Free Radical Biology & Medicine, Vol. 13, pp. 17-20, 1992 0891-5849/92 $5.00 + .00 Printed in the USA. All rights reserved. Copyright © 1992 Pergamon Press Ltd.

Original Contribution

O X I D A T I O N O F 3 - H Y D R O X Y K Y N U R E N I N E C A T A L Y Z E D BY M E T H E M O G L O B I N W I T H H Y D R O G E N P E R O X I D E

TOSHIHIRO ISHII, t HIDEO IWAHASHI, ;t RYOJIN SUGATA, t and RYO KIDO *+

Departments of *Biochemistry and *Chemistry, Wakayama Medical College, 27 Kyu-bancho, Wakayama, 640 Japan

(Received 25 July 1991; Revised 15 November 1991; Accepted 19 December 1991 )

Abstract--Methemogiobin (metHb) with H202 catalyzed the oxidation of 3-hydroxykynurenine (3-HKY) in the reaction mixture of metHb, 3-HKY, and H202. The spectrophotometric experiments suggest the following mechanism for the 3-HKY oxidation by metHb with H202. MetHb first reacts with H202 to form the ferryl complex of Hb. This species then oxidizes 3-HKY, while it returns to metHb. 3-HKY was more reactive with the ferryl complex than glutathione but less reactive than ascorbic acid. Scavengers of the hydroxyl radical, dimethyl sulfoxide and ethanol, scarcely inhibited the 3-HKY oxidation by metHb with H202. Desferrioxamine, a metal chelator, hardly suppressed the 3-HKY oxidation. These results indicate that the hydroxyl radical is not involved in the 3-HKY oxidation by metHb with H202.

Keywords--3-Hydroxykynurenine, Methemoglobin, Oxidation, Hydrogen peroxide, Free radicals

INTRODUCTION

3-Hydroxykynurenine (3-HKY) is a tryptophan me- tabolite that occurs in the kynurenine pathway. It is known to be carcinogenic and neurotoxic. Bladder cancers were induced by 3-HKY in mice. 1,2 3-HKY was toxic to a neuronally derived hybrid cell line and oxidative stress was supposed to be critical for the tox- icity. 3 In contrast to these deleterious effects, 3-HKY has been reported to have antioxidative activity. 3- HKY was shown to have a scavenging activity for the superoxide radical and exert the role in Malpighian tubes of insects. 4 3-HKY protected B-phycoerythrin from peroxyl radical-mediated oxidative damage/ Because the oxidation of 3-HKY is supposed to be involved in these toxic effects and antioxidative activ- ity, it is important to examine details of the oxidation of 3-HKY. 3-HKY is easily oxidized to xanthomma- tin (XA) by potassium fen-icyanide. 6-8 We here report the oxidation of 3-HKY by hemoglobin (Hb) with hydrogen peroxide, which is known to undergo oxida- tion of phenols 9 and peroxidation of fatty acids. I°,11 We also examine the involvement of the hydroxyl rad- ical in the 3-HKY oxidation by Hb with hydrogen

Address correspondence to: Ryo Kido.

peroxide. Involvement of iron released from Hb for a Fenton reaction seems to be a subject of dispute.l°,12,13

MATERIALS AND METHODS

Materials

DL-3-Hydroxykynurenine (3-HKY) was prepared according to the method of Kotake et al. 14 with slight modification. Human hemoglobin (Hb) was pur- chased from Sigma Chemical Co. (St. Louis, MO, USA). Hb was oxidized with potassium ferricyanide or reduced with sodium hydrosulfite under aerobic conditions, and then purified by gel filtration of Seph- adex G-25.15 Heme concentration was determined by the pyridine hemochromogen method by using ~557 nm -- 33.4 mM -1" cm-I. 16 Desferrioxamine (Desferal)was from Ciba-Geigy Japan (Takarazuka, Japan). All other chemicals used were commercial products of the highest grade available.

Methods

The standard reaction mixture consisted of metHb (50 gM in heme), 3-HKY (0.2 mM), and H202 (0.2 mM) in sodium phosphate buffer (50 mM, pH 7.4). The reactions were initiated by adding H202 and were carried out at 22°C. Spectrophotometric measure-

17

18 1. ISHi! el ~1[.

0,5

0.4

0.3 E

o 0.2 i n

<] 0.1

0.0

i I , I i I , i *

0 1 2 3 4 5

Time (rain)

Fig. 1. Oxidation of 3-HKY by metHb with H202. The oxidation of 3-HKY was followed by the increase in absorbance at 450 nm caused by the oxidized product, xanthommatin. The standard reac- tion mixture contained metHb (50#M in berne), 3-HKY (0.2 raM), and H202 (0.2 mM) in the sodium phosphate buffer (50 mM, pH 7.4). The reactions were initiated by adding H202 and carried out at 22°C. (O), Complete: (e), without metHb: (A), without H202.

with the results that Hb or H,O, alone did not oxidizc 3-HKY (Fig. 1).

Next, 3-HKY was added to the species I rain after H202 and metHb had been mixed. 3-HKY reacted with the species to increase the absorbance at 450 nm (data not shown). However, the increase in absor- bance at 450 nm was 60'~i lower than that for tile standard reaction where H~O, was added to the reac- tion mixture containing 3-HKY and metHb. After the reaction of the species with 3-HKY was com- pleted, a spectrum similar to that of metHb was left (Fig. 3). Almost the same spectrum was obtained after the completion of the standard reaction where H,O, was added to the reaction mixture containing 3-HKY and metHb.

From the results described earlier, the following mechanism is assumed for the 3-HKY oxidation in the reaction mixture of metHb, 3-HKY, and H202. MetHb reacts first with H202 to t'orm a reactive spe- cies, which then oxidizes 3-HKY to xanthommatin, while the reactive species returns to metHb.

merits were carried out using a Shimadzu UV 160 spec- t rophotometer (Kyoto, Japan) with a 10-mm light path cuvette. The oxidation of 3-HKY was followed by the increase in absorbance at 450 nm caused by the oxidized product, xanthommatin. 6-8

RESULTS AND DISCUSSION

For the reaction mixture of metHb, 3-HKY, and H202, the absorbance at 450 nm increased (Fig. 1). indicating that 3-HKY was oxidized to xanthomma- tin. 6-8 The oxidation was completed in 2 rain. With- out metHb or H202, the oxidation did not proceed (Fig. 1).

The interactions of 3-HKY, H202 and 3-HKY were examined separately. For the mixture of metHb and H202, the absorbance spectrum of metHb changed (Fig. 2), indicating that metHb reacted with H202 to form a species that showed a different spec- t rum from that of metHb or oxyHb. A shoulder at 590 nm in the spectrum of the species is characteris- tic. This reaction was completed in 1 min, and the spectrum of the species remained unchanged several minutes. For the mixture of 3-HKY and H 2 0 2 , there were no changes in the absorbance spectrum of 3- H K Y from 300 to 500 nm in 10 min. For the mixture of 3-HKY and metHb, 3-HKY did not affect the ab- sorbance spectrum of metHb from 500 to 700 nm in 10 min. These results indicate that 3-HKY does not react with H202 or with metHb. They correspond

MetHb + H~O~ --* reactive species.

Reactive species + 3-HKY --~

metHb + xanthommatin.

It was reported that metHb reacted with H 2 0 2 tO pro- duce a metHb-peroxide compound.~S The reaction of H2O 2 with hemoproteins has been known to generate a high oxidative state of the heine iron, the ferryl com- plex Fe(IV) - O. ~9-2~ The complex (and/or its free-

0.8 ¸

0.6

0.4

0.2

0.0

-,o,,= 3

, I i

5 0 0 6 0 0 7 0 0

Wavelength (nm)

Fig. 2. Change of the absorbance spectrum of metHb in the reaction with H202. Line 1: the absorbance spectrum of metHb (55 #M in heme) in the phosphate buffer. Line 2:H202 (0.22 mM at the final) was added to the metHb solution. The absorbance spectrum was taken 1 min after the addition of H~02, when the spectral changes stopped. Line 3: the absorbance spectrum of oxyHb (55 uM in heme) in the phosphate buffer.

Oxidation of 3-HKY 19

0.8

0.6

O t-- m .o 0.4 0

.Q <

0.2 1

0.0 , i , 5 0 0 6 0 0 7 0 0

Wave length (nm)

Fig. 3. Change of the absorbance spectrum of the species in the reaction with 3-HKY. Line 1: the absorbance spectrum of the spe- cies formed by adding H202 (0.22 mM at the final) to metHb (55 uM in heme). The absorbance spectrum was taken 1 min after the addition ofH202. Line 2: 3-HKY solution was added to the species 1 rain after the reaction ofmetHb with H202, yielding final concen- trations of 0.2 mM for 3-HKY and H202 and 50 uM in heme for metHb. The absorbance spectrum was taken 3 min after the addi- tion of 3-HKY.

radical form) has been implicated in the oxidation of phenols, 9 fl-carotene, ascorbic acid, 21 lipid, l 1.2 ] salicy- lateY cholesterol, 22 and glutathione. 23 The spectrum of the reactive species (Fig. 2) is in accordance with the spectrum of ferryl complex. 24,25 The reactive spe- cies in the study here is the ferryl complex of Hb.

Ascorbic acid and glutathione were reported to react with the ferryl complex] 1,23 Ascorbic acid or glu- tathione was competed with 3-HKY in the reaction with the ferryl complex of l ib (Table 1). Ascorbic acid of the same molar suppressed the 3-HKY oxidation by metHb with H202 more than 50%, indicating that ascorbic acid reacts with the ferryl complex of Hb more easily than 3-HKY. However, the inhibition by glutathione (reduced) was weak, suggesting that the

Table 1. Effects of Ascorbic Acid and Glutathione on the 3-HKY Oxidation by metHb with H202

Reagent Added A 450 nm

(% of Standard Reaction)

None (standard reaction) 100 Ascorbic acid (0.2 mM) 24 Glutathione (0.4 mM) 79 Glutathione (2 mM) 58

Ascorbic acid or glutathione (reduced) was contained in the standard reaction mixture of metHb (50 #M in heme), 3-HKY (0.2 mM), and H202 (0.2 mM). The reactions were initiated by adding H202 and carded out at 22°C. The in- crease in absorbance at 450 nm was measured in the first 3 min and was compared with that of the standard reaction.

Table 2. Effects of Desferal, EDTA, DMSO, and Ethanol on the 3-HKY Oxidation by metHb with H202

A 450 nm Reagent Added (% of Standard Reaction)

None (standard reaction) 100 Desferal ( l mM) 85 EDTA (1 mM) 101 DMSO (5% v/v) 86 Ethanol (5% v/v) 87

Desferal, EDTA, dimethyl sulfoxide (DMSO), or ethanol was contained in the standard reaction mixture of metHb (50 uM in heme), 3-HKY (0.2 mM), and H2Oz (0.2 mM). The reactions were initiated by adding H202 and carried out at 22°C. The increase in absorbance at 450 nm was mea- sured in the first 3 min and was compared with that of the standard reaction.

reactivity of glutathione with the ferryl complex is lower than 3-HKY.

It has been reported that the hydroxyl radical is generated from free iron ion released from Hb in the reaction of Hb and H202 and that Desferal suppresses the hydroxyl radical generation. 1°'13 Desferal hardly suppressed the 3-HKY oxidation by metHb with H202 (Table 2). EDTA did not affect the 3-HKY oxi- dation at all. Dimethyl sulfoxide (DMSO) and eth- anol, scavengers of the hydroxyl radical, scarcely in- hibited the 3-HKY oxidation (Table 2). These results suggest that the hydroxyl radical is not involved in the 3-HKY oxidation by metHb with H202. For the iron release from hemoprotein by H202, large excess of H202 over the protein and hour-time exposure to H202 were needed. 1o,13,26,27 In the experiments here, a four-fold molar excess 0fH202 was used, and the reac- tions were completed in several minutes. The iron release seems to be slight, and the generation of the hydroxyl radical might be scarce.

Hb with H202 oxidized 3-HKY. From another point of view, 3-HKY decomposed H202 with the aid of Hb. From this point, 3-HKY is a possible antioxi- dant against H202.3-HKY might attenuate the toxic- ity of H202.3-HKY was more reactive with the ferryl complex than glutathione, suggesting that the antioxi- dative efficiency is better than glutathione. The amount of 3-HKY in human bodies is scarce. How- ever, 3-HKY is abundant in Malpighian tubes of in- sects and was reported to work as a major antioxidant in the tubes. 4 It is of interest to study further the an- tioxidative activity of 3-HKY in biological systems.

R E F E R E N C E S

1. Allen, M. J.; Boyland, E.; Dukes, C. E.; Horning, E. S.; Watson, J. G. Cancer of the urinary bladder induced in mice with metab-

20 T. ISHII e: al.

olites of aromatic amines and tryptophan. Br. J. Cancer 11:212-230; 1957.

2. Bryan, G. T.; Brown, R. R.: Price, J. M. Mouse bladder carcino- genicity of certain tryptophan metabolites and other aromatic nitrogen compounds suspended in cholesterol. Cancer Re.~. 24:596-602; 1964.

3. Eastman, C. L.: Guilarte, T. R. Cytotoxicity of3-hydroxy kyn- urenine in a neural hybrid cell line. Brain Res. 495:225-231: 1989.

4. Goshima, N.; Wadano, A.; Miura, K. 3-Hydroxykynurenine as O2- scavenger in the blowfly, AIdrichina grahami. Biochem Biophys. Res. Commun. 139:666-672; 1986.

5. Christen, S.: Peterhans, E.; Stocker, R. Antioxidant activities of some tryptophan metabolites: Possible implication for inflam- matory diseases. Proc. Natl. Acad. Sci. USA 87:2506-2510; 1990.

6. Butenandt, A.: Schiedt, U.; Biekert, E.; Kornmann, P. 0ber Ommochrome, I. Mitteilung: Isolierung yon Xanthommatin, Rhodommatin und Ommatin C aus den Schlupfsekreten yon Vanessa urticae. Liebigs Ann. ('hem. 586:21%228; 1954.

7. Butenandt, A.; Schiedt, U.: Biekert, E. Ober Ommochrome, 1II. Mitteilung: Synthese des Xanthommalins. Liebi,~s Ann ('hem. 588:106-116; 1954.

8. Butenandt, A.; Schiedt, U.; Biekert, E.; Cromartie, R. J. Y. Ober Ommochrome, IV. Mitteilung: Konstitution des Xanth- ommatins. Liebigs Ann. ('hem. 590:75-90; 1954.

9. Shiga, T.: lmaizumi, K. Electron spin resonance study on per- oxidase- and oxidase- reactions of horseradish peroxidase and methemoglobin. Arch. Biochem. Biophys. 167:469-479: 1975.

10. Gutteridge, J. M. C. Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by perox- ides. FFBS Lett. 201:291-295:1986.

11. Kanner, J.; Hard, S. Inhibition of membranal lipid peroxida- tion by activated metmyoglobin and methemoglobin. Arch. Biochem. Biophys. 237:314-321 ; 1985.

12. Sadrzadeh, S. M. H.; Graf, E.; Panter, S. S.; Hallaway, P. E.; Eaton, J. W. Hemoglobin, a biologic Fenton reagent. J. Biol. Chem. 259:14354-14356; 1984.

13. Puppo, A.; Halliwell, B. Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Is haemoglobin a biological Fenton reagent? Biochem. J. 249:185-190; 1988.

14. Kotake, M.; Sakan, T.; Senoh, S. Studies on amino acids. IV. The synthesis of 3-hydroxykynurenine. J. Am. Chem. Soc. 73:1832-1834; 1951.

15. Mieyal, J. J.; Blumer, J. L. Acceleration of the autooxidation of human oxyhemoglobin by aniline and its relation to hemoglo- bin-catalyzed aniline hydroxylation. J. Biol. Chem. 251:3442- 3446; 1976.

16. Paul, K. G.; Theorell, H.; Akeson, A. The molar light absorp-

tion ofpyridine ferroprotoporphyrin (pyridine haemochromo- gen). Acta (,hem. Stand. 7:1284-1287:1953.

17. Keilin, D.; Hartree, E. F. Reaction of methaemoglobin with hydrogen peroxide. Nature 166:513-514; 1950.

18. George, P.; Irvine, D. H. The reaction between metm~oglobin and hydrogen peroxide. Biochem. J. 52:51 I-517: 1952.

19. Kelso King, N.: Winfield, M. E. The mechanism of metmyo- globin oxidation. J. Biol. ('hem. 238:1520-1528: 1963.

20. La Mar, G. N.: de Ropp, J. S.: katos-Grazynski, k.; Balch, A. L.: Johnson, R. B.: Smith, K. M.; Parish, D. W.: Cheng, R.-J. Proton NMR characterization of the ferryl group in model heme complexes and hemoproteins: Evidence for the Fe TM = O group in ferryl myoglobin and compound 11 of horse- radish peroxidase..I..Ira. (Twin. Soc. 105:782-787: 1983.

21. Kanner, J.: Harel, S. Lipid peroxidation and oxidation of'sex- eral compounds by H202 activated metmyoglobin. Lipids 20:625-628: 1985.

22. Galaris, D.: Mira, D.: Scvanian, A.: ('adenas, E.: ttochstein, P. Cooxidation of salicylate and cholesterol during the oxidation of metmyoglobin by H202. , t rch. Biochem. Biophys. 262:221- 231: 1988.

23. Galaris, D.: Cadenas, E.: Hochstein, P. Glutathione-dependent reduction of peroxides during fer~l- and met-myoglobin inter- conversion: A potential protective mechanism in muscle. Frec Rad. Biol. Med. 6:473-478; 1989.

24. Winterbourn, C. C. Oxidative reactions of hemoglobin. In: Packer, L.; Glazer, A. N.. eds. Methods' in enzymology. Vol. 186 San Diego: Academic Press: 1990:265-272.

25. Puppo, A.: Cecchini, R.: Aruoma, O. I.: Bolli. R.; Halliwell, B. Scavenging of hypochlorous acid and of myoglobin-derived oxidants by the cardioprotective agent mercaptopropionylgly- cine. Free Radio. Re.s. ('ommun. 10:371-381: 1990,

26. Rice-Evans, C.: Okunade, G.: Khan, R. The suppression of iron release from activated myoglobin by physiological elec- tron donors and by desferrioxamine. Free Radio. Res. Corn- man. 7:45-54- 1989.

27. Puppo, A.: Halliwell, B. Formation of hydroxyl radicals in bio- logical systems. Does myoglobin stimulate hydroxyl radical tbrmation from hydrogen peroxide'? Free Radio. Res. Corn- mtm. 4:415-422:1988.

ABBREVIATIONS

3-HKY--3-hydroxykynurenine Hb--hemoglobin DMSO--dimethyl sulfoxide EDTA--ethylenediaminetetraacetic acid