- Email: [email protected]
Glutathione-Linked Thiol Peroxidase Activity of Human Serum Albumin: A Possible Antioxidant Role of Serum Albumin in Blood Plasma
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
222, 619–625 (1996)
0793
Glutathione-Linked Thiol Peroxidase Activity of Human Serum Albumin: A Possible Antioxidant Role of Serum Albumin in Blood Plasma Mee-Kyung Cha and Il-Han Kim1 Department of Biochemistry, Pai-Chai University, Taejon 302-735, Korea Received April 1, 1996 A 65-kDa molecular mass of thiol-specific antioxidant protein was purified from human plasma and identified as human serum albumin (HSA) by the analysis of amino-terminal amino acid sequence. This protein exhibited the preventive effects against the inactivation of glutamine synthetase activity and the peroxidation of lipid by a metal-catalyzed oxidation system. These antioxidant activities were supported by a thiol-reducing equivalent such as DTT and reduced glutathione. The thiol-specific antioxidant activity of HSA was greatly activated by halide ion, especially by chloride ion. HSA showed a significant capability to destroy H2O2 in the presence of reduced glutathione, resulting in the production of oxidized glutathione. Both the preventive activity against the glutamine synthetase inactivation and the peroxidase activity were completely abolished by the reactions of HSA with N-ethylmaleimide and iodoacetate, chemical modification agents for sulfhydryl of protein, only in the presence of thiol-reducing equivalent such as DTT. These results suggest that serum albumin acts as a major and predominate antioxidant exerting a glutathione-linked thiol peroxidase activity which removes reactive oxygen species such as H2O2 within blood plasma. © 1996 Academic Press, Inc.
It is increasingly recognized that reactive oxygen species play a significant role in the pathogenesis of certain human disease such as atherosclerosis, cancer, and rheumatoid arthritis (1–3). Blood plasma is exposured to reactive oxygen species which are generated from activated phagocytes and the reactions of oxygen with a great variety of compounds derived from cellular metabolism, dietay uptake, and xenobiotics. As results of this oxidative stress to human blood there is a growing effort to find out antioxidant enzymes such as glutathione peroxidase, superoxide dismutase, and catalase in blood plasma (4–7). Recently, a novel type of antioxidant enzyme was found in from prokaryotes (8) and to eukaryotes (9–11). These intracellular antioxidant proteins exerted the preventive activity against glutamine synthetase inactivation by a metal-catalyzed oxidation system and the peroxidase activity. These activities were supported by thioredoxin. This antioxidant protein was, thus, named “thioredoxin peroxidase” (12–14). We reported the another type of a peroxidase, which exists in the periplasmic spaces of E. coli (15). This type of peroxidase was proved to have a functional cysteine as a nucleophile in the catalytic reaction, thus named “thiol peroxidase”. Thiol peroxidase shares a similar catalytic activity to that of thioredoxin peroxidase in that thiol peroxidase activity was also linked to thioredoxin. However, the deduced amino acid sequence of thiol peroxidase is completely different from that of thioredoxin peroxidase, suggesting the possibility that various types of peroxidase family having functional cysteine remain to be undiscovered. It is generally believed that blood plasma is exposed to more severe oxidative stress than intracellular fluids, but the level of antioxidant enzymes in blood plasma is much lower than the intracellular level (7). The sulfhydryl groups of serum proteins including serum albumin have been suggested to be a “sacrificial” antioxidant in plasma and the extravascular space (16). However, 1
Corresponding author: Department of Biochemistry, Pai-Chai University, Doma-2-Dong 439-6, Seo-Gu, Taejon 302735, Korea: Fax: 82-42-520-5379. The abbreviations used are: DTT, dithiothreitol; TSA, thiol-specific antioxidant: MCO, metal-catalyzed oxidation; GSH, reduced form of glutathione; GSSG, oxidized form of GSH; HSA, human serum albumin; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis. 619 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
none of serum proteins showing a enzymatic mechanism to remove reactive oxygen species such as H2O2 has not been reported. In this study, we purified a 65-kDa protein showing an enzymatic antioxidant activity (i.e., thiol specific antioxidant activity and thiol peroxidase activity) from human plasma, and identified as a human serum albumin (HSA). Our results suggest that HSA as a thiol peroxidase could be a major antioxidant enzyme of extracellular fluids such as human blood plasma. MATERIALS AND METHODS Purification of 65-kDa protein showing thiol-specific antioxidant activity from Human blood plasma. Human blood plasmas were obtained from freshly drawn heparinized blood. Human plasma proteins were subjected to ammonium sulfate precipitation (70%). The precipitate was dissolved and extensively dialyzed against DEAE column equilibrium buffer (50 mM Tris-HCl buffer, pH 7.6). After centrifugation, the clear supernatant was loaded into a DEAE column. The column was washed with equilibrium buffer and eluted with a linear KCl gradient (0–400 mM). The presence of thiol-specific antioxidant (TSA) activity was assayed throughout the purification by monitoring its ability to prevent the inactivation of glutamine synthetase by the thiol-MCO system. The peak fractions of TSA activity eluted at a KCl concentration of 250 mM were pooled and precipitated with 70% ammonium sulfate. The dissolved ammonium sulfate-precipitates were applied to a G-75 gel permeation column previously equilibrated with 100 mM Hepes buffer, pH 7.4, containing 100 mM KCl. The ammonium sulfate precipitate of the fractions exerting was dissolved in 100 mM Hepes-NaOH buffer, pH 7.4, containing 1.0 M ammonium sulfate, and applied to phenyl sepharose CL-4B column previously equilibrated with 100 mM HepesNaOH buffer (pH 7.4) containing 1.0 M ammonium sulfate. Proteins were eluted with a linear gradient from 1.0 M ammonium sulfate to the buffer containing no salt. The TSA activity was eluted between 0.7 and 0.5 M of the salt. The fractions showing TSA activities were precipitated, dissolved, and applied to G-100 gel permeation column. The major peaks for TSA activity gave a 65-kDa of homogeneous protein. Determination of thiol-specific antioxidant (TSA) activity. TSA activity of 65-kDa protein was determined by monitoring its activity to inhibit the inactivation of E. coli glutamine synthetase by a metal catalyzed thiol MCO system (DTT, Fe3+, O2) (17) as described by Kim et al. (9). Instead of DTT ascorbate was included as a non-thiol reducing equivalent (non-thiol MCO system). Fifty ml of reaction mixture containing 5 mg of glutamine synthetase, 3 mM FeCl3, 10 mM DTT or ascorbate, antioxidant enzyme, 100 mM NaCl, and 100 mM Hepes-NaOH (pH 7.0) was incubated at 37°C. The remaining activity of glutamine synthetase was measured by addition of 5 ml of the reaction mixture to 2 ml of g-glutamyltransferase assay mixture as described (18). Determination of peroxidase activity of the 65-kDa protein in the presence of GSH. To determine the peroxidase activity of the enzymes, the reaction was started by the addition of 0.5 mM of H2O2 into the 50 ml of reaction mixture containing 5 mM GSH, 100 mM NaCl, and 50 mM Hepes-NaOH, pH 7.0, and then incubated at 37°C. At appropriate reaction time, 20 ml of the reaction mixture was added to 0.8 ml of TCA solution (12.5%, w/v) to stop the reaction, followed by the addition of 0.2 ml of 10 mM Fe(NH4)2(SO4)2 and 0.1 ml of 2.5 N KSCN to develop the complex, giving a purple color. The concentration of H2O2 was monitored by measurement of the decrease in absorbance at 480 nm. The rate of production of GSSG by HSA was measured in the presence of excess glutathione reductase (0.5 unit) by following the rate of NADPH oxidation (19). Determination of lipid peroxidation. The peroxidation of RBC (red blood cell)-membrane lipids by a thiol-MCO system were determined by measurement of TBAR (thiobarbituric acid-reactive products). In the presence of the thiol-MCO system, one ml of reaction mixture containing 0.6 M of membrane lipid in term of inorganic phosphate and 100 mM NaCl was incubated for one hour at 37°C. Whole reaction mixture was boiled for 20 min with thiobarbituric acid and trichloroacetic acid, in the presence of 0.01% butylated hydroxyltoluene, as described by Buege and Aust (20). After centrifugation at 2,000 × g, the malonyldialdehyde content of supernatant was then determined by their absorbance at 535 nm. Protein concentration was determined by using the Bio-Rad Protein Assay kit based on the method of Bradford (21). SDS-PAGE was performed by the method of Laemmli (22). Chemical modification of sulfhydryl group of the 65-kDa protein. The 65-kDa protein was preincubated in the presence of 2 mM DTT and 100 mM NaCl for 20 min at 30°C, and then reacted with N-ethylmaleimide (10 mM) and iodoacetate (10 mM) at 30°C for 2 hrs. The unreacted reagent was washed out using Centricon 30 (Amicon). Free sulfhydryl group of the protein was determined by the measurement of the release of 2-nitro-5-mercaptobenzoic acid at 412 nm during the reaction with sulfhydryl group in protein (100 mM) with 5,5-dithiobis-(2-nitrobenzoic), DTNB, (1 mM) (23).
RESULTS AND DISCUSSION Purification of a thiol-specific antioxidant protein from human plasma and its identification as human serum albumin. We purified a thiol-specific antioxidant (TSA) protein from human blood plasma. This antioxidant protein was purified to homogeneity by four sequential chromatographic steps on DEAE, G-75, phenyl Sepharose CL-4B, and G-100 (Fig. 1). The molecular mass of this 620
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. SDS–PAGE analysis of 65-kDa thiol-specific antioxidant protein at different stages of purification. Protein sample from different stages of purifications were electrophoresed in 8% SDS-PAGE gel with b-mercaptoethanol. Lane 1 contains 50-mg sample from plasma. Lane 2, 30-mg sample from DEAE chromatography. Lane 3, 20-mg sample from G-75 gel permeation chromatography. Lane 4, 10-mg sample from phenyl Sepharose CL-4B chromatography. Lane 5, 2-mg sample from G-100 gel permeation chromatography. Lane 7, the nonreducing electrophoresis of the 2-mg of sample from the G-100 chromatography. Lane 6, size markers. The molecular masses, from the bottom, are 14.4, 21.5, 31, 45, 66.2, and 97.4 kDa.
protein was determined to be 65-kDa on reducing SDS-PAGE (8%) with b-mercaptoethanol. The protein band migrated faster on the non-reducing gel, indicating that the protein has intradisulfide linkage(s) (lane 6, 7 of Fig. 1). The purity of 65-kDa TSA protein was confirmed by obtaining the first peak corresponding to Asp in the N-terminal amino acid analysis as single peak (data not shown). The analysis of N-terminal amino acid sequence of 65-kDa TSA protein (18 residues) was determined as that of HSA (DAHKSEVAHRFKDLGEEN). Fig. 2 shows the thiol-specific antioxidant activity of HSA. HSA exerted the preventive activity against the inactivation of glutamine synthetase by thiol MCO system, but did not by non-thiol MCO system (ascorbate MCO system) in which DTT was replaced by ascorbate. Requirement for a thiol-reducing equivalent in the antioxidant activity of HSA suggests that the HSA could function as an extracellular antioxidant enzyme having functional cysteine (e.g., thiol peroxidase). Activation of TSA activity of HSA by chloride ion. We found that the TSA activity was increased
FIG. 2. Protection of HSA against the inactivation of glutamine synthetase (GS) caused by MCO system. The inactivation mixture contained 5 mg of E. coli glutamine synthetase, 100 mM NaCl, 10 mM DTT for a thiol MCO system or 10 mM ascorbate for a non-thiol MCO system, 3 mM FeCl3, 50 mM Hepes, pH 7.0, in 50 ml of reaction volume. At indicated times, aliquots (5 ml) were removed and assayed 10 min at 37°C for remaining glutamine synthetase activity. Each inactivation-reaction mixture contained as follows: curve 1, the thiol MCO system plus 2 mg/ml HSA; curve 2, the non-thiol MCO system plus 2 mg/ml of HSA; curve 3, the non-thiol MCO system; curve 4, the thiol MCO system; curve 4, the thiol MCO system. 621
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by KCl. To determine which ion have a capability to activate the activity, we examined the effects of various salts consisting monovalent cation and halide ion. All salts containing chloride anion activated the activity regardless of the counterpart cations (lane 1–9, Fig. 3-A), but the salts lacking chloride anion such as sodium acetate and potassium acetate did not elevate the activity (lane 7,8 of Fig. 3-A). The extent of the activation by the various halide anions from fluoride to iodide (lane 9–12 of Fig. 3-A) revealed that chloride ion was the most effective halide ion to activate the TSA activity of HSA. Fig. 3-B showed the TSA activity as the function of concentration of chloride ion, indicating 100 mM of chloride ion is necessary for the full activation of the enzyme. The TSA activity of HSA was about five-fold increased by chloride ion with comparison to that in the absence of the ion (Fig. 3-C). The effect of chloride ion on the conformation of serum albumin was well known. Zurawski and Foster (24) showed evidence for the existence of two distinguishable conformations of serum albumin, so called N and B forms (25). In addition, the conformation of HSA has been reported to be influenced by physiologically important chloride ion (26). Thus, the activation of TSA activity of HSA by chloride ion can be interpreted in term of an effect of chloride ion on the conformational change of the protein. In vivo thiol equivalent linked to HSA. We searched for an enzyme or in vivo thiol reducing equivalent capable of supporting the TSA activity of HSA. An enzymatic thiol regenerating system (thioredoxin / thioredoxin reductase / NADPH system) did not provide HSA with the capability to protect the inactivation of glutamine synthetase by a non-thiol MCO system in which DTT was replaced by ascorbate (data not shown). Glutamine synthetase was also inactivated by a thiol MCO system (GSH MCO system) in which DTT was replaced by GSH, known as an in vivo thiol reducing equivalent (curve 2 of Fig. 4-A). This inactivation was completely protected by HSA (curve 1 of Fig. 4-A). The lipid peroxidation was caused by two GSH MCO systems (GSH + Fe3+ + O2; GSH + hemoglobin + O2). Fig. 4-B shows the increase of protection against lipid peroxidation as a function of HSA concentration. The capabilities of HSA preventing these oxidations by the GSH MCO system capable of generating reactive oxygen species may reveal the in vivo antioxidant activity of HSA in human blood plasma containing GSH. The concentration of GSH in whole blood was reported to be 1.7 mM (27). A GSH-linked peroxidase activity of HSA. We examined HSA for peroxidase activity by measuring the decrease of H2O2 in the presence of GSH. The time-coursed removal of H2O2 showed a first-order kinetics. The velocity of the removal of H2O2 increased as a function of HSA concentration, showing a saturation pattern (Fig. 5-A). The activity for removal of H2O2 by HSA
FIG. 3. Activation of thiol-specific antioxidant activity of HSA by chloride ion. (A) The effect of various salts. The inactivation mixture contained 10 mM DTT, 3 mM of FeCl3, 5 mg of glutamine synthetase (GS), 1 mg of HSA, and 100 mM of various salts as follow: lane 1–12; no salt, LiCl, NaCl, KCl, RbCl, CsCl, sodium acetate, potassium acetate, NaF, NaCl, NaBr, and Nal, respectively. (B) The activation of HSA as a function of concentration of NaCl. The inactivation mixture contained 5 mg of E. coli glutamine synthetase, 1 mg/ml HSA, 10 mM DTT, 3 mM FeCl3, and 50 mM Hepes, pH 7.0. (C) The thiol-specific antioxidant activity as a function of the concentration of HSA. The inactivation mixture contained 5 mg of E. coli glutamine synthetase, 10 mM DTT, 3 mM FeCl3, and 50 mM Hepes, pH 7.0, and 100 mM NaCl for curve 1 or without NaCl for curve 2. After 30 min inactivation, 5 ml of aliquot was removed and assayed for the remaining glutamine synthetase activity. 622
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Protections of HSA against the inactivation of glutamine synthetase (GS) (A) and the lipid peroxidation (B) caused by thiol MCO system in which DTT was replaced by GSH. The GS-inactivation mixture contained 5 mg of E. coli glutamine synthetase, 20 mM GSH, 3 mM FeCl3, 100 mM NaCl, 50 mM Hepes, pH 7.0, and 1 mg/ml HSA for curve 1, or without HSA for curve 2. The lipid-peroxidation mixture contained 0.6 M of red blood cell membrane in term of phospholipid, 20 mM GSH, the indicated concentration of HSA, and 400 mM of FeCl3 for curve 1 or 2 mg/ml of hemoglobin as an iron source for curve 2. After one hour incubation of one ml of the reaction mixture at 37°C, malonyldialdehyde was determined by measuring the absorbance at 535 nm.
in the presence of glutathione reductase and NADPH can be measured indirectly by following the decrease in A340 resulted from the oxidation of NADPH by the glutathione reductase reaction (i.e., NADPH + GSSG → NADP+ + GSH) (Fig. 5-B). The continuous oxidation of NADPH to NADP+ in this system indicates the production of GSSG from GSH by the reaction of HSA (i.e., H2O2 + GSH → H2O + GSSG). These results suggest a peroxidase activity of HSA supported by GSH. Functional group of HSA. HSA consists of 585 amino acids. Its amino-acid sequence contains a total of 17 disulfide linkages and one free thiol (Cys-34) (28). When a thiol reducing equivalent was replaced with another non-thiol electron donor such as ascorbate, HSA no longer protected the inactivation of glutamine synthetase by the ascorbate MCO system (Fig. 1). The addition of DTT to the ascorbate MCO system significantly restored the antioxidant activity of HSA (data not shown). These results support a catalytic cycle of functional cysteine of HSA as shown in Fig. 7. To examine this, HSA was reacted with N-ethylmaleimide and iodoacetate, cysteine-specific modification reagents, with or without DTT. HSA was preincubated with 2 mM DTT and then reacted with 10 mM N-ethylmaleimide or iodoacetate. The resulting HSAs did not prevent the
FIG. 5. Glutathione (GSH)-linked peroxidase activity of HSA. (A) Removal of H2O2 by HSA supported by GSH. Peroxidase reaction was carried out in a 50-ml reaction mixture containing 0.5 mM H2O2, 5 mM GSH, 50 mM Hepes, pH 7.0, and 0, 15 mM (1 mg/ml), 30 mM, and 45 mM of HSA for curves 1, 2, 3, and 4, respectively. At indicated time, 20-ml aliquot was removed and assayed for remaining H2O2 according to the method described under Materials and Method. (B) The production of oxidized glutathione (GSSG) by a peroxidase activity of HSA supported by GSH. Assay mixture for GSSG contained 2 mM GSH, 0.5 mM H2O2, 0.5 unit (mmole/min) glutathione reductase, 2 mM NADPH and 15 mM (1 mg/ml) HSA for curve 2 or without HSA for curve 1. The reaction mixture was preincubated in the absence of H2O2 for 5 min. and then 0.5 mM of H2O2 was added into the mixture to start the peroxidation reaction of HSA. The decrease of NADPH was spectrophotometrically monitored at 340 nm. 623
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 6. Inactivation of HSA by N-ethylmaleimide (NEM). Inactivation of thiol-specific antioxidant activity of HSA by NEM was measured in both terms of the protection activity against the inactivation of glutamine synthetase caused by DTT MCO system (A) and the H2O2-removal activity supported by GSH (B). Curves A-1 and B-4, the control HSA without the inactivation by NEM. Curves A-2 and B-3, the HSA which was reacted with NEM in the absence of DTT. Curves A-3 and B-2, the HSA which was reacted with NEM in the presence of DTT. Details are given under Materials and Methods.
inactivation of glutamine synthease and did not exert a GSH-linked peroxidase. However, these antioxidant activities of HSA were not inhibited by these reagents in the absence of DTT (Fig. 6-A, B) (data for iodoacetate not shown). To exclude a possible pathway | denoted in Fig. 7, we modified the thiol of Cys-34, the single free thiol of HSA (28), with 5,5-dithiobis-(2-nitrobenzoic) in the absence of DTT. The chemical modification of the sulfhydryl group of Cys-34 in HSA was confirmed by observing the release of 2-nitro-5-mercaptobenzoic acid at 412 nm (data not shown). This modification did not affect the antioxidant activity of HSA (data not shown), removing the possibility of pathway I. These results indicate that a peroxidase activity of HSA should be resulted from the catalytic cycle (pathway II) shown in Fig. 7, which shares the similar catalytic cycle of glutathione peroxidase (29). Human blood plasma contains a variety of antioxidant biomolecules that can potentially protect circulating lipids, proteins and cells as well as the vasculature-lining endothelial cells from oxidative damage caused by activated oxygen species. Enzymatic antioxidants are present only in small concentrations in plasma (7) and their physiological function remains unclear. For this reason, we searched for a new type of antioxidant enzyme in human blood plasma and purified a 65-kDa protein exerting TSA activity. This TSA protein was proved to be HSA. HSA as the major soluble protein consituents of human blood plasma has many physiological functions such as the regulation of blood pressure and the transport of many endogenous and exogenous ligands. In addition to blood plasma, serum albumin was also found in tissue and bodily secretions throughout the body. The extravascular protein comprises 60% of the total albumin. In this paper, we suggest the potential importance of HSA as a extracellular enzymatic antioxidant, named “GSH-linked thiol peroxidase”, not as a “sacrificial” antioxidant (16) to protect oxidative damages associated with
FIG. 7. Mechanism for the reduction of H2O2 by HSA with glutathione (GSH) as an in vivo electron donor. 624
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
oxidative stress. Further study will be required to define which sulfhydryl group(s) from 17 disulfide linkages in HSA acts as a functional nucleophile in the peroxidation reaction. ACKNOWLEDGMENTS This work was supported by grants from BSRI-96-3434 and Pai-Chai University (1996). We are grateful to Dr. KyungHoon Suh for critical reading of this manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
Sies, H. (Ed.) (1985) Oxidative Stress, Academic Press, New York London. Halliwell, B., and Gutteridge, J. M. C. (1985) Free Radicals in Biology and Medicine, Oxford Univ. Press, London. Halliwell, B., and Gutteridge, J. M. C. (1986) Arch. Biochem. Biophys. 246, 501–504. Karlsson, K., and Marklund, S. L. (1987) Biochem. J. 242, 55–59. Marklund, S. L. (1987) Proc. Natl. Acad. Sci. USA 84, 6634–6638. Frei, B., Yamamoto, Y., Niclas, D., and Ames, B. N. (1988) Anal. Biochem. 175, 120–130. Lentner, C. (Ed.) (1984) Geigy Scientific Tables, Vol. 3, 8th revised and enlarged edition, Ciba-Geigy, Basel. Jacobson, F. S., Morgan, R., Christman, M. F., and Ames, B. N. (1989) J. Biol. Chem. 264, 1488–1496. Kim, K., Kim, I. H., Lee, K. Y., Rhee, S. G., and Stadtman, E. R. (1988) J. Biol. Chem. 263, 4704–4711. Lim, Y. S., Cha, M. K., Kim, H. K., and Kim, I. H. (1994) Gene (Amst.) 140, 279–284. Lim, Y. S., Cha, M. K., Yun, C. H., Kim, H. K., and Kim, I. H. (1994) Biochem. Biophys. Res. Commun. 199, 199–206. Lim, Y. S., Cha, M. K., Kim, H. K., Uhm, T. B., Park, J. W., Kim, K., and Kim, I. H. (1993) Biochem. Biophys. Res. Commun. 192, 273–280. Kwon, S. J., Park, J. W., Choi, W. K., Kim, I. H., and Kim, K. (1994) Biochem. Biophys. Res. Commun. 201, 8–15. Chae, H. Z., and Rhee, S. G. (1994) Biofactors 4, 177–180. Cha, M. K., and Kim, I. H. (1995) J. Biol. Chem. 270, 28635–28641. Halliwell, B. (1988) Biochem. Pharmacol. 37, 569–571. Stadtman, E. R., and Oliver, C. N. (1991) J. Biol. Chem. 266, 2005–2008. Rhee, S. G., Chock, P. B., and Stadtman, E. R. (1985) Methods Enzymol. 113, 213–241. Tappel, A. L. (1978) Methods Enzymol. 52, 506–513. Buege, J. A., and Aust, A. D. (1978) Methods Enzymol. 52, 302–310. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254. Laemmli, U. K. (1970) Nature 227, 680–685. Ogilvie, J. W. (1980) Biochim. Biophys. Acta 622, 277–284. Zurawski, V. R. Jr. and Foster, J. F. (1974) Biochemistry 10, 3465–3471. Wallevik, K. (1973) J. Biol. Chem. 248, 2650–2655. Wilting, J., van der Giesen, W. F., Jassen, L. H. M., Weideman, M. M., Otagiri, M., and Perrin, J. H. (1980) J. Biol. Chem. 255, 3032–3037. Mason, R. P., Cha, G. H., Gorrie, G. H., Babcock, E. E., and Antich, P. P. (1993) FEBS Lett. 318, 30–34. He, X. M., and Carter, D. C. (1992) Nature 358, 209–215. Ladenstein, R., Epp, O., Bartels, K., Jones, A., Huber, R., and Wendel, A. (1979) J. Mol. Biol. 134, 199–218.
625
222, 619–625 (1996)
0793
Glutathione-Linked Thiol Peroxidase Activity of Human Serum Albumin: A Possible Antioxidant Role of Serum Albumin in Blood Plasma Mee-Kyung Cha and Il-Han Kim1 Department of Biochemistry, Pai-Chai University, Taejon 302-735, Korea Received April 1, 1996 A 65-kDa molecular mass of thiol-specific antioxidant protein was purified from human plasma and identified as human serum albumin (HSA) by the analysis of amino-terminal amino acid sequence. This protein exhibited the preventive effects against the inactivation of glutamine synthetase activity and the peroxidation of lipid by a metal-catalyzed oxidation system. These antioxidant activities were supported by a thiol-reducing equivalent such as DTT and reduced glutathione. The thiol-specific antioxidant activity of HSA was greatly activated by halide ion, especially by chloride ion. HSA showed a significant capability to destroy H2O2 in the presence of reduced glutathione, resulting in the production of oxidized glutathione. Both the preventive activity against the glutamine synthetase inactivation and the peroxidase activity were completely abolished by the reactions of HSA with N-ethylmaleimide and iodoacetate, chemical modification agents for sulfhydryl of protein, only in the presence of thiol-reducing equivalent such as DTT. These results suggest that serum albumin acts as a major and predominate antioxidant exerting a glutathione-linked thiol peroxidase activity which removes reactive oxygen species such as H2O2 within blood plasma. © 1996 Academic Press, Inc.
It is increasingly recognized that reactive oxygen species play a significant role in the pathogenesis of certain human disease such as atherosclerosis, cancer, and rheumatoid arthritis (1–3). Blood plasma is exposured to reactive oxygen species which are generated from activated phagocytes and the reactions of oxygen with a great variety of compounds derived from cellular metabolism, dietay uptake, and xenobiotics. As results of this oxidative stress to human blood there is a growing effort to find out antioxidant enzymes such as glutathione peroxidase, superoxide dismutase, and catalase in blood plasma (4–7). Recently, a novel type of antioxidant enzyme was found in from prokaryotes (8) and to eukaryotes (9–11). These intracellular antioxidant proteins exerted the preventive activity against glutamine synthetase inactivation by a metal-catalyzed oxidation system and the peroxidase activity. These activities were supported by thioredoxin. This antioxidant protein was, thus, named “thioredoxin peroxidase” (12–14). We reported the another type of a peroxidase, which exists in the periplasmic spaces of E. coli (15). This type of peroxidase was proved to have a functional cysteine as a nucleophile in the catalytic reaction, thus named “thiol peroxidase”. Thiol peroxidase shares a similar catalytic activity to that of thioredoxin peroxidase in that thiol peroxidase activity was also linked to thioredoxin. However, the deduced amino acid sequence of thiol peroxidase is completely different from that of thioredoxin peroxidase, suggesting the possibility that various types of peroxidase family having functional cysteine remain to be undiscovered. It is generally believed that blood plasma is exposed to more severe oxidative stress than intracellular fluids, but the level of antioxidant enzymes in blood plasma is much lower than the intracellular level (7). The sulfhydryl groups of serum proteins including serum albumin have been suggested to be a “sacrificial” antioxidant in plasma and the extravascular space (16). However, 1
Corresponding author: Department of Biochemistry, Pai-Chai University, Doma-2-Dong 439-6, Seo-Gu, Taejon 302735, Korea: Fax: 82-42-520-5379. The abbreviations used are: DTT, dithiothreitol; TSA, thiol-specific antioxidant: MCO, metal-catalyzed oxidation; GSH, reduced form of glutathione; GSSG, oxidized form of GSH; HSA, human serum albumin; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis. 619 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
none of serum proteins showing a enzymatic mechanism to remove reactive oxygen species such as H2O2 has not been reported. In this study, we purified a 65-kDa protein showing an enzymatic antioxidant activity (i.e., thiol specific antioxidant activity and thiol peroxidase activity) from human plasma, and identified as a human serum albumin (HSA). Our results suggest that HSA as a thiol peroxidase could be a major antioxidant enzyme of extracellular fluids such as human blood plasma. MATERIALS AND METHODS Purification of 65-kDa protein showing thiol-specific antioxidant activity from Human blood plasma. Human blood plasmas were obtained from freshly drawn heparinized blood. Human plasma proteins were subjected to ammonium sulfate precipitation (70%). The precipitate was dissolved and extensively dialyzed against DEAE column equilibrium buffer (50 mM Tris-HCl buffer, pH 7.6). After centrifugation, the clear supernatant was loaded into a DEAE column. The column was washed with equilibrium buffer and eluted with a linear KCl gradient (0–400 mM). The presence of thiol-specific antioxidant (TSA) activity was assayed throughout the purification by monitoring its ability to prevent the inactivation of glutamine synthetase by the thiol-MCO system. The peak fractions of TSA activity eluted at a KCl concentration of 250 mM were pooled and precipitated with 70% ammonium sulfate. The dissolved ammonium sulfate-precipitates were applied to a G-75 gel permeation column previously equilibrated with 100 mM Hepes buffer, pH 7.4, containing 100 mM KCl. The ammonium sulfate precipitate of the fractions exerting was dissolved in 100 mM Hepes-NaOH buffer, pH 7.4, containing 1.0 M ammonium sulfate, and applied to phenyl sepharose CL-4B column previously equilibrated with 100 mM HepesNaOH buffer (pH 7.4) containing 1.0 M ammonium sulfate. Proteins were eluted with a linear gradient from 1.0 M ammonium sulfate to the buffer containing no salt. The TSA activity was eluted between 0.7 and 0.5 M of the salt. The fractions showing TSA activities were precipitated, dissolved, and applied to G-100 gel permeation column. The major peaks for TSA activity gave a 65-kDa of homogeneous protein. Determination of thiol-specific antioxidant (TSA) activity. TSA activity of 65-kDa protein was determined by monitoring its activity to inhibit the inactivation of E. coli glutamine synthetase by a metal catalyzed thiol MCO system (DTT, Fe3+, O2) (17) as described by Kim et al. (9). Instead of DTT ascorbate was included as a non-thiol reducing equivalent (non-thiol MCO system). Fifty ml of reaction mixture containing 5 mg of glutamine synthetase, 3 mM FeCl3, 10 mM DTT or ascorbate, antioxidant enzyme, 100 mM NaCl, and 100 mM Hepes-NaOH (pH 7.0) was incubated at 37°C. The remaining activity of glutamine synthetase was measured by addition of 5 ml of the reaction mixture to 2 ml of g-glutamyltransferase assay mixture as described (18). Determination of peroxidase activity of the 65-kDa protein in the presence of GSH. To determine the peroxidase activity of the enzymes, the reaction was started by the addition of 0.5 mM of H2O2 into the 50 ml of reaction mixture containing 5 mM GSH, 100 mM NaCl, and 50 mM Hepes-NaOH, pH 7.0, and then incubated at 37°C. At appropriate reaction time, 20 ml of the reaction mixture was added to 0.8 ml of TCA solution (12.5%, w/v) to stop the reaction, followed by the addition of 0.2 ml of 10 mM Fe(NH4)2(SO4)2 and 0.1 ml of 2.5 N KSCN to develop the complex, giving a purple color. The concentration of H2O2 was monitored by measurement of the decrease in absorbance at 480 nm. The rate of production of GSSG by HSA was measured in the presence of excess glutathione reductase (0.5 unit) by following the rate of NADPH oxidation (19). Determination of lipid peroxidation. The peroxidation of RBC (red blood cell)-membrane lipids by a thiol-MCO system were determined by measurement of TBAR (thiobarbituric acid-reactive products). In the presence of the thiol-MCO system, one ml of reaction mixture containing 0.6 M of membrane lipid in term of inorganic phosphate and 100 mM NaCl was incubated for one hour at 37°C. Whole reaction mixture was boiled for 20 min with thiobarbituric acid and trichloroacetic acid, in the presence of 0.01% butylated hydroxyltoluene, as described by Buege and Aust (20). After centrifugation at 2,000 × g, the malonyldialdehyde content of supernatant was then determined by their absorbance at 535 nm. Protein concentration was determined by using the Bio-Rad Protein Assay kit based on the method of Bradford (21). SDS-PAGE was performed by the method of Laemmli (22). Chemical modification of sulfhydryl group of the 65-kDa protein. The 65-kDa protein was preincubated in the presence of 2 mM DTT and 100 mM NaCl for 20 min at 30°C, and then reacted with N-ethylmaleimide (10 mM) and iodoacetate (10 mM) at 30°C for 2 hrs. The unreacted reagent was washed out using Centricon 30 (Amicon). Free sulfhydryl group of the protein was determined by the measurement of the release of 2-nitro-5-mercaptobenzoic acid at 412 nm during the reaction with sulfhydryl group in protein (100 mM) with 5,5-dithiobis-(2-nitrobenzoic), DTNB, (1 mM) (23).
RESULTS AND DISCUSSION Purification of a thiol-specific antioxidant protein from human plasma and its identification as human serum albumin. We purified a thiol-specific antioxidant (TSA) protein from human blood plasma. This antioxidant protein was purified to homogeneity by four sequential chromatographic steps on DEAE, G-75, phenyl Sepharose CL-4B, and G-100 (Fig. 1). The molecular mass of this 620
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. SDS–PAGE analysis of 65-kDa thiol-specific antioxidant protein at different stages of purification. Protein sample from different stages of purifications were electrophoresed in 8% SDS-PAGE gel with b-mercaptoethanol. Lane 1 contains 50-mg sample from plasma. Lane 2, 30-mg sample from DEAE chromatography. Lane 3, 20-mg sample from G-75 gel permeation chromatography. Lane 4, 10-mg sample from phenyl Sepharose CL-4B chromatography. Lane 5, 2-mg sample from G-100 gel permeation chromatography. Lane 7, the nonreducing electrophoresis of the 2-mg of sample from the G-100 chromatography. Lane 6, size markers. The molecular masses, from the bottom, are 14.4, 21.5, 31, 45, 66.2, and 97.4 kDa.
protein was determined to be 65-kDa on reducing SDS-PAGE (8%) with b-mercaptoethanol. The protein band migrated faster on the non-reducing gel, indicating that the protein has intradisulfide linkage(s) (lane 6, 7 of Fig. 1). The purity of 65-kDa TSA protein was confirmed by obtaining the first peak corresponding to Asp in the N-terminal amino acid analysis as single peak (data not shown). The analysis of N-terminal amino acid sequence of 65-kDa TSA protein (18 residues) was determined as that of HSA (DAHKSEVAHRFKDLGEEN). Fig. 2 shows the thiol-specific antioxidant activity of HSA. HSA exerted the preventive activity against the inactivation of glutamine synthetase by thiol MCO system, but did not by non-thiol MCO system (ascorbate MCO system) in which DTT was replaced by ascorbate. Requirement for a thiol-reducing equivalent in the antioxidant activity of HSA suggests that the HSA could function as an extracellular antioxidant enzyme having functional cysteine (e.g., thiol peroxidase). Activation of TSA activity of HSA by chloride ion. We found that the TSA activity was increased
FIG. 2. Protection of HSA against the inactivation of glutamine synthetase (GS) caused by MCO system. The inactivation mixture contained 5 mg of E. coli glutamine synthetase, 100 mM NaCl, 10 mM DTT for a thiol MCO system or 10 mM ascorbate for a non-thiol MCO system, 3 mM FeCl3, 50 mM Hepes, pH 7.0, in 50 ml of reaction volume. At indicated times, aliquots (5 ml) were removed and assayed 10 min at 37°C for remaining glutamine synthetase activity. Each inactivation-reaction mixture contained as follows: curve 1, the thiol MCO system plus 2 mg/ml HSA; curve 2, the non-thiol MCO system plus 2 mg/ml of HSA; curve 3, the non-thiol MCO system; curve 4, the thiol MCO system; curve 4, the thiol MCO system. 621
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by KCl. To determine which ion have a capability to activate the activity, we examined the effects of various salts consisting monovalent cation and halide ion. All salts containing chloride anion activated the activity regardless of the counterpart cations (lane 1–9, Fig. 3-A), but the salts lacking chloride anion such as sodium acetate and potassium acetate did not elevate the activity (lane 7,8 of Fig. 3-A). The extent of the activation by the various halide anions from fluoride to iodide (lane 9–12 of Fig. 3-A) revealed that chloride ion was the most effective halide ion to activate the TSA activity of HSA. Fig. 3-B showed the TSA activity as the function of concentration of chloride ion, indicating 100 mM of chloride ion is necessary for the full activation of the enzyme. The TSA activity of HSA was about five-fold increased by chloride ion with comparison to that in the absence of the ion (Fig. 3-C). The effect of chloride ion on the conformation of serum albumin was well known. Zurawski and Foster (24) showed evidence for the existence of two distinguishable conformations of serum albumin, so called N and B forms (25). In addition, the conformation of HSA has been reported to be influenced by physiologically important chloride ion (26). Thus, the activation of TSA activity of HSA by chloride ion can be interpreted in term of an effect of chloride ion on the conformational change of the protein. In vivo thiol equivalent linked to HSA. We searched for an enzyme or in vivo thiol reducing equivalent capable of supporting the TSA activity of HSA. An enzymatic thiol regenerating system (thioredoxin / thioredoxin reductase / NADPH system) did not provide HSA with the capability to protect the inactivation of glutamine synthetase by a non-thiol MCO system in which DTT was replaced by ascorbate (data not shown). Glutamine synthetase was also inactivated by a thiol MCO system (GSH MCO system) in which DTT was replaced by GSH, known as an in vivo thiol reducing equivalent (curve 2 of Fig. 4-A). This inactivation was completely protected by HSA (curve 1 of Fig. 4-A). The lipid peroxidation was caused by two GSH MCO systems (GSH + Fe3+ + O2; GSH + hemoglobin + O2). Fig. 4-B shows the increase of protection against lipid peroxidation as a function of HSA concentration. The capabilities of HSA preventing these oxidations by the GSH MCO system capable of generating reactive oxygen species may reveal the in vivo antioxidant activity of HSA in human blood plasma containing GSH. The concentration of GSH in whole blood was reported to be 1.7 mM (27). A GSH-linked peroxidase activity of HSA. We examined HSA for peroxidase activity by measuring the decrease of H2O2 in the presence of GSH. The time-coursed removal of H2O2 showed a first-order kinetics. The velocity of the removal of H2O2 increased as a function of HSA concentration, showing a saturation pattern (Fig. 5-A). The activity for removal of H2O2 by HSA
FIG. 3. Activation of thiol-specific antioxidant activity of HSA by chloride ion. (A) The effect of various salts. The inactivation mixture contained 10 mM DTT, 3 mM of FeCl3, 5 mg of glutamine synthetase (GS), 1 mg of HSA, and 100 mM of various salts as follow: lane 1–12; no salt, LiCl, NaCl, KCl, RbCl, CsCl, sodium acetate, potassium acetate, NaF, NaCl, NaBr, and Nal, respectively. (B) The activation of HSA as a function of concentration of NaCl. The inactivation mixture contained 5 mg of E. coli glutamine synthetase, 1 mg/ml HSA, 10 mM DTT, 3 mM FeCl3, and 50 mM Hepes, pH 7.0. (C) The thiol-specific antioxidant activity as a function of the concentration of HSA. The inactivation mixture contained 5 mg of E. coli glutamine synthetase, 10 mM DTT, 3 mM FeCl3, and 50 mM Hepes, pH 7.0, and 100 mM NaCl for curve 1 or without NaCl for curve 2. After 30 min inactivation, 5 ml of aliquot was removed and assayed for the remaining glutamine synthetase activity. 622
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Protections of HSA against the inactivation of glutamine synthetase (GS) (A) and the lipid peroxidation (B) caused by thiol MCO system in which DTT was replaced by GSH. The GS-inactivation mixture contained 5 mg of E. coli glutamine synthetase, 20 mM GSH, 3 mM FeCl3, 100 mM NaCl, 50 mM Hepes, pH 7.0, and 1 mg/ml HSA for curve 1, or without HSA for curve 2. The lipid-peroxidation mixture contained 0.6 M of red blood cell membrane in term of phospholipid, 20 mM GSH, the indicated concentration of HSA, and 400 mM of FeCl3 for curve 1 or 2 mg/ml of hemoglobin as an iron source for curve 2. After one hour incubation of one ml of the reaction mixture at 37°C, malonyldialdehyde was determined by measuring the absorbance at 535 nm.
in the presence of glutathione reductase and NADPH can be measured indirectly by following the decrease in A340 resulted from the oxidation of NADPH by the glutathione reductase reaction (i.e., NADPH + GSSG → NADP+ + GSH) (Fig. 5-B). The continuous oxidation of NADPH to NADP+ in this system indicates the production of GSSG from GSH by the reaction of HSA (i.e., H2O2 + GSH → H2O + GSSG). These results suggest a peroxidase activity of HSA supported by GSH. Functional group of HSA. HSA consists of 585 amino acids. Its amino-acid sequence contains a total of 17 disulfide linkages and one free thiol (Cys-34) (28). When a thiol reducing equivalent was replaced with another non-thiol electron donor such as ascorbate, HSA no longer protected the inactivation of glutamine synthetase by the ascorbate MCO system (Fig. 1). The addition of DTT to the ascorbate MCO system significantly restored the antioxidant activity of HSA (data not shown). These results support a catalytic cycle of functional cysteine of HSA as shown in Fig. 7. To examine this, HSA was reacted with N-ethylmaleimide and iodoacetate, cysteine-specific modification reagents, with or without DTT. HSA was preincubated with 2 mM DTT and then reacted with 10 mM N-ethylmaleimide or iodoacetate. The resulting HSAs did not prevent the
FIG. 5. Glutathione (GSH)-linked peroxidase activity of HSA. (A) Removal of H2O2 by HSA supported by GSH. Peroxidase reaction was carried out in a 50-ml reaction mixture containing 0.5 mM H2O2, 5 mM GSH, 50 mM Hepes, pH 7.0, and 0, 15 mM (1 mg/ml), 30 mM, and 45 mM of HSA for curves 1, 2, 3, and 4, respectively. At indicated time, 20-ml aliquot was removed and assayed for remaining H2O2 according to the method described under Materials and Method. (B) The production of oxidized glutathione (GSSG) by a peroxidase activity of HSA supported by GSH. Assay mixture for GSSG contained 2 mM GSH, 0.5 mM H2O2, 0.5 unit (mmole/min) glutathione reductase, 2 mM NADPH and 15 mM (1 mg/ml) HSA for curve 2 or without HSA for curve 1. The reaction mixture was preincubated in the absence of H2O2 for 5 min. and then 0.5 mM of H2O2 was added into the mixture to start the peroxidation reaction of HSA. The decrease of NADPH was spectrophotometrically monitored at 340 nm. 623
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 6. Inactivation of HSA by N-ethylmaleimide (NEM). Inactivation of thiol-specific antioxidant activity of HSA by NEM was measured in both terms of the protection activity against the inactivation of glutamine synthetase caused by DTT MCO system (A) and the H2O2-removal activity supported by GSH (B). Curves A-1 and B-4, the control HSA without the inactivation by NEM. Curves A-2 and B-3, the HSA which was reacted with NEM in the absence of DTT. Curves A-3 and B-2, the HSA which was reacted with NEM in the presence of DTT. Details are given under Materials and Methods.
inactivation of glutamine synthease and did not exert a GSH-linked peroxidase. However, these antioxidant activities of HSA were not inhibited by these reagents in the absence of DTT (Fig. 6-A, B) (data for iodoacetate not shown). To exclude a possible pathway | denoted in Fig. 7, we modified the thiol of Cys-34, the single free thiol of HSA (28), with 5,5-dithiobis-(2-nitrobenzoic) in the absence of DTT. The chemical modification of the sulfhydryl group of Cys-34 in HSA was confirmed by observing the release of 2-nitro-5-mercaptobenzoic acid at 412 nm (data not shown). This modification did not affect the antioxidant activity of HSA (data not shown), removing the possibility of pathway I. These results indicate that a peroxidase activity of HSA should be resulted from the catalytic cycle (pathway II) shown in Fig. 7, which shares the similar catalytic cycle of glutathione peroxidase (29). Human blood plasma contains a variety of antioxidant biomolecules that can potentially protect circulating lipids, proteins and cells as well as the vasculature-lining endothelial cells from oxidative damage caused by activated oxygen species. Enzymatic antioxidants are present only in small concentrations in plasma (7) and their physiological function remains unclear. For this reason, we searched for a new type of antioxidant enzyme in human blood plasma and purified a 65-kDa protein exerting TSA activity. This TSA protein was proved to be HSA. HSA as the major soluble protein consituents of human blood plasma has many physiological functions such as the regulation of blood pressure and the transport of many endogenous and exogenous ligands. In addition to blood plasma, serum albumin was also found in tissue and bodily secretions throughout the body. The extravascular protein comprises 60% of the total albumin. In this paper, we suggest the potential importance of HSA as a extracellular enzymatic antioxidant, named “GSH-linked thiol peroxidase”, not as a “sacrificial” antioxidant (16) to protect oxidative damages associated with
FIG. 7. Mechanism for the reduction of H2O2 by HSA with glutathione (GSH) as an in vivo electron donor. 624
Vol. 222, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
oxidative stress. Further study will be required to define which sulfhydryl group(s) from 17 disulfide linkages in HSA acts as a functional nucleophile in the peroxidation reaction. ACKNOWLEDGMENTS This work was supported by grants from BSRI-96-3434 and Pai-Chai University (1996). We are grateful to Dr. KyungHoon Suh for critical reading of this manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
Sies, H. (Ed.) (1985) Oxidative Stress, Academic Press, New York London. Halliwell, B., and Gutteridge, J. M. C. (1985) Free Radicals in Biology and Medicine, Oxford Univ. Press, London. Halliwell, B., and Gutteridge, J. M. C. (1986) Arch. Biochem. Biophys. 246, 501–504. Karlsson, K., and Marklund, S. L. (1987) Biochem. J. 242, 55–59. Marklund, S. L. (1987) Proc. Natl. Acad. Sci. USA 84, 6634–6638. Frei, B., Yamamoto, Y., Niclas, D., and Ames, B. N. (1988) Anal. Biochem. 175, 120–130. Lentner, C. (Ed.) (1984) Geigy Scientific Tables, Vol. 3, 8th revised and enlarged edition, Ciba-Geigy, Basel. Jacobson, F. S., Morgan, R., Christman, M. F., and Ames, B. N. (1989) J. Biol. Chem. 264, 1488–1496. Kim, K., Kim, I. H., Lee, K. Y., Rhee, S. G., and Stadtman, E. R. (1988) J. Biol. Chem. 263, 4704–4711. Lim, Y. S., Cha, M. K., Kim, H. K., and Kim, I. H. (1994) Gene (Amst.) 140, 279–284. Lim, Y. S., Cha, M. K., Yun, C. H., Kim, H. K., and Kim, I. H. (1994) Biochem. Biophys. Res. Commun. 199, 199–206. Lim, Y. S., Cha, M. K., Kim, H. K., Uhm, T. B., Park, J. W., Kim, K., and Kim, I. H. (1993) Biochem. Biophys. Res. Commun. 192, 273–280. Kwon, S. J., Park, J. W., Choi, W. K., Kim, I. H., and Kim, K. (1994) Biochem. Biophys. Res. Commun. 201, 8–15. Chae, H. Z., and Rhee, S. G. (1994) Biofactors 4, 177–180. Cha, M. K., and Kim, I. H. (1995) J. Biol. Chem. 270, 28635–28641. Halliwell, B. (1988) Biochem. Pharmacol. 37, 569–571. Stadtman, E. R., and Oliver, C. N. (1991) J. Biol. Chem. 266, 2005–2008. Rhee, S. G., Chock, P. B., and Stadtman, E. R. (1985) Methods Enzymol. 113, 213–241. Tappel, A. L. (1978) Methods Enzymol. 52, 506–513. Buege, J. A., and Aust, A. D. (1978) Methods Enzymol. 52, 302–310. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254. Laemmli, U. K. (1970) Nature 227, 680–685. Ogilvie, J. W. (1980) Biochim. Biophys. Acta 622, 277–284. Zurawski, V. R. Jr. and Foster, J. F. (1974) Biochemistry 10, 3465–3471. Wallevik, K. (1973) J. Biol. Chem. 248, 2650–2655. Wilting, J., van der Giesen, W. F., Jassen, L. H. M., Weideman, M. M., Otagiri, M., and Perrin, J. H. (1980) J. Biol. Chem. 255, 3032–3037. Mason, R. P., Cha, G. H., Gorrie, G. H., Babcock, E. E., and Antich, P. P. (1993) FEBS Lett. 318, 30–34. He, X. M., and Carter, D. C. (1992) Nature 358, 209–215. Ladenstein, R., Epp, O., Bartels, K., Jones, A., Huber, R., and Wendel, A. (1979) J. Mol. Biol. 134, 199–218.
625