Characterization of One- and Two-electron Oxidations of Glutathione Coupled with Lactoperoxidase and Thyroid Peroxidase Reactions*

Transcription

1 ~ ~ ~~~~ THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1986 by The American Society of Biological Chemists. Inc. Vol. 261, No. 30, Issue of October 25, pp ,1986 Printed in U.S.A. Characterization of One- and Two-electron Oxidations of Glutathione Coupled with Lactoperoxidase and Thyroid Peroxidase Reactions* (Received for publication, March 31,1986) Masao Nakamura, Isao Yamazaki, Sachiya OhtakiS, and Shingo NakarnuraO From the Biophysics Division, Research Institute of Applied Electricity, Hokkaido University, Sapporo 060, Japan, the $Central Laboratory of Clinical Investigation, Miyazaki Medical College Hospital, Kiyotake, Miyazaki , Japan, and the $Faculty of Agriculture, Hirosaki University, Hirosaki 036, Japan Glutathione (GSH) was oxidized to GSSG in thepres- been confirmed by the spin-trapping ESR technique for the ence of H202, tyrosine,andperoxidase.duringthe thiol oxidation reactions promoted by peroxidase and pros- GSH oxidation catalyzed by lactoperoxidase, 0, was taglandin synthetase (7-11). Therefore, reactions of thiyl consumed and the formation of glutathione free radical radicals with 0, could be physiologically important (5, 8, 12, was confirmed by ESR of its 5,5 -dimethyl-l-pyrro- 13). line-n-oxide adduct. When lactoperoxidase was re- GSH is oxidized in biological systems mostly by the glutaplaced by thyroid peroxidase in the reaction system, thione peroxidase reaction that serves as a defense system the consumption of Oz and the formation of the free against hydroperoxides (14-16). This GSH oxidation appears radical became negligibly small. These results led us to to occur by way of a two-electron transfer (1, 16). However, conclude that, in the presence of H,O, and tyrosine, no proper experimental data have been presented for comparlactoperoxidase and thyroid peroxidase caused the ison of one-electron and two-electron oxidations of one-electron and two-electron oxidations of GSH, re- GSH. spectively. It was assumed that GSH is oxidized by Careful consideration is needed in order to conclude that an primary oxidation products of tyrosine, which are oxidation-reduction reaction is a two-electron type (17). As phenoxyl free radicals in lactoperoxidase reactions and reported previously (la), thyroid peroxidase selects either onephenoxyl cations in thyroid peroxidase reactions. electron or two-electron oxidation mechanisms of phenols, When tyrosine was replacedbydiiodotyrosine or depending on their substituents, while lactoperoxidase and 2,6-dichlorophenol, the difference in the mechanism horseradish peroxidase invariably catalyze one-electron oxibetween lactoperoxidase and thyroid peroxidase dis- dation of phenols. Since GSH was found to be oxidized by the appeared and both caused the one-electron oxidation primary oxidation products of phenols (M), we thought that of GSH. Iodides also served as an effective mediator of these reaction systems might be used to characterize one- GSH oxidation coupled with the peroxidase reactions. electron and two-electron oxidations of GSH. In this case the two peroxidases both caused the twoelectron oxidation of GSH. MATERIALS AND METHODS GSH plays a role in the protection of cellular constituents against oxidative damage and its redox property is an important subject studied by many workers. In general the oxidation of thiols can be divided formally into two classes; one-electron and two-electron reactions (1). In both cases the product appears to be the disulfide. For one-electron oxidation, For two-electron oxidation, RSH - RS. + H i e- (1) 2 RS. RSSR (2) RSH - RS + H + 2e- (3) RSH + RS - RSSR + H (4) The technique of pulse radiolysis has been successfully used for kinetic studies of thiyl radical reactions (2-4). The involvement of thiyl radicals has been suggested in the autoxidation of thiols (5, 6). Recently, the formation of thiyl radicals has *This research was supported by Grants-in-Aid and for Scientific Research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Thyroid peroxidase used in these experiments was purified from hog thyroid microsomes by immunoaffinity chromatography without the procedure of trypsin digestion (19). Lactoperoxidase was prepared from fresh bovine milk by the method of Morrison and Hultquist (20). The ratio of A113nm to Am nm was 0.24 for thyroid peroxidase and 0.92 for lactoperoxidase. The concentration of these peroxidases was calculated on the basis of a value of 114 for t mm at 413 nm (21). GSH, GSSG, DMPO, bovine catalase, and superoxide dismutase were purchased from Sigma; L-tyrosine, monoiodotyrosine, diiodotyrosine, 5,5 -dithiobis-(2-nitrobenzoic acid), penicillamine, and dithioerythritol from Nakarai (Kyoto); 2,6-dichlorophenol from Wako Pure Chemical Industry (Osaka); glutathione reductase from Oriental Co. (Osaka); and NADPH from Boehringer Mannheim. The 02 concentration was measured polarographically with a Clark-type 0 2 electrode. The oxidation product of GSH was measured with a Hitachi 655 HPLC system equipped with a Merck Lichrosorb NH, column (22). The ESR spectra were recorded on a Varian E- 109B spectrometer equipped with an aqueous flat cell. The reactions were carried out in 0.1 hi potassium phosphate (ph 7.4) at 25 C RESULTS Lactoperoxidase and thyroid peroxidase both catalyze HaOz-dependent oxidation of tyrosine to dityrosine, which can be measured from the increase of absorbance at 310 nm (23, 24). Although these peroxidases were unable to catalyze directly the oxidation of GSH, the formation of dityrosine was inhibited by GSH and the inhibition was removed after a certain lag time which depended on the amount of added The abbreviations used are: DMPO, 5,5 -dimethyl-l-pyrroline- N-oxide; HPLC, high pressure liquid chromatography.

2 13924 One-electron Two-electron and ; o. 2 $ q c 0 a Q GSH (JJM) OO Time (s) Time (s) FIG. 1. The effect of GSH on the peroxidase-catalyzed oxidation of tyrosine. The concentrations were: 500 p~ tyrosine, 100 p~ H202, and 170 nm lactoperoxidase (A) or 82 nm thyroid peroxidase (B). The GSH concentration is indicated on the figure. A Enzyme FIG. 2. The 02 consumption during GSH oxidation mediated by tyrosine or 2,6-dichlorophenol in peroxidase systems. A, 200 NM tyrosine, 20 pm H202, 1.0 mm GSH, and 170 nm lactoperoxidase (LPO) or 82 nm thyroid peroxidase (TPO). B, 40 p~ 2,6- &chlorophenol, 20 pm HzO,, 1.0 mm GSH, and 80 nm lactoperoxidase or 82 nm thyroid peroxidase. GSH (Fig. 1). Since the final oxidation product of tyrosine did not oxidize GSH, we concluded that GSH was oxidized by an intermediate oxidation product of tyrosine (18). The time course of tyrosine oxidation was monophasic for lactoperoxidase and diphasic for thyroid peroxidase (Fig. 1). The reason for this will be discussed later. No essential difference was seen in the inhibitory pattern of GSH for the two peroxidase reactions. A remarkable difference was observed between the two reactions when the O2 consumption was measured in the presence of a large amount of GSH. As can be seen in Fig. 24, a considerable amount of O2 was consumed when lactoperoxidase was added to a GSH solution containing H202 and tyrosine, but the O2 consumption became negligibly small when lactoperoxidase was replaced by thyroid peroxidase. This difference between the two peroxidases disappeared when tyrosine was replaced by 2,6-dichlorophenol (Fig. 2B). The O2 consumption appeared to occur invariably in the phenol-mediated oxidation of GSH catalyzed by lactoperoxidase, but it depended on phenol molecules when thyroid peroxidase was used as catalyst (Table I). The concentrations of the two peroxidases were chosen so as to give a similar rate of oxidation of a phenol. This classification of phenols was in accord with that determined from analysis of rate-determining intermediates of the enzyme during catalytic reactions (18). Table 11 shows that the molar ratio of GSSG formed per H202 was nearly 1.0 when H202 was added at concentrations B Oxidations of GSH TABLE r Activity of phenols and iodide as a mediator for the 02-consuming oxidation of GSH catalyzed by peroxidases The reactions were carried out under experimental conditions described in the legends to Figs. 2 and 8. The 0, uptake was negligibly slow in the absence of enzyme (see Footnote b). Lacto- Thyroid peroxidase peroxidase Tyrosine ++ a Monoiodotyrosine 2,4-Dimethylphenol 2,4-Dichlorophenol + - Diiodotyrosine + + 2,6-Dibromophenol ,4,6-Tribromophenol ,6-Dich1orophenol6 ++ -I-+ Potassium iodide - - This sign means that no significant Oz uptake was detected. ' In this case a slow 02 uptake was seen in the absence of enzyme, but the addition of enzyme markedly promoted the reaction (see Fig. 2B). TABLE I1 Stoichiometry of the tyrosine-mediated oxidation of GSH catalyzed by lactoperoxidase and thyroid peroxidase Aliquots (0.1 ml) of the reaction solutions were assayed after 30 min incubation under aerobic conditions unless otherwise noted. GSH was measured from the increase of absorbance at 412 nm upon the addition of 200 &M 5,5'-dithiobis-(2-nitrobenzoic acid), and GSSG was from the decrease of absorbance at 340 nm observed 10 min after the addition of 0.6 unit of glutathione reductase and p~ NADPH. Both measurements were carried out in a final volume of 2 ml. Thyroid peroxidase" Lactoperoxidaseb 20 Anaerobic 20 Anaerobic 80 Hz02 GSH GSSG added oxidized formed " mm GSH, 100 pm tyrosine, and 80 nm thyroid peroxidase. 1.1 mm GSH, 200 pm tyrosine, and 170 nm lactoperoxidase. below 80 p~ in the tyrosine-mediated oxidation of GSH catalyzed by thyroid peroxidase. At higher concentrations of HzOz, catalytic decomposition of H202 became appreciable and the ratio of [GSSG]/[H,O,] was decreased. In the case of lactoperoxidase t.he 1:l stoichiometry was observed only under anaerobic conditions (Table 11). Under aerobic conditions GSH was oxidized at the expense of 02. Relation between the disappearance of GSH and the formation of GSSG is shown in Fig. 3 and also in Table IT. The results indicated that GSH was oxidized mostly to GSSG in the tyrosine-mediated 0,- consuming oxidation of GSH. Wefers and Sies (12) reported the formation of about 10% glutathione sulfonate in the 0,-consuming oxidation of GSH occurring during the xanthine oxidase reaction. Using HPLC we could confirm their results and also found that the oxidation product of GSH in our reactions was only GSSG (Fig. 4). Glutathione sulfonate was less than 1% of GSSG formed. Enzymatic formation of glutathione free radical has recently been confirmed by ESR of its DMPO adduct. Fig. 5 shows a big difference in the formation of glutathione free radical in the tyrosine-mediated oxidations of GSH catalyzed by lactoperoxidase and thyroid peroxidase. The ESR signal accorded with that of the DMPO-glutathione thiyl radical adduct (25).

3 One-electron and Two-electron Oxidations of GSH GSH (mm) GSSG (mm) IO 15 Time (mln) FIG. 3. Time courses of GSH disappearance and GSSG formation in the tyrosine-mediated oxidation of GSH catalyzed by lactoperoxidase. The concentrations were: 200 pm tyrosine, 20 p~ H202, 1.1 mm GSH, and 170 nm lactoperoxidase. Aliquots (0.1 ml for GSH determination and 0.2 ml for GSSG determination) were withdrawn for assay at times indicated. GSH was measured by the addition of 5,5 -dithiobis-(2-nitrobenzoic acid) (0.2 mm) and GSSG was measured as described in Table 11, both in a final volume of 2 ml. FIG. 5. The ESR spectrum of DMPO-GSH free radical adduct produced in the tyrosine-mediated oxidation of GSH. The spectrum was recorded with the modulation amplitude of 0.4 G about 1 min after 50 pm HzOz was added to a solution containing 1 mm GSH, 200 p~ tyrosine, 100 mm DMPO, and 170 nm lactoperoxidase (A) or 150 nm thyroid peroxidase (B). It took 2 min to scan 80 G and a slight decrease of the ESR signal was seen in the second scan. Penicillamine * Retention Time (min) FIG. 4. Assay of oxidation products of GSH by HPLC. The products were assayed 40 min after the reactions were started according to method of Wefers and Sies (12). The positions indicated represent: 1, N-ethylsuccinimidyl GSH 2, glutathione sulfonate; and 3, GSSG. The others are attributed to impurities contained in the GSH and GSSG preparations used. A, a mixture of 1.1 mm GSH and 0.25 mm GSSG (control). B, 2.2 mm GSH, 1.0 mm xanthine, and 200 nm xanthine oxidase under 02-saturated conditions. C, 1.1 mm GSH, 200 p~ tyrosine, 20 PM H2O2, and 170 nbf lactoperoxidase under aerobic conditions. A very small amount of glutathione free radical was formed in the reaction of thyroid peroxidase. This fact appeared to correspond with the previous finding (18) that the formation of a small amount of benzosemiquinone was observed although thyroid peroxidase catalyzes the oxidation of hydroquinone mostly to benzoquinone. DMPO inhibited the tyrosine-mediated Op-consuming oxidation of GSH caused by lactoperoxidase. The concentration of DMPO to give 50% inhibition was 18 mm. The tyrosine-mediated 02-consuming oxidation was not specific for GSH; other sulfhydryl compounds such as cysteine, penicillamine, and dithioerythritol were also oxidized by O2 in the presence of Hz02, tyrosine, and lactoperoxidase (Fig. 6). Hp02 was necessary for the reaction to proceed, but in some cases it was not necessary to be added to the reaction solution, particularly when 2,6-dichlorophenol served as the mediator (Fig. 7). In a solution containing GSH and 2,6- dichlorophenol, the O2 consumption occurred slowly in the $1 N I O S - FIG. 6. Tyrosine-mediated 02-consuming oxidation of sulfhydryl compounds catalyzed by lactoperoxidase. The concentrations were:1.0 mm sulfhydryl compounds (indicated on the figure), 200 pm tyrosine, 20 pm H2O2, and 170 nm lactoperoxidase. The enzyme was added at arrows. absence of lactoperoxidase probably because 2,6-dichlorophenol was autoxidized to produce its free radical and superoxide, which caused chain reactions of GSH oxidation. The O2 uptake burst upon the addition of the enzyme and was inhibited by catalase and superoxide dismutase. The mechanism will be discussed later. It has been reported (26) that the aerobic oxidation of dithiothreitol is catalyzed by peroxidases in the absence of mediator. In our cases the reaction was negligibly slow without a mediator. We have reported (27) that the oxidation of iodide is very fast in the reactions of lactoperoxidase and thyroid peroxidase and also that GSH is a good acceptor for the enzyme-bound oxidized iodide. Fig. 8 shows that a small amount of iodide greatly accelerated the oxidation of GSH. In this reaction, the iodide acted as the mediator and only GSSG was detected as the oxidation product of GSH by means of HPLC. No O2 consumption was observed in the iodide-mediated oxidation

4 13926 One-electron and Two-electron Oxidations of GSH Catalase (j", I 1 min,310 FIG. 7. The effect of catalase andsuperoxidedismutase (SOD) on the aerobic oxidation of GSHin the presence of 2,6- dichlorophenol and lactoperoxidase.the concentrations were: 1.1 mm GSH, 50 FM dichlorophenol, and 170 nm lactoperoxidase. The concentrations of catalase and superoxide dismutase were indicated on the figure. I E I Time (min) FIG. 8. Iodide-mediatedoxidation of GSH inperoxidase systems. The concentrations were: 1.05 mm GSH, 95 1M HzOZ, 2.5 PM KI (A, 0) or 7.5 PM KI (A, O), and 65 nm lactoperoxidase (A, A) or 22 nm thyroid peroxidase (0, 0). The chain line denotes control without KI and with lactoperoxidase or thyroid peroxidase. TABLE I11 Rate constants for reactions of GSH and HZ02 with Compound I (EO) and the assumed enzyme-bound wdinium cation (EOz-I+) of lactoperoxidase and thyroid peroxidase, Rate constants for the reaction of EO with H2OZ were measured from the initial rate of 0 2 evolution by assuming that the reaction was rate-determining. Rate constants for the reactions of E02-I+ with Hz02 and GSH were measured from the kinetic analysis of the effects of iodide and iodide + GSH on the rate of 0 2 evolution, respectively. EO E02-I' M' s -' Lactoperoxidase GSH X lo6 H ~ O ~ 2.1 X X lo6 Thyroid peroxidase GSH X 107 H,O, 5.3 x x 106 a This sign means that catalytic oxidation of GSH was negligibly small. DISCUSSION From the spectrophotometric analysis of catalytic intermediates of thyroid peroxidase, we have concluded (18) that thyroid peroxidase catalyzes two-electron oxidation of some phenolic molecules and one-electron oxidation of the other. This classification is in good accord with that listed in Table I. These results will be explained as follows. Here, we tentatively use chemical formula of 4-O+ and GS+ for two-electron oxidation products of phenols and GSH, respectively, to simplify the discussion. The oxidation of phenols (+OH) catalyzed by peroxidases will be; for one-electron oxidation, peroxidase + Hz02 L Hz0 (5) and for two-electron oxidation, + Hz Hz0 (6) GSH seems to be oxidized by these primary oxidation products of phenols as follows. GSH GS. (7) GSH GS' (8) Finally, GS. and GS' are both converted to GSSG through Reactions 2 or 4. This implies that there is no apparent difference in the final products between the above two reactions under anaerobic conditions. Little experimental evidence has been reported on the existence ofgs' as an intermediate of GSH oxidation. It is reasonable to assume that no reaction occurs between GS' and 02. On the other hand, special attention has been devoted to the reactivity of GS. particularly under aerobic conditions (10,12,13,31) and the following reactions have been presented. GS. + GSH + GSSG' + H+ (9) GSSG' GSSG + 02 (10) GS GSOO. (11) The rate constant for GSSGT formation (Reaction 9) has been reported to be 6.2 X 10' M" s-' (2). A final product of Reaction 11 might be glutathione sulfonate as reported by Wefers and Sies (12), who have found that GSH is oxidized to a mixture of GSSG with 6-10% glutathione sulfonate when GSH is present in the xanthine oxidase reaction. We also confirmed their results under similar conditions but could not detect the formation of glutathione sulfonate in the tyrosinemediated Op-consuming oxidation of GSH catalyzed by lactoperoxidase. This difference is not yet explained. We assume that tyrosine is oxidized by the thyroid peroxidase system through Reaction 6. However, there is some complication in the two-electron oxidation catalyzed by thyroid peroxidase. Even though tyrosine reduces Compound I compulsorily to the ferric state, the one-electron reduction of Compound I to Compound I1 will take place slowly at the expense of unidentified endogenous electron donors (32). This one-electron reduction of Compound 1 will result in the formation of a small amount of glutathione free radical as seen in Fig. 5. Since Compound I1 of thyroid peroxidase is much of GSH even when the reaction was caused either by lactoperoxidase or thyroid peroxidase under aerobic conditions. less reactive toward tyrosine and iodide (18), its accumulation Hp02 itself was reported to be oxidized by the oxidized iodide will slow down the oxidation of tyrosine (Fig. 1B). (28-30) and the rate of the reaction of H202 with the oxidized The peroxidase-catalyzed aerobic oxidation of GSH appears iodide was measured kinetically from the effect of HpOp con- to occur through a free-radical chain reaction consisting of centration upon the rate of 0, evolution at varied iodide Reactions 9, 10, and 12. concentrations. The rate constant was compared with that GSH + 0; + H+- GS. + HZ02 (12) for GSH in Table 111, which showed that the oxidized iodide reacted mostly with GSH rather than HpOz under the present Reaction 12 has been reported (12, 33) and is a key reaction experimental conditions. to be involved in most peroxidase-catalyzed aerobic oxidations

5 One-electron Two-electron and Oxidations of GSH (34), which are inhibited by catalase and superoxide dismu- 6. Saez, G., Thornalley, P. J., Hill, H. A. O., Hems, R., and Bannistase. ter, J. V. (1982) Biochim. Biophys. Acta 719, Ross, D., Albano, E., Nilsson, U., and Moldeus, P. (1984) Iodide is a good electron donor in the reactions of lactoper- Bwchem. Bwphys. Res. Commun. 125, oxidase and thyroid peroxidase, and is oxidized by Compound 8. Harman, L. S., Mottley, C., and Mason, R. P. (1984) J. Biol. I (EO) of these peroxidases to an iodinium cation, which is Chem. 259, assumed to be present as an enzyme-bound form (29,35). 9. Eling, T. E., Mason, R. P., and Sivarajah, K. (1985) J. Bwl. Chem. EO + I- - E02T (13) This iodhium cation reacts with various reductants (27). When GSH and H202 are used as the reductant, the reactions will be the following. EOZ-I+ + GSH + H' - E + Hz0 + GSI (14) GSI + GSH - GSSG + I- + H' (15) EOz-I+ + HZ02 E I- + Hz0 (16) Through these reactions the iodide may serve as an effective mediator in the peroxidase-catalyzed oxidation of GSH (36) and decomposition of H2O2 (29). These reactions are concluded to occur by way of two-electron transfer. The marked difference between GSH and H202 is that Compound I of lactoperoxidase and thyroid peroxidase reacts directly with H202 but not with GSH (Table 111). It is of interest that these peroxidases act as an effective GSH peroxidase in the presence of a small amount of iodide. In this case no free radicals are formed and O2 is not consumed during the oxidation of GSH even under aerobic conditions. In this sense this reaction system is similar to that of selenium glutathione peroxidase. After completion of this work, a publication appeared from Harman et al. (37), who reported one- and two-electron oxidation of GSH by peroxidases. 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