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Hydrogen peroxide-mediated Cu,Zn-superoxide dismutase fragmentation: protection by carnosine, homocarnosine and anserine
Biochimica et Biophysica Acta 1472 (1999) 651^657 www.elsevier.com/locate/bba
Hydrogen peroxide-mediated Cu,Zn-superoxide dismutase fragmentation: protection by carnosine, homocarnosine and anserine Soo Young Choi a , Hyeok Yil Kwon b , Oh Bin Kwon c , Jung Hoon Kang
c;
*
a c
Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon 200-702, South Korea b Department of Physiology, College of Medicine, Hallym University, Chunchon 200-702, South Korea Department of Genetic Engineering, Division of Natural Sciences, Chongju University, Chongju 360-764, South Korea Received 12 May 1999; received in revised form 8 September 1999; accepted 20 September 1999
Abstract The fragmentation of human Cu,Zn-superoxide dismutase (SOD) was observed during incubation with H2 O2 . Hydroxyl radical scavengers such as sodium azide, formate and mannitol protected the fragmentation of Cu,Zn-SOD. These results suggested that OH was implicated in the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation. Carnosine, homocarnosine and anserine have been proposed to act as anti-oxidants in vivo. We investigated whether three compounds could protect the fragmentation of Cu,Zn-SOD induced by H2 O2 . The results showed that carnosine, homocarnosine and anserine significantly protected the fragmentation of Cu,Zn-SOD. All three compounds also protected the loss of enzyme activity induced by H2 O2 . Carnosine, homocarnosine and anserine effectively inhibited the formation of OH by the Cu,ZnSOD/H2 O2 system. These results suggest that carnosine and related compounds can protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation through the scavenging of OH. ß 1999 Elsevier Science B.V. All rights reserved. b
b
b
Keywords: Copper,zinc-superoxide dismutase; Fragmentation; Hydrogen peroxide; Carnosine
1. Introduction Cu,Zn-superoxide dismutase (SOD) is an antioxidant enzyme in tissues. However, the overexpression of Cu,Zn-SOD among others has been correlated to cell killing [1], increased lipid peroxidation [2], reduced transport of biogenic amines [3] and decomposition of brain neuron [4]. In addition to the usual superoxide dismutase activity, Cu,Zn-SOD has a free radical-generating function that utilizes its own product, H2 O2 as a substrate [5,6]. If OH radicals are generated by the Cu,Zn-SOD and H2 O2 system in b
* Corresponding author. Fax: +82-431-229-8432; E-mail: [email protected]
biological cells, it could react with its own molecule (SOD) and other molecules in the vicinity of its generation sites. Involvement of the free radical-generating function of Cu,Zn-SOD in human disease has been suggested [7^9]. Carnosine (L-alanyl-L-histidine) and related compounds such as anserine (L-alanyl-1-methyl-L-histidine) and homocarnosine (Q-amino-butyryl-L-histidine) are present in several mammalian tissues, including skeletal muscle and brain at high concentrations (up to 20 mM in humans) [10^13]. Carnosine has been postulated to act as a bu¡er to neutralize lactic acid produced in skeletal muscle that is undergoing anaerobic glycolysis [14]. Carnosine and anserine have been shown to be e¤cient copper-chelating agents and it has been suggested that they may play
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a role in copper metabolism in vivo [15]. Carnosine also protects rabbit heart from reperfusion injury after ischemia [16]. Many biochemical studies have suggested that carnosine possesses anti-oxidant and free radical scavenging functions which may partly explain its apparent homeostatic function [17,18]. In this study, we examined the oxidative damage of Cu,Zn-SOD caused by H2 O2 . Our results suggest that the fragmentation of Cu,Zn-SOD induced by H2 O2 is due to the oxidative damage resulting from OH radicals generated by a combination of the free radical-generating function of Cu,Zn-SOD and the Fenton-like reaction of free copper ions released from oxidatively damaged SOD. We also investigated the e¡ects of carnosine, homocarnosine and anserine on the fragmentation of Cu,Zn-SOD induced by H2 O2 . Our results show that three compounds can protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation through the scavenging of OH radicals. b
b
2. Materials and methods 2.1. Materials Carnosine, homocarnosine, anserine, 2-deoxy-D-ribose, diethylenetriaminepenta-acetic acid (DTPA), thiobarbituric acid and trichloroacetic acid were purchased from Sigma (St. Louis, MO, USA). Chelex 100 resin (sodium form) was obtained from BioRad Lab (Hercules, CA, USA). Recombinant human Cu,Zn-SOD was prepared as described previously [19]. 2.2. Analysis of Cu,Zn-SOD fragmentation by H2 O2 Cu,Zn-SOD (25 WM) was incubated with H2 O2 (10 mM) in 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6) at 37³C during various incubation periods. After stopping the reaction by adding catalase to a ¢nal concentration of 100 Wg/ml, aliquots were diluted 4U with concentrated sample bu¡er (0.25 mM TrisHCl, 8% SDS, 40% glycerol, 20% L-mercaptoethanol, 0.01% bromophenol blue) and were boiled at 100³C for 10 min before electrophoresis. An aliquot
of each sample was subjected to SDS-PAGE as described by Laemmli [20], using a slab gel (stacking gel, 2.5% acrylamide and a separating gel, 18% acrylamide). The gels were stained with 0.15% Coomassie brilliant blue R-250. The amount of protein was quantitated by the staining intensity as judged by densitometric scanning with the £ying spot scanner CS-9000 (Shimadzu, Kyoto, Japan). Protein concentration was determined by the BCA method [21]. 2.3. SOD activity assay The activity was measured by monitoring their capacities to inhibit the reduction of ferricytochrome C by xanthine/xanthine oxidase as described by McCord and Fridovich [22]. Activity staining of Cu,Zn-SOD was performed by electrophoresis in 10% native polyacrylamide gels and visualized as described by Beauchamp and Fridovich [23]. Brie£y, the gel was soaked in 2.45 mM nitroblue tetrazolium solution for 15 min, followed by 30 min in 28 mM N,N,NP,NP-tetramethylethylene diamine and 28 WM ribo£avin in 0.36 mM potassium phosphate bu¡er (pH 7.8). The gel was then exposed to a £uorescence light source until the bands showed maximum resolution. 2.4. Measurement of OH production b
Detection of OH formation was determined by measuring thiobarbituric acid reactive 2-deoxy-D-ribose oxidation products [24]. The assay mixture contained 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6), 10 mM 2-deoxy-D-ribose, 10 mM H2 O2 and 5 WM enzyme in a total volume of 100 Wl. The reaction was initiated by addition of H2 O2 and incubated for 1 h at 37³C. Reaction was stopped by addition of 2.8% trichloroacetic acid (200 Wl), PBS (200 Wl)and 1% thiobarbituric acid (200 Wl) and boiled at 100³C for 15 min. After the samples were cooled and centrifuged at 15 000 rpm for 10 min. Results were read by a UV/vis spectrophotometer (Shimadzu, UV1601) at 532 nm. All solutions used in the present experiments were treated with Chelex 100 and prepared by bubbling a 5% CO2 , 95% N2 gas mixture [5].
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3. Results and discussion Incubation of Cu,Zn-SOD with 10 mM H2 O2 for 0^180 min at 37³C resulted in a time-dependent decrease in the amount of intact enzyme. SDS-PAGE showed a gradual decrease in the intensity of the original band (Fig. 1A). The kinetics of the decrease of the intact Cu,Zn-SOD was assessed by densitometric scanning of staining intensity on SDS-PAGE (Fig. 1B). When Cu,Zn-SOD was incubated with various concentrations of H2 O2 , the frequency of the protein fragmentation increased in a hydrogen peroxide concentration-dependent manner (Fig. 2A and B). Incubation of Cu,Zn-SOD with 0.1 mM
Fig. 2. E¡ect of H2 O2 concentrations on the fragmentation of Cu,Zn-SOD. 25 WM Cu,Zn-SOD was incubated with various concentrations of H2 O2 in 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6) at 37³C for 12 h. Reactions were stopped by adding catalase to a ¢nal concentration of 100 Wg/ml and an aliquot was analyzed by SDS-PAGE. (A) Coomassie-stained gel. (B) densitometric analysis of Coomassie-stained gel. Data represent the means þ S.D. (n = 3^5).
H2 O2 for 12 h at 37³C resulted in about 80% proteins remaining. The H2 O2 production, mainly via O3 2 , has been frequently observed in mitochondria and microsomes. There are also several enzymes that produce H2 O2 without the intermediacy of O3 2 . It has been reported that at least 0.1 mM H2 O2 can be produced continuously under physiological conditions and at a much higher rate under adverse conditions such as hyperoxia or ischemia and reperfusion [32]. Our results suggest that the fragmentation of Cu,Zn-SOD may occur at the physiological concentration of H2 O2 . During incubation of Cu,ZnSOD with H2 O2 , the enzyme activity gradually decreased and simultaneously OH formation increased (Fig. 3). The formation of OH in the Cu,Zn-SOD and H2 O2 system was paralleled with the loss of enzyme activity. The results suggest that the fragb
b
Fig. 1. Fragmentation of Cu,Zn-SOD after the incubation with H2 O2 . 25 WM Cu,Zn-SOD was incubated with 10 mM H2 O2 in 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6) at 37³C during various incubation periods. Reactions were stopped at the time indicated by adding catalase to a ¢nal concentration of 100 Wg/ml and an aliquot was analyzed by SDS-PAGE. (A) Coomassiestained gel. (B) Densitometric analysis of Coomassie-stained gel (A), performed data were expressed as a percentage of the relative optical density. Values in (B) represent the means þ S.D. (n = 3^5).
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Fig. 3. Inactivation of Cu,Zn-SOD during incubation with H2 O2 . After incubation for the appropriate time, an aliquot was taken and Cu,Zn-SOD activity (b) and the formation of OH (F) was determined as described under Section 2. Data represent the means þ S.D. (n = 3^5).
Fig. 4. E¡ect of radical scavengers on the fragmentation of Cu,Zn-SOD by H2 O2 . Cu,Zn-SOD was incubated with 10 mM H2 O2 in absence or presence of radical scavengers at 37³C for 3 h. Lane 1, Cu,Zn-SOD control; lane 2, incubated with H2 O2 ; lane 3, lane 2 plus 100 mM azide; lane 4, lane 2 plus 100 mM formate; lane 5, lane 2 plus 100 mM mannitol. Data shown are representative of three experiments.
b
mentation is related to the generation of OH in the Cu,Zn-SOD/H2 O2 system. The participation of OH in the fragmentation of Cu,Zn-SOD by H2 O2 was studied by examining the protective e¡ects of the hydroxyl radical scavengers such as azide, formate and mannitol. When Cu,ZnSOD was incubated with H2 O2 in the presence of various hydroxyl radical scavengers at 37³C for 3 h, all scavengers prevented the fragmentation of Cu,ZnSOD (Fig. 4). This suggests that OH may participate in the mechanism of the hydrogen peroxidemediated Cu,Zn-SOD fragmentation. Trace metals such as iron and copper, which are variously present in biological systems, may interact with active oxygen species, ionizing radiation or microwave radiation to damage macromolecules [25^ 28]. The cleavage of the metalloproteins by oxidative damage may lead to increases in the level of metal ions in some cells [29]. The release of copper ions from Cu,Zn-SOD could be induced by the free radical generating function of the enzyme [5]. It has been proposed that hydroxyl radicals were generated by Cu,Zn-SOD in two distinct stages. In the early stage, bound copper ion of the enzyme reacted with H2 O2 and produced the radical enzymatically (the free radical-generating activity). In this stage, two distinct fates for the hydroxyl radicals were described; ¢rst, the hydroxyl radicals may exit from the active site channel, leading to the oxidative damage of other molecules within the short range of hydroxyl radical b
b
b
di¡usion. Second, the hydroxyl radicals may react directly with Cu,Zn-SOD, irreversibly inactivating the enzyme and leading to copper ion release from the damaged enzyme. In the late stage, the Fentonlike reaction may occur non-enzymatically by the reaction of the released copper ions with H2 O2 . The participation of copper ions in the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation was investigated by an examination of the protective effects of the copper chelator DTPA. The inhibition of fragmentation by 10 mM DTPA (Fig. 5) indicates that copper ions are involved. 10 mM DTPA protected 50% of intact protein for 3 h as determined by densitometric scanning of staining intensity on SDSPAGE (data not shown). The released copper ions from the oxidatively damaged Cu,Zn-SOD can enhance the Fenton-like reaction to produce hydroxyl radicals which amplify damage to the protein. Taken
Fig. 5. E¡ect of DTPA on the fragmentation of Cu,Zn-SOD by H2 O2 . Cu,Zn-SOD was incubated with 10 mM H2 O2 in the presence of 10 mM DTPA at 37³C for various incubation periods.
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together these results suggest that the fragmentation of Cu,Zn-SOD by H2 O2 is due to the oxidative damage resulting from OH radicals generated by a combination of the free radical-generating function of Cu,Zn-SOD and the Fenton-like reaction of free copper ions released from oxidatively damaged SOD. Many functions have previously been proposed for carnosine; these include anti-oxidant and free radical scavenger, physiological bu¡er, neurotransmitter, radioprotectant, metal chelator and wound healing agent [30,31]. It has been shown that carnosine is an e¤cient singlet-oxygen scavenger, quenching singlet-oxygen more e¡ectively than histidine and that carnosine and anserine protect phages against Q-irradiation, which gives rise to oxidative DNA damage [16,17]. We investigated whether carnosine, homocarnosine and anserine could protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation. Carnosine, homocarnosine and anserine showed a concentration-dependent inhibition of the Cu,ZnSOD fragmentation induced by H2 O2 (Fig. 6). Three compounds also protected the loss of Cu,Zn-SOD activity (Fig. 7). These results indicate that carnosine, homocarnosine and anserine can prevent oxidative damage of Cu,Zn-SOD mediated by H2 O2 . The ability of carnosine, homocarnosine and anserine to inhibit the generation of OH in the Cu,Zn-SOD/H2 O2 system was examined. The generation of OH was measured by using the deoxyribose assay. The degradation of 2-deoxy-D-ribose in the Cu,Zn-SOD/ H2 O2 system was inhibited approximately 70, 60, 80% by 10 mM carnosine, 10 mM homocarnosine and 10 mM anserine, respectively (Table 1). These results suggests that carnosine and the related compounds can e¡ectively remove OH radicals generated in the Cu,Zn-SOD/H2 O2 system. One of the mechanisms by which anti-oxidants can protect their biological targets against oxidative stress is the chelation of transition metals such as copper and iron, preventing them from participating in the deleterious Fenton reaction. Carnosine and anserine have been shown to be very e¤cient copper chelating agents [14]. These compounds might be able to bind Cu2 and prevent some Cu2 -dependent radical reaction [10]. However, Aruoma et al. [18] reported that such a binding is insu¤ciently strong to prevent reactivity of Cu2 in the phenanthroline assay. They also demonstrated that carnosine, homob
b
b
b
Fig. 6. E¡ects of carnosine, homocarnosine and anserine on the fragmentation of Cu,Zn-SOD by H2 O2 . Control reaction mixture containing 25 WM Cu,Zn-SOD and 10 mM H2 O2 in 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6). Various concentrations of carnosine (A), homocarnosine (B) and anserine (C) were added into the control reaction mixture. Reaction mixtures were incubated at 37³C for 3 h and an aliquot was analyzed by SDSPAGE. Lane 1, Cu,Zn-SOD control ; lane 2, incubated with 10 mM H2 O2 ; lane 3, lane 2 plus 1 mM reagents; lane 4, lane 2 plus 5 mM reagents; lane 5, lane 2 plus 10 mM reagents; lane 6, lane 2 plus 20 mM reagents. Relative staining intensity of SDS-PAGE gels (A,B and C) was analyzed by densitometric scanning (D). Values in (D) represent the means þ S.D. (n = 3^ 5).
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therapeutic agents for the oxidative damage of protein mediated by active oxygen species. Acknowledgements Fig. 7. Protections of carnosine, homocarnosine and anserine on the loss of Cu,Zn-SOD activity during incubation with H2 O2 . Cu,Zn-SOD was incubated with 10 mM H2 O2 in the presence of reagents as described below at 37³C for 3 h and an aliquot was analyzed by activity staining as described under Section 2. Lane 1, Cu,Zn-SOD control; lane 2, incubated with 10 mM H2 O2 ; lane 3, lane 2 plus 20 mM carnosine; lane 4, lane 2 plus 20 mM homocarnosine; lane 5, lane 2 plus 20 mM anserine.
carnosine and anserine did not react with O3 or 2 with H2 O2 at signi¢cant rates [18]. In the present study, we detected that none of them could react copper ions, nor could remove O3 and H2 O2 2 (data not shown). Carnosine is active electrochemically as a reducing agent in cyclic voltammetric measurements, donating a hydrogen atom to the peroxyl radical [33]. It has been reported that carnosine and related compounds quench 50^95% of hydroxyl radicals produced in the Fenton reaction [34]. Therefore, it can be assumed that carnosine, homocarnosine and anserine may protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation through the scavenging of OH radicals rather than through chelation of copper ions. From the present results, we suggest that carnosine and related compounds may be explored as potential b
b
b
This work was supported by a grant from the Hallym Academy Sciences in 1999. References [1] P. Amstad, R. Moret, P. Cerutti, J. Biol. Chem. 269 (1994) 1606^1609. [2] O. Elory-Stein, Y. Bernstein, Y. Groner, EMBO J. 5 (1986) 615^622. [3] O. Elory-Stein, Y. Groner, Cell 52 (1988) 257^259. [4] I. Ceballos-Picot, A. Nichole, P. Briand, G. Grimber, A. Delacourte, A. Defossez, F. Javoy-Agid, M. Lafon, J.L. Blouin, P.M. Sinet, Brain Res. 552 (1991) 198^214. [5] M.B. Yim, P.B. Chock, E.R. Stadman, J. Biol. Chem. 268 (1993) 4099^4105. [6] S.M. Kim, J.H. Kang, Mol. Cell 7 (1997) 120^124. [7] J.H. Kang, S.M. Kim, Mol. Cell 7 (1997) 777^782. [8] G. Multhaup, A. Schlicksupp, L. Hesse, D. Beher, T. Ruppert, C. Masters, K. Beyreuther, Science 271 (1996) 1406^ 1409. [9] M.B. Yim, J.H. Kang, H.-S. Yim, H.-S. Kawk, P.B. Chock, E.R. Stadtman, Proc. Natl. Acad. Sci. USA 93 (1996) 5709^ 5714. [10] R. Kohn, Y. Yamamoto, K.C. Cundy, B.N. Ames, Proc. Natl. Acad. Sci. USA 85 (1988) 3175^3179. [11] J.J. O'Dowd, D.J. Robins, D.J. Miller, Biochem. Biophys. Acta 967 (1988) 241^249. [12] M.T. Cairns, D.J. Miller, J.J. O'Dowd, J. Physiol. (Lond.) 407 (1988) 51P.
Table 1 E¡ects of carnosine, homocarnosine and anserine on the formation of OH by the Cu,Zn-SOD and hydrogen peroxide system b
Additive None Carnosine Homocarnosine Anserine
Concentration
5 mM 10 mM 5 mM 10 mM 5 mM 10 mM
Deoxyribose (DR) degradation nmol MDA/mg DR
%
5.8 þ 0.48 3.0 þ 0.25 1.6 þ 0.33 4.4 þ 0.56 2.3 þ 0.43 3.5 þ 0.22 1.2 þ 0.21
100 60 28 76 40 52 21
The reaction mixture contained 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6), 10 mM 2-deoxy-D-ribose, 10 mM H2 O2 and 5 WM enzyme in a total volume of 100 Wl. The reaction was initiated by addition of H2 O2 and incubated without or with carnosine and related compounds for 1 h at 37³C. The control reaction was performed in the reaction mixture without the enzyme. Data represent means þ S.D. (n = 3^5)
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S.Y. Choi et al. / Biochimica et Biophysica Acta 1472 (1999) 651^657 [13] C.L. Davey, Arch. Biochem. Biophys. 89 (1960) 296^299. [14] C.E. Brown, J. Theor. Biol. 88 (1981) 296^299. [15] G. Gercken, H. Bischo¡, M. Trotz, Arzneim.forsch. 30 (1980) 2140^2143. [16] T.A. Dahl, W.R. Midden, P.E. Hartman, Photochem. Photobiol. 47 (1988) 357^362. [17] P.E. Hartman, K.T. Ault, Photochem. Photobiol. 51 (1990) 59^66. [18] O.I. Auroma, M.J. Laughton, B. Halliwell, Biochem. J. 264 (1989) 863^869. [19] J.H. Kang, B.J. Choi, S.M. Kim, J. Biochem. Mol. Biol. 30 (1997) 60^65. [20] U.K. Laemmli, Nature 27 (1970) 680^685. [21] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Hallia, F.-H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Anal. Biochem. 150 (1985) 76^85. [22] J.M. MacCord, I. Fridovich, J. Biol. Chem. 244 (1969) 6049^6055. [23] C. Beauchamp, I. Fridovich, Anal. Biochem. 44 (1971) 276^ 287.
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[24] B. Halliwell, J.M.C. Gutteridge, FEBS Lett. 128 (1981) 347^ 352. [25] S. Goldstein, G. Czapski, J. Am. Chem. Soc. 108 (1986) 2244^2250. [26] J.L. Sagripanti, M.L. Swicord, C.C. Davis, Radiat. Res. 110 (1987) 219^231. [27] J.A. Imlay, S.M. Chin, S. Linn, Science 240 (1988) 640^642. [28] J.L. Sagripanti, K.H. Kraemer, J. Biol. Chem. 264 (1989) 1729^1734. [29] M.J. O'Connell, T.J. Peters, Chem. Phys. Lipids 45 (1987) 24^249. [30] A.A. Boldyrev, V.E. Formazyuk, V.I. Sergienko, Sov. Sci. Rev. D. Physicochem. Biol. 13 (1994) 1^60. [31] P.J. Quinn, A.A. Boldyrev, V.E. Formazyuk, Mol. Asp. Med. 13 (1992) 379^444. [32] A. Boveries, N. Oshino, B. Chance, Biochem. J. 128 (1972) 617^630. [33] R. Kohen, Y. Yamamoto, K.C. Cundy, B.N. Ames, Proc. Natl. Acad. Sci. USA 85 (1988) 3175^3179. [34] W.K.M. Chan, E.A. Decker, J.B. Lee, D.A. Butter¢eld, J. Agric. Food Chem. 42 (1994) 1407^1410.
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Hydrogen peroxide-mediated Cu,Zn-superoxide dismutase fragmentation: protection by carnosine, homocarnosine and anserine Soo Young Choi a , Hyeok Yil Kwon b , Oh Bin Kwon c , Jung Hoon Kang
c;
*
a c
Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon 200-702, South Korea b Department of Physiology, College of Medicine, Hallym University, Chunchon 200-702, South Korea Department of Genetic Engineering, Division of Natural Sciences, Chongju University, Chongju 360-764, South Korea Received 12 May 1999; received in revised form 8 September 1999; accepted 20 September 1999
Abstract The fragmentation of human Cu,Zn-superoxide dismutase (SOD) was observed during incubation with H2 O2 . Hydroxyl radical scavengers such as sodium azide, formate and mannitol protected the fragmentation of Cu,Zn-SOD. These results suggested that OH was implicated in the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation. Carnosine, homocarnosine and anserine have been proposed to act as anti-oxidants in vivo. We investigated whether three compounds could protect the fragmentation of Cu,Zn-SOD induced by H2 O2 . The results showed that carnosine, homocarnosine and anserine significantly protected the fragmentation of Cu,Zn-SOD. All three compounds also protected the loss of enzyme activity induced by H2 O2 . Carnosine, homocarnosine and anserine effectively inhibited the formation of OH by the Cu,ZnSOD/H2 O2 system. These results suggest that carnosine and related compounds can protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation through the scavenging of OH. ß 1999 Elsevier Science B.V. All rights reserved. b
b
b
Keywords: Copper,zinc-superoxide dismutase; Fragmentation; Hydrogen peroxide; Carnosine
1. Introduction Cu,Zn-superoxide dismutase (SOD) is an antioxidant enzyme in tissues. However, the overexpression of Cu,Zn-SOD among others has been correlated to cell killing [1], increased lipid peroxidation [2], reduced transport of biogenic amines [3] and decomposition of brain neuron [4]. In addition to the usual superoxide dismutase activity, Cu,Zn-SOD has a free radical-generating function that utilizes its own product, H2 O2 as a substrate [5,6]. If OH radicals are generated by the Cu,Zn-SOD and H2 O2 system in b
* Corresponding author. Fax: +82-431-229-8432; E-mail: [email protected]
biological cells, it could react with its own molecule (SOD) and other molecules in the vicinity of its generation sites. Involvement of the free radical-generating function of Cu,Zn-SOD in human disease has been suggested [7^9]. Carnosine (L-alanyl-L-histidine) and related compounds such as anserine (L-alanyl-1-methyl-L-histidine) and homocarnosine (Q-amino-butyryl-L-histidine) are present in several mammalian tissues, including skeletal muscle and brain at high concentrations (up to 20 mM in humans) [10^13]. Carnosine has been postulated to act as a bu¡er to neutralize lactic acid produced in skeletal muscle that is undergoing anaerobic glycolysis [14]. Carnosine and anserine have been shown to be e¤cient copper-chelating agents and it has been suggested that they may play
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a role in copper metabolism in vivo [15]. Carnosine also protects rabbit heart from reperfusion injury after ischemia [16]. Many biochemical studies have suggested that carnosine possesses anti-oxidant and free radical scavenging functions which may partly explain its apparent homeostatic function [17,18]. In this study, we examined the oxidative damage of Cu,Zn-SOD caused by H2 O2 . Our results suggest that the fragmentation of Cu,Zn-SOD induced by H2 O2 is due to the oxidative damage resulting from OH radicals generated by a combination of the free radical-generating function of Cu,Zn-SOD and the Fenton-like reaction of free copper ions released from oxidatively damaged SOD. We also investigated the e¡ects of carnosine, homocarnosine and anserine on the fragmentation of Cu,Zn-SOD induced by H2 O2 . Our results show that three compounds can protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation through the scavenging of OH radicals. b
b
2. Materials and methods 2.1. Materials Carnosine, homocarnosine, anserine, 2-deoxy-D-ribose, diethylenetriaminepenta-acetic acid (DTPA), thiobarbituric acid and trichloroacetic acid were purchased from Sigma (St. Louis, MO, USA). Chelex 100 resin (sodium form) was obtained from BioRad Lab (Hercules, CA, USA). Recombinant human Cu,Zn-SOD was prepared as described previously [19]. 2.2. Analysis of Cu,Zn-SOD fragmentation by H2 O2 Cu,Zn-SOD (25 WM) was incubated with H2 O2 (10 mM) in 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6) at 37³C during various incubation periods. After stopping the reaction by adding catalase to a ¢nal concentration of 100 Wg/ml, aliquots were diluted 4U with concentrated sample bu¡er (0.25 mM TrisHCl, 8% SDS, 40% glycerol, 20% L-mercaptoethanol, 0.01% bromophenol blue) and were boiled at 100³C for 10 min before electrophoresis. An aliquot
of each sample was subjected to SDS-PAGE as described by Laemmli [20], using a slab gel (stacking gel, 2.5% acrylamide and a separating gel, 18% acrylamide). The gels were stained with 0.15% Coomassie brilliant blue R-250. The amount of protein was quantitated by the staining intensity as judged by densitometric scanning with the £ying spot scanner CS-9000 (Shimadzu, Kyoto, Japan). Protein concentration was determined by the BCA method [21]. 2.3. SOD activity assay The activity was measured by monitoring their capacities to inhibit the reduction of ferricytochrome C by xanthine/xanthine oxidase as described by McCord and Fridovich [22]. Activity staining of Cu,Zn-SOD was performed by electrophoresis in 10% native polyacrylamide gels and visualized as described by Beauchamp and Fridovich [23]. Brie£y, the gel was soaked in 2.45 mM nitroblue tetrazolium solution for 15 min, followed by 30 min in 28 mM N,N,NP,NP-tetramethylethylene diamine and 28 WM ribo£avin in 0.36 mM potassium phosphate bu¡er (pH 7.8). The gel was then exposed to a £uorescence light source until the bands showed maximum resolution. 2.4. Measurement of OH production b
Detection of OH formation was determined by measuring thiobarbituric acid reactive 2-deoxy-D-ribose oxidation products [24]. The assay mixture contained 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6), 10 mM 2-deoxy-D-ribose, 10 mM H2 O2 and 5 WM enzyme in a total volume of 100 Wl. The reaction was initiated by addition of H2 O2 and incubated for 1 h at 37³C. Reaction was stopped by addition of 2.8% trichloroacetic acid (200 Wl), PBS (200 Wl)and 1% thiobarbituric acid (200 Wl) and boiled at 100³C for 15 min. After the samples were cooled and centrifuged at 15 000 rpm for 10 min. Results were read by a UV/vis spectrophotometer (Shimadzu, UV1601) at 532 nm. All solutions used in the present experiments were treated with Chelex 100 and prepared by bubbling a 5% CO2 , 95% N2 gas mixture [5].
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3. Results and discussion Incubation of Cu,Zn-SOD with 10 mM H2 O2 for 0^180 min at 37³C resulted in a time-dependent decrease in the amount of intact enzyme. SDS-PAGE showed a gradual decrease in the intensity of the original band (Fig. 1A). The kinetics of the decrease of the intact Cu,Zn-SOD was assessed by densitometric scanning of staining intensity on SDS-PAGE (Fig. 1B). When Cu,Zn-SOD was incubated with various concentrations of H2 O2 , the frequency of the protein fragmentation increased in a hydrogen peroxide concentration-dependent manner (Fig. 2A and B). Incubation of Cu,Zn-SOD with 0.1 mM
Fig. 2. E¡ect of H2 O2 concentrations on the fragmentation of Cu,Zn-SOD. 25 WM Cu,Zn-SOD was incubated with various concentrations of H2 O2 in 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6) at 37³C for 12 h. Reactions were stopped by adding catalase to a ¢nal concentration of 100 Wg/ml and an aliquot was analyzed by SDS-PAGE. (A) Coomassie-stained gel. (B) densitometric analysis of Coomassie-stained gel. Data represent the means þ S.D. (n = 3^5).
H2 O2 for 12 h at 37³C resulted in about 80% proteins remaining. The H2 O2 production, mainly via O3 2 , has been frequently observed in mitochondria and microsomes. There are also several enzymes that produce H2 O2 without the intermediacy of O3 2 . It has been reported that at least 0.1 mM H2 O2 can be produced continuously under physiological conditions and at a much higher rate under adverse conditions such as hyperoxia or ischemia and reperfusion [32]. Our results suggest that the fragmentation of Cu,Zn-SOD may occur at the physiological concentration of H2 O2 . During incubation of Cu,ZnSOD with H2 O2 , the enzyme activity gradually decreased and simultaneously OH formation increased (Fig. 3). The formation of OH in the Cu,Zn-SOD and H2 O2 system was paralleled with the loss of enzyme activity. The results suggest that the fragb
b
Fig. 1. Fragmentation of Cu,Zn-SOD after the incubation with H2 O2 . 25 WM Cu,Zn-SOD was incubated with 10 mM H2 O2 in 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6) at 37³C during various incubation periods. Reactions were stopped at the time indicated by adding catalase to a ¢nal concentration of 100 Wg/ml and an aliquot was analyzed by SDS-PAGE. (A) Coomassiestained gel. (B) Densitometric analysis of Coomassie-stained gel (A), performed data were expressed as a percentage of the relative optical density. Values in (B) represent the means þ S.D. (n = 3^5).
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Fig. 3. Inactivation of Cu,Zn-SOD during incubation with H2 O2 . After incubation for the appropriate time, an aliquot was taken and Cu,Zn-SOD activity (b) and the formation of OH (F) was determined as described under Section 2. Data represent the means þ S.D. (n = 3^5).
Fig. 4. E¡ect of radical scavengers on the fragmentation of Cu,Zn-SOD by H2 O2 . Cu,Zn-SOD was incubated with 10 mM H2 O2 in absence or presence of radical scavengers at 37³C for 3 h. Lane 1, Cu,Zn-SOD control; lane 2, incubated with H2 O2 ; lane 3, lane 2 plus 100 mM azide; lane 4, lane 2 plus 100 mM formate; lane 5, lane 2 plus 100 mM mannitol. Data shown are representative of three experiments.
b
mentation is related to the generation of OH in the Cu,Zn-SOD/H2 O2 system. The participation of OH in the fragmentation of Cu,Zn-SOD by H2 O2 was studied by examining the protective e¡ects of the hydroxyl radical scavengers such as azide, formate and mannitol. When Cu,ZnSOD was incubated with H2 O2 in the presence of various hydroxyl radical scavengers at 37³C for 3 h, all scavengers prevented the fragmentation of Cu,ZnSOD (Fig. 4). This suggests that OH may participate in the mechanism of the hydrogen peroxidemediated Cu,Zn-SOD fragmentation. Trace metals such as iron and copper, which are variously present in biological systems, may interact with active oxygen species, ionizing radiation or microwave radiation to damage macromolecules [25^ 28]. The cleavage of the metalloproteins by oxidative damage may lead to increases in the level of metal ions in some cells [29]. The release of copper ions from Cu,Zn-SOD could be induced by the free radical generating function of the enzyme [5]. It has been proposed that hydroxyl radicals were generated by Cu,Zn-SOD in two distinct stages. In the early stage, bound copper ion of the enzyme reacted with H2 O2 and produced the radical enzymatically (the free radical-generating activity). In this stage, two distinct fates for the hydroxyl radicals were described; ¢rst, the hydroxyl radicals may exit from the active site channel, leading to the oxidative damage of other molecules within the short range of hydroxyl radical b
b
b
di¡usion. Second, the hydroxyl radicals may react directly with Cu,Zn-SOD, irreversibly inactivating the enzyme and leading to copper ion release from the damaged enzyme. In the late stage, the Fentonlike reaction may occur non-enzymatically by the reaction of the released copper ions with H2 O2 . The participation of copper ions in the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation was investigated by an examination of the protective effects of the copper chelator DTPA. The inhibition of fragmentation by 10 mM DTPA (Fig. 5) indicates that copper ions are involved. 10 mM DTPA protected 50% of intact protein for 3 h as determined by densitometric scanning of staining intensity on SDSPAGE (data not shown). The released copper ions from the oxidatively damaged Cu,Zn-SOD can enhance the Fenton-like reaction to produce hydroxyl radicals which amplify damage to the protein. Taken
Fig. 5. E¡ect of DTPA on the fragmentation of Cu,Zn-SOD by H2 O2 . Cu,Zn-SOD was incubated with 10 mM H2 O2 in the presence of 10 mM DTPA at 37³C for various incubation periods.
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together these results suggest that the fragmentation of Cu,Zn-SOD by H2 O2 is due to the oxidative damage resulting from OH radicals generated by a combination of the free radical-generating function of Cu,Zn-SOD and the Fenton-like reaction of free copper ions released from oxidatively damaged SOD. Many functions have previously been proposed for carnosine; these include anti-oxidant and free radical scavenger, physiological bu¡er, neurotransmitter, radioprotectant, metal chelator and wound healing agent [30,31]. It has been shown that carnosine is an e¤cient singlet-oxygen scavenger, quenching singlet-oxygen more e¡ectively than histidine and that carnosine and anserine protect phages against Q-irradiation, which gives rise to oxidative DNA damage [16,17]. We investigated whether carnosine, homocarnosine and anserine could protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation. Carnosine, homocarnosine and anserine showed a concentration-dependent inhibition of the Cu,ZnSOD fragmentation induced by H2 O2 (Fig. 6). Three compounds also protected the loss of Cu,Zn-SOD activity (Fig. 7). These results indicate that carnosine, homocarnosine and anserine can prevent oxidative damage of Cu,Zn-SOD mediated by H2 O2 . The ability of carnosine, homocarnosine and anserine to inhibit the generation of OH in the Cu,Zn-SOD/H2 O2 system was examined. The generation of OH was measured by using the deoxyribose assay. The degradation of 2-deoxy-D-ribose in the Cu,Zn-SOD/ H2 O2 system was inhibited approximately 70, 60, 80% by 10 mM carnosine, 10 mM homocarnosine and 10 mM anserine, respectively (Table 1). These results suggests that carnosine and the related compounds can e¡ectively remove OH radicals generated in the Cu,Zn-SOD/H2 O2 system. One of the mechanisms by which anti-oxidants can protect their biological targets against oxidative stress is the chelation of transition metals such as copper and iron, preventing them from participating in the deleterious Fenton reaction. Carnosine and anserine have been shown to be very e¤cient copper chelating agents [14]. These compounds might be able to bind Cu2 and prevent some Cu2 -dependent radical reaction [10]. However, Aruoma et al. [18] reported that such a binding is insu¤ciently strong to prevent reactivity of Cu2 in the phenanthroline assay. They also demonstrated that carnosine, homob
b
b
b
Fig. 6. E¡ects of carnosine, homocarnosine and anserine on the fragmentation of Cu,Zn-SOD by H2 O2 . Control reaction mixture containing 25 WM Cu,Zn-SOD and 10 mM H2 O2 in 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6). Various concentrations of carnosine (A), homocarnosine (B) and anserine (C) were added into the control reaction mixture. Reaction mixtures were incubated at 37³C for 3 h and an aliquot was analyzed by SDSPAGE. Lane 1, Cu,Zn-SOD control ; lane 2, incubated with 10 mM H2 O2 ; lane 3, lane 2 plus 1 mM reagents; lane 4, lane 2 plus 5 mM reagents; lane 5, lane 2 plus 10 mM reagents; lane 6, lane 2 plus 20 mM reagents. Relative staining intensity of SDS-PAGE gels (A,B and C) was analyzed by densitometric scanning (D). Values in (D) represent the means þ S.D. (n = 3^ 5).
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therapeutic agents for the oxidative damage of protein mediated by active oxygen species. Acknowledgements Fig. 7. Protections of carnosine, homocarnosine and anserine on the loss of Cu,Zn-SOD activity during incubation with H2 O2 . Cu,Zn-SOD was incubated with 10 mM H2 O2 in the presence of reagents as described below at 37³C for 3 h and an aliquot was analyzed by activity staining as described under Section 2. Lane 1, Cu,Zn-SOD control; lane 2, incubated with 10 mM H2 O2 ; lane 3, lane 2 plus 20 mM carnosine; lane 4, lane 2 plus 20 mM homocarnosine; lane 5, lane 2 plus 20 mM anserine.
carnosine and anserine did not react with O3 or 2 with H2 O2 at signi¢cant rates [18]. In the present study, we detected that none of them could react copper ions, nor could remove O3 and H2 O2 2 (data not shown). Carnosine is active electrochemically as a reducing agent in cyclic voltammetric measurements, donating a hydrogen atom to the peroxyl radical [33]. It has been reported that carnosine and related compounds quench 50^95% of hydroxyl radicals produced in the Fenton reaction [34]. Therefore, it can be assumed that carnosine, homocarnosine and anserine may protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation through the scavenging of OH radicals rather than through chelation of copper ions. From the present results, we suggest that carnosine and related compounds may be explored as potential b
b
b
This work was supported by a grant from the Hallym Academy Sciences in 1999. References [1] P. Amstad, R. Moret, P. Cerutti, J. Biol. Chem. 269 (1994) 1606^1609. [2] O. Elory-Stein, Y. Bernstein, Y. Groner, EMBO J. 5 (1986) 615^622. [3] O. Elory-Stein, Y. Groner, Cell 52 (1988) 257^259. [4] I. Ceballos-Picot, A. Nichole, P. Briand, G. Grimber, A. Delacourte, A. Defossez, F. Javoy-Agid, M. Lafon, J.L. Blouin, P.M. Sinet, Brain Res. 552 (1991) 198^214. [5] M.B. Yim, P.B. Chock, E.R. Stadman, J. Biol. Chem. 268 (1993) 4099^4105. [6] S.M. Kim, J.H. Kang, Mol. Cell 7 (1997) 120^124. [7] J.H. Kang, S.M. Kim, Mol. Cell 7 (1997) 777^782. [8] G. Multhaup, A. Schlicksupp, L. Hesse, D. Beher, T. Ruppert, C. Masters, K. Beyreuther, Science 271 (1996) 1406^ 1409. [9] M.B. Yim, J.H. Kang, H.-S. Yim, H.-S. Kawk, P.B. Chock, E.R. Stadtman, Proc. Natl. Acad. Sci. USA 93 (1996) 5709^ 5714. [10] R. Kohn, Y. Yamamoto, K.C. Cundy, B.N. Ames, Proc. Natl. Acad. Sci. USA 85 (1988) 3175^3179. [11] J.J. O'Dowd, D.J. Robins, D.J. Miller, Biochem. Biophys. Acta 967 (1988) 241^249. [12] M.T. Cairns, D.J. Miller, J.J. O'Dowd, J. Physiol. (Lond.) 407 (1988) 51P.
Table 1 E¡ects of carnosine, homocarnosine and anserine on the formation of OH by the Cu,Zn-SOD and hydrogen peroxide system b
Additive None Carnosine Homocarnosine Anserine
Concentration
5 mM 10 mM 5 mM 10 mM 5 mM 10 mM
Deoxyribose (DR) degradation nmol MDA/mg DR
%
5.8 þ 0.48 3.0 þ 0.25 1.6 þ 0.33 4.4 þ 0.56 2.3 þ 0.43 3.5 þ 0.22 1.2 þ 0.21
100 60 28 76 40 52 21
The reaction mixture contained 23.5 mM NaHCO3 /CO2 bu¡er (pH 7.6), 10 mM 2-deoxy-D-ribose, 10 mM H2 O2 and 5 WM enzyme in a total volume of 100 Wl. The reaction was initiated by addition of H2 O2 and incubated without or with carnosine and related compounds for 1 h at 37³C. The control reaction was performed in the reaction mixture without the enzyme. Data represent means þ S.D. (n = 3^5)
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S.Y. Choi et al. / Biochimica et Biophysica Acta 1472 (1999) 651^657 [13] C.L. Davey, Arch. Biochem. Biophys. 89 (1960) 296^299. [14] C.E. Brown, J. Theor. Biol. 88 (1981) 296^299. [15] G. Gercken, H. Bischo¡, M. Trotz, Arzneim.forsch. 30 (1980) 2140^2143. [16] T.A. Dahl, W.R. Midden, P.E. Hartman, Photochem. Photobiol. 47 (1988) 357^362. [17] P.E. Hartman, K.T. Ault, Photochem. Photobiol. 51 (1990) 59^66. [18] O.I. Auroma, M.J. Laughton, B. Halliwell, Biochem. J. 264 (1989) 863^869. [19] J.H. Kang, B.J. Choi, S.M. Kim, J. Biochem. Mol. Biol. 30 (1997) 60^65. [20] U.K. Laemmli, Nature 27 (1970) 680^685. [21] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Hallia, F.-H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Anal. Biochem. 150 (1985) 76^85. [22] J.M. MacCord, I. Fridovich, J. Biol. Chem. 244 (1969) 6049^6055. [23] C. Beauchamp, I. Fridovich, Anal. Biochem. 44 (1971) 276^ 287.
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