 7.4), in an EPR thin tube and incubation for 0.5 h at a constant 37 C, then the spin adduct DMPO-OH was detected on EPR
instrument. For GC method mixed 1 ml of all above reactant except replacing DMPO with MBA (4 mM), the produced amount of propylene and methane was measured and the suppression ratio for OH was calculated from the following expression [13]: M0 ÿ M Suppression ratio 100 ; M0 where M; M0 stands for content of propylene and methane in the presence and absence of complexes to test the quenching action of TEMP radical (20 mM) by EPR method using thiolate compounds as inhibitor was similarly carried out only by replacing (Fe2 -EDTA)/H2 O2 / L -ascorbic acid and DMPO with TEMP. 3. Results and discussion Chemical characterization: Three thiolate complexes were prepared and characterized by elemental analyses, IR, and 1 H NMR spectra, the structure of complex 3 was solved by X-ray crystal structure analysis in our previous work [14]. The elemental analyses data (Table 1) are in good agreement with the calculated values. IR spectra of the complexes are similar. Some characteristic bands of phenyl ring (dCH 743; UC±C 440 of ortho-disubstituted phenyl ring of 1,2 and d@CH 774, 702 of monosubsititued phenyl ring of 3), and C±S bond (vC±S 675 cmÿ1 ) appeared in their IR spectra, reveals that thiolate ligand are all coordinated to metal center. 1 H NMR spectra of the complexes are simple, as there are only three kinds of hydrogen in the complexes: action (MMe4 ), phenyl ring and substituents on it (CH3 or CH2 S). Scavenging radical reaction: Upon incubation of hydrogen peroxide with ferrous salt and ascorbic acid, hydroxyl radicals are generated in Fenton-type reaction [12]. Their production can be easily monitored by the hydroxyl radical spin adduct DMPO-OH. The maximum concentration of DMPO-OH in the absence and presence of thiolate complexes or NMT was detected. The results (Fig. 1) showed that they have the inhibiting action for OH radicals. The hydroxyl radicals spin adduct DMPO-OH was characterized by g 2.0054 and hyper®ne splitting constants aN arH 1:49 mT, while for TEMP g 2.0060 and aN 1.58 mT. In contrast to hydroxyl radical, the scavenging eect on the same molar concentration of TEMP, had been smaller, and quenching order: 1 > 3 > NMT > 2, which diers from
Table 1 Elemental analyses data and melting point of the complexes Compound no. Found
1 2 3
Melting point ( C)
Calc.
C%
H%
N%
S%
C%
H%
N%
S%
56.82 51.18 47.69
5.52 5.28 4.48
1.75 1.53 1.04
19.35 17.50 23.84
57.06 50.96 47.80
5.73 5.14 4.67
1.71 1.53 1.21
19.55 17.53 23.60
196±197 132±133 >320(dec.)
S.L. Guo et al. / J. Inorg. Biochem. 70 (1998) 7±10
Fig. 1. The EPR spectra of the (Fe2 -EDTA)/H2 O2 / L -ascorbic acid reaction in the presence of the spin trapping agent DMPO and scavenger with a (control-no further additions), b (+0.3 mM complex 2), c (+0.3 mM complex 1), d (+0.3 mM of NMT), e (+0.3 mM complex 3). Spectra a±e are plotted in the same x-axis scale, but they are arbitrarily shifted from each other.
that for OH radicals: 3 > NMT > 1 2, such suppression selectivity may be attributed to TEMP radicals being more stable than DMPO-OH analogue. The complex 3 has best scavenging eect on hydroxyl radicals, the native MT has second one, however, the suppression action of tetranuclear complexes 1 and 2 are the weakest of all tested scavenger (see Fig. 2). Hydroxyl radicals had ever been detected by their reaction with methional to give ethylene [15]. Dierent from this, we developed a new method which employed 3-methylthiobutyraldehyde as a probe, the OH radicals concentration was measured indirectly from content of propylene and methane (very little amount of ethylene being neglected) given by the reaction of OH radicals with 3-methylthiobutyraldehyde (Eq. (1)). C H3 SHCH2 CHO OH
CH3
! CH3 CH@CH2 CH4 CH2 @CH2 90%
9%
1%
1
The result obtained from this method is identical with that of the result from EPR technique, see Table 2. Thus, it is seemed that the scavenging eects of complexes on hydroxyl radicals had mainly relevance to the mole number of metal atom of the complexes, since there are seven mole Zn atom per mole of native MT, e.g., the scavenging eect decrease across the series [Zn8 ] (3) > [Zn7 ](NMT) > Zn4 (1) [Cd4 ](2). P.J. Thornalley et al. [16] had reported their studying result that the orders of quenching of DMPO-OH adduct production was: Cd±MT Zn±Mt bovine
9
Fig. 2. The EPR spectra of TEMP radical in the absence and presence of scavenger, the same denotation of a±e as Fig. 1. Spectra a±e are plotted in the same x-axis scale, but they are arbitrarily shifted from each other.
Table 2 Data of scavenging eect on OH of the complexes and NMT Compound no.
Complex conc. (mM)
Average suppression ratio(%) (Based on propylene only
(Propylene methane)
1 2 3 NMT
0.05 0.05 0.05 0.05
38.5 35.1 84.0 64.0
37.4 34.6 83.8 62.1
serum albumin. Our observance that complex 3 had a scavenging capacity towards hydroxyl radicals even greater than Zn±MT, may implicate some interrelation between the unusual structure of complex 3 and its reactivity of scavenging OH free radicals. References [1] J.H.R. K agi, M. Nordberg (Eds.), Metallothionein; Birkh auser, Basel, Switzerland, 1979. [2] D.C. Dalgarno, I.M. Armitage, in: G.L. Eichorn, L. Marzilli (Eds.), Advances in Inorganic Biochemistry, Elsevier, New York 6 (1984) 113. [3] K.B. Nielson, C.L. Atkin, D.R. Winge, J. Biol. Chem. 260 (1985) 5342.
10
S.L. Guo et al. / J. Inorg. Biochem. 70 (1998) 7±10
[4] J.H.R. Kagi, M. Vas ak, K. Lerch, D.E.O. Gilg, P. Hunziker, W.R. Bernhard, M. Good, Environ. Health Perspect. 54 (1984) 93. [5] W.F. Furey, A.H. Robbins, L.L. Clancy, D.R. Winge, B.C. Wang, C.D. Stout, Science 231 (1986) 704. [6] A. Bakka, A.S. Johnson, L. Endressen, H.E. Rugstad, Experientia 38 (1982) 381. [7] I.G. Dance, J. Amer. Chem. Soc. 101 (1979) 6264. [8] A. Choy, D. Craig, I.G. Dance, M.L. Scudder, J. Chem. Soc. Chem. Commun. 1246 (1982). [9] I.G. Dance, A. Choy, M.L. Scudder, J. Amer. Chem. Soc. 106 (1984) 6285.
[10] Sumitomo Chemical Co., Ltd. Fr.1, 520, 328(Cl. C07 C), 05, Apr. 1968, Japan Appl. 25, Apr. 1966. [11] K.S. Hagen, D.W. Stephan, R.H. Holml, Inorg. Chem. 21 (1982) 3928. [12] C.C. Winterbourn, Biochem. J. 198 (1981) 125. [13] K.W. Yang, L.F. Wang, J.G. Wu, F. Dong, J. Inorg. Biochem. 52 (1993) 145. [14] S.L. Guo, X.M. Liu, Y.Q. Yin, Chinese J. Struct. Chem. 16 (1997) 349. [15] C. Beauchamp, I. Fridovich, J. Biol. Chem. 245 (1970) 4641. [16] P.J. Thornalley, M. Vas ak, Biochem. Biophys. Acta 827 (1985) 36.