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Home , Catalase, Horseradish peroxidase, Myeloperoxidase

J. Biochem. 101, 1407-1412 (1987)

Reaction of Human with

and Its True Activity1

Hiroyuki IWAMOTO,* Takashi KOBAYASHI,** Eiichi HASEGAWA,** and Yuhei MORITA*,2

*Research Institute for Food Science, Kyoto University, Uji, Kyoto 611; and **Green Cross Co., Joto-ku, Osaka, Osaka 534

Received for publication, December 23, 1986

The instability of human myeloperoxidase [EC 1.11.1.7] compound 1, which was spontaneously reduced to compound II, and the abnormal stoichiometry of the

reaction of myeloperoxidase with H2O2 were investigated. As to the former, a

pretreatment of myeloperoxidase with H2O2 did not stabilize compound I, and no difference in its stability was observed between native (ƒ¿2ƒÀ2) and hemi (ƒ¿ƒÀ) myelo

. From these results, it was thought that the instability of compound I was caused by neither the presence of endogenous donors nor the intramolecular

reduction of compound I to compound II by the other in the native

molecule. As for the latter, true catalase activity of myeloperoxidase was demon

strated by monitoring 02 evolution after the injection of H2O2 into the enzyme

solution. Myeloperoxidase compound I reacted with H2O2 and returned to the

ferric state with concomitant evolution of an 02 molecule. Accordingly, the abnor

mal stoichiometry of the reaction with H2O2 and a part of the instability of com

pound I can probably be ascribed to this true catalase activity.

Myeloperoxidase (MPO) is a heme-containing Previous studies have shown that MPO is a glycoprotein located in the azurophilic granules component of the and halogen-dependent of polymorphonuclear (1). This en microbicidal system of leukocytes (4, 5). In the zyme is composed of two large and two small stage of , MPO catalyzes the oxidation subunits and contains two molecules of heme, of Cl to OCL-, one of the agents responsible for the whose chemical structure has not been elucidated oxidative degradation of invading microbes and yet (2). As reported in our previous paper, we other biological substances, at the expense of crystallized human MPO and separated three H2O2 generated during the (6, 7). multiple forms of this enzyme (3). This enzyme also catalyzes halogen -inde pendent peroxidation of various hydrogen donors 1 This study was supported in part by Grant-in-Aid for such as guaiacol, which is a typical reaction of all Scientific Research (No. 61470128) from the Ministry of other . Harrison et al. (8) demon Education. Science and Culture of Japan. strated that this reaction proceeded via compound 2 To whom correspondence should be addressed. Abbreviations: MPO, myeloperoxidase; HRP, horse I and compound 11, the primary and the secondary radish peroxidase; MCD, monochlorodimedone. intermediates, which have similar optical spectra

Vol. 101, No. 6, 1987 1407 1408 H . IWAMOTO, T. KOBAYASHI, E. HASEGAWA, and Y. MORITA

to those of (HRP), using 17,700M-1•Ecm-1 at 290 nm (15) were employed

a stopped flow/rapid scan instrument (Reactions for the determination of H2O2 and MCD concen

1-3). tration, respectively. All experiments in this study

were carried out in 50mM sodium phosphate buff MPO+H2O2 •¨ compound I (1) er, pH 7.0, at 25•Ž, unless otherwise mentioned. compound I+AH •¨compound II+A. (2) Hydrogen Peroxide Titration-A hydrogen compound II+AH •¨ MPO+A (3) peroxide titration was carried out by the addition where AH is a one-electron hydrogen donor. of 1.0mM H2O2 to 1.0 ml of 4.05ƒÊM enzyme Compound I is the sole catalytic intermediate of solution in a quartz microcell with a light-path the peroxidation of Cl to OCI as indicated in length of 1.0 cm. Difference spectra were recorded Reaction 4 (9). immediately after the addition of H2O2 on a Shimadzu UV-260 spectrophotometer and the compound I + C.- •¨ MPO+OCI- (4) absorbance changes at 456 nm were plotted against Horseradish peroxidase compound I can be the ratio of the concentration of H2O2 to the en prepared in a relatively stable state with a half-life zyme. of 15-20 min by the addition of equimolar H2O2 Stopped-Flow Analysis-The compound II (10). On the other hand, compound I of MPO formation rates were determined with a Union is very unstable, having a half-life of 100 ms, and Giken stopped-flow rapid reaction analyzer (model being spontaneously reduced to compound II (8). RA-401 and RA-601) equipped with a 2 mm reac In addition, more than 10-fold excess of H2O2 is tion cell. The increase of the absorbance at 456 nm, required to convert the ferric enzyme to compound the isosbestic point between the ferric enzyme and II (11). These two points are unique features of compound I (8), was followed. One driving syringe

the reaction of MPO with H2O2 in comparison of this apparatus contained 1.98ƒÊM enzyme solu with HRP. However, their mechanisms have not tion and the other contained 30ƒÊM H2O2 solution. been elucidated. Determination of 02 Concentration-The cata The present investigation was centered on the lase activity of MPO or HRP was measured with above two problems, the instability of compound a Hansatech oxygen electrode unit. The 02 evolu I and the abnormal stoichiometry of the reaction tion after the injection of 1.0mM H2O2 into 1.0 with H2O2, and demonstrated that the latter was ml of the enzyme solution was monitored. Oxygen due to the true catalase activity of MPO. concentration was determined from a calibration curve constructed with 300 Sigma units of bovine MATERIALS AND METHODS catalase.

Materials-Human native MPO-III, Hemi RESULTS AND DISCUSSION MPO-III, and HRP C-1 used in these studies were purified as reported previously (3, 12). These Figure 1 shows the difference spectra in the titra preparations exhibited absorption ratios A429/A280 tion of MPO with H2O2, which resemble those of of 0.82 for MPOs and A403/A280of 3.20 for HRP. the canine enzyme reported by Agner (11). These

Twice-recrystallized bovine liver catalase and mon spectra reveal well-defined isosbestic points be ochlorodimedone (MCD) were purchased from tween the ferric enzyme and compound II , and Sigma Chemical Company (St. Louis, U.S.A.). the formation of compound II was completed

Hydrogen peroxide and all other chemicals were when a 13-fold excess of H2O2 was added. In of analytical grade and were obtained from Wako the course of this reaction , Harrison et al. (8) Pure Chemical, Ltd. (Osaka, Japan). reported a primary intermediate , compound I, The concentration of the was deter having similar Soret and visible spectra to those of mined spectrophotometrically using millimolar HRP compound I. We obtained a second-order absorption coefficients of 89mM-1•Ecm-1 per heme rate constant of the order of 107M-1•Es-1 for the at 428 nm for MPO (13) and 102mM-1•Ecm-1 at formation of compound I (Reaction 1) and a value

403 nm for HRP (12). The molar absorption of about 1.8s-1 for compound II formation by coefficients of 39.4M-1•Ecm-1 at 240 nm (14) and the stopped-flow method.

J. Biochem. REACTION OF MYELOPEROXIDASE WITH H2O2 1409

Fig. 1. Soret and visible difference spectra between ferric myeloperoxidase and its compound 11. The spectra were recorded after the successive addition of 1.0mM H2O2 solution to 1.0 ml of 4.05 ƒÊM enzyme solution in a quartz micro

cell. Hydrogen peroxide concentration at each step was as follows: a, 0.0 Jim; b, 2.0 ƒÊM; c, 5.0 ƒÊM; d, 8.9 ƒÊM; e, 16.7 ƒÊM; f, 26ƒÊM; g, 36 ƒÊM; h, 51 ƒÊM; i, 72 ƒÊM.

Odajima and Yamazaki (16) reported that MPO compound II had one oxidizing equivalent above the ferric enzyme. Hence, the presence of a hydrogen donor is required for the spontaneous reduction of compound I to compound IL Endo genous hydrogen donors are well known to affect the stability of HRP compound h Thus, we first assumed that the presence of some endogenous donor(s) was responsible for the spontaneous re duction of compound I to compound 11 and the abnormal stoichiometry of the reaction with 1420, Since myeloperoxidase compound II slowly re turned to the ferric enzyme, with a half-life of about 20 min, the enzyme solution titrated with Fig. 2, Hydrogen peroxide titration curves of myelo H2O2 was allowed to stand overnight (the absorp peroxidase. The titration conditions were the same tion ratio A429/A280fell from 0.82 to about 0.71), as those of Fig. 1. Absorbance changes at 456 nm and retitrated on the next day. Figure 2 shows corresponding to the formation of compound II were the titration curves on which the absorbance plotted against the ratio of the concentration of H2O2 to the enzyme. The enzyme solution titrated with H2O2 changes at 456 nm corresponding to compound II was allowed to stand overnight and retitrated as de formation are plotted against the ratio of the scribed in the text. •›, the first titration (H2O2 un concentration of H2O2 to the enzyme. We re treated); ??, the second titration; •œ the third titration. peated this retitration 3 or 4 times, but we could not obtain the compound I spectrum, and the MPO + compound I •¨ 2 compound II (5) abnormal stoichiometry of the reaction still re mained. These results indicate that the above two Myeloperoxidase is composed of two half-enzymes features were not caused by the presence of endo and each of them contains one heme (2). Hence, genous donor. it could be thought that one of the two Next we assumed the following mechanism: first reacted with H2O2 and then the other home

Vol. 101, No. 6, 1987 1410 H. IWAMOTO, T. KOBAYASHI, E. HASEGAWA, and Y. MORITA

Fig. 3. Recorder traces of 0, evolution after the successive injection of 1.0 mM H2O2 solution into 1.0 ml of (A) catalase (300 Sigma units), (B) 7.96ƒÊM myelo

peroxidase, and (C) 7.09ƒÊM horseradish peroxidase solution. The arrows indicate H2O2 injections and H2O2 concentration at each step was as follows: a, 5.0 p M; b, 9.9 ƒÊM; c, 19.6 ƒÊM; d, 29.1 ƒÊM; e, 48 ƒÊM.

reduced the formed compound I by one electron to compound II. We carried out H2O2 titrations and measured the compound II formation rates for the native and hemi enzymes in 0.2 M phos phate buffer, pH 7,0, under which conditions hemi enzyme did not associate to the native form (3). However, we obtained nearly the same titration curves for the two enzymes and similar compound II formation rates, 1.78 s-1 for the native enzyme and 1.73 s-1 for the hemi enzyme. Based on the two half-enzymes structure, it seems unlikely that compound I formed is intermolecularly reduced by another enzyme molecule. Accordingly, we con cluded that Reaction 5 did not take place and Fig. 4. Plots of the evolved 02 after the successive was not responsible for the spontaneous reduction injection of H2O2 into myeloperoxidase and horseradish of compound I. peroxidase solution against the concentration of H2O2. The other question concerning the reaction of The enzyme concentration and other experimental con MPO with H2O2 is why more than 10-fold excess ditions were the same as those of Fig. 3. Oxygen con centration was determined from a calibration curve of H202 is required for the formation of com constructed with bovine liver catalase. •›, myelo pound II. With respect to this abnormal stoichio peroxidase; •œ horseradish peroxidase; -.-.-, 02 metry of the reaction, Bolscher and Wever (17) evolution when injected H2O2 is completely decom assumed that Reaction 1 was reversible for MPO posed to 02. and expressed this abnormal stoichiometry in terms of an equilibrium reaction. This explanation, how ever, is unlikely because compound I of other H2O2 into catalase, MPO, and HRP solution in peroxidases is not a Michaelis-Menten type com an oxygen electrode cell. As can be seen from plex and Reaction 1 is irreversible (18). these traces, MPO and catalase evolved 02 im We followed 02 evolution using an oxygen mediately after the injection of H2O2, whereas 02 electrode to determine whether the excess H2O2 evolution from HRP solution was relatively slow. added to the enzyme solution remained intact or Figure 4 shows plots of evolved 02 against the was decomposed to 02. Figure 3 shows the traces concentration of HO, added to the enzyme solu of 02 evolution after the successive injection of tion. In these plots, MPO did not exhibit any

J. Biochem. REACTION OF MYELOPEROXIDASE WITH H2O2 1411

lag in the 02 evolution, i.e. O2 evolution was ob served on addition of even less than equimolar H2O2. On the other hand, HRP showed an ob vious lag phase due to the formation of compound I. These findings combined with the results in Fig. I show that MPO, contrary to HRP, can decompose 14202 to 02 before the formation of compound II. This means that MPO has true

catalase activity as below: Fig. 5. Recorder traces of OZ evolution after the injec tion of H2O2 into 2.5ƒÊM myeloperoxidase solution in compound I + H2O2 •¨ MPO + O2 + H2O the absence or presence of NaCl. Injections of 14202 (6) are indicated by the arrows and the final concentration was 50 FEM. A, without NaCl and monochlorodime In Fig. 4, the differences between the amount of done; B, with 0.1 M NaCl and 50ƒÊM monochloro 02 evolved from MPO or HRP solution and the dimedone; C, with 50ƒÊM monochlorodimedone. In complete decomposition of H2O2 to 02 were prob the course of the reaction (C), 300 Sigma units of ably due to the consumption of added H2O2 for catalase were injected at the position indicated by the arrow. the formation of compound I or compound II or the oxidation of the protein moiety. This true catalase activity was not due to seconds during incubation with H2O2 and Cl (20). contamination of catalase for the fol Figure 5 shows that the catalase activity is com lowing reasons: 1) Catalase is an acidic protein pletely inhibited after a slight and immediate 02 whose pl is around 5.5, unlike MPO which is a evolution by the presence of 0.1M Cl-, which is strongly basic protein (pI>11), so catalase would near to the intracellular concentration in human have been removed by cation exchange chro leukocytes (21) and to the dissociation constant matography in the course of the purification; 2) of the MPO-CI complex (22). The amount of this true catalase activity was inhibited by the evolved 02 of Fig. 5B was about 20% of that of presence of more than 1mM H2O2 with the break Fig. 5A and it might be caused by the reaction down of the Soret spectra of MPO. of OCl- with H2O2. Such a halogen ion-dependent Catalase activity of peroxidases such as HRP pseudocatalatic reaction has also been reported is well known, and previous studies have reported for (23). In the reaction of Fig. that of MPO (11). Harrison (9) proposed a Cl- 5B, the Soret band of the enzyme was decreased dependent catalatic mechanism involving the reac by only a few percent and 50-60% of MCD was tion of H2O2 with OCl- formed by the peroxidatic observed to be chlorinated to dichlorodimedone. reaction of MPO. Winterbourn et al. (15) sug These findings indicated that the inhibition of the true catalase activity was not caused by inactiva gested a halogen ion-independent catalatic mech anism, where compound III played an important tion of the enzyme. role. These catalase activities, however, proceeded Even in the absence of Cl-, the true catalase by a pseudocatalatic mechanism. In this study, activity appeared to be slightly inhibited by the we experimentally demonstrated the true catalase presence of 50ƒÊM MCD (Fig. 5C). When cata activity of MPO, which was predominant in the lase was injected in the course of this reaction, range of less than several-fold excess of H2O2. the amount of 02 evolution recovered to the con As expressed in Reaction 4, MPO compound trol level. In addition, only a 1.5-fold excess of I oxidizes Cl to OCl-. We examined whether 14202 was sufficient to convert MPO to compound the presence of CI inhibited the true catalase II in the presence of 50 ƒÊM MCD. Next, we activity of MPO or not. The reaction mixture measured the compound II formation rates at contained 50ƒÊM MCD, which is an efficient OC various MCD concentrations by the stopped-flow method, and we observed that they were directly l- scavenger and is stoichiometrically chlorinated to dichlorodimedone (19), because the heme groups proportional to the MCD concentration. Based on these results, MCD seemed to play a role as and oxidizable residues of the enzyme a hydrogen donor for compound I. have been reported to be damaged within a few

Vol. 101, No. 6, 1987 1412 H. IWAMOTO, T. KOBAYASHI, E. HASEGAWA, and Y. MORITA

REFERENCES 1. Cramer, E., Pryzwansky, K.B., Villeval, J.-L., Testa, U., & Breton-Gorius, J. (1985) Blood 65, 423-432 2. Andrews, P.C. & Krinsky, N.I. (1981) J. Biol. Chem. 256, 4211-4218 3. Morita, Y., Iwamoto, H., Aibara, S., Kobayashi, T., & Hasegawa, E. (1986) J. Biochem. 99, 761-770 4. Klebanoff, S.J. (1968) J. Bacteriol. 95, 2131-2138 5. Lehrer, R.I. (1969) J. Bacteriol. 99, 361-365 6. Albrich, J.M., McCarthy, C.A., & Hurst, J.K. We summarize our present results and other (1981) Proc. Natl. Acad. Sci. U.S. 78, 210-214 7. Foote, C.S., Goyne, T.E., & Lehrer, R.I. (1983) studies in Scheme 1. The mechanism of the Nature 301, 715-716 spontaneous conversion of compound I to com 8. Harrison, J.E., Araiso, T., Palcic, M.M., & Dun pound II has not been elucidated yet. In this ford, H.B. (1980) Biochem. Biophys. Res. Commun. connection, the hydrogen donor represented by 94,34-40 XH in the Scheme might be H2O2, because MPO 9. Harrison, J.E. (1982) in Oxidases and Related compound I had such a high potential to Redox Systems (King, T.E., Mason, H.S., & oxidize Cl- to OCl-. If so, compound I is reduced Morrison, M., eds.) pp. 717-732, Pergamon Press, to compound II by H2O2 with concomitant for Oxford mation of one 02 molecule. 10. Dunford, H.B. & Nadezhdin, A.D. (1982) in In the reaction of MPO with H2O2, we as Oxidases and Related Redox Systems (King, T.E.,

sumed two different mechanisms for 02 evolution. Mason, H.S., & Morrison, M., eds.) pp. 653-670, Pergamon Press, Oxford When less than several-fold excess of H2O2 was 11. Agner, K. (1963) Acta Chem. Scand. 17, S332-S338 injected into the enzyme solution in the absence 12. Aibara, S., Yamashita, H., Mori, E., Kato, M., of halogen , excess H2O2 was decomposed to & Morita, Y. (1982) J. Biochem. 92, 531-539 02 and H2O mainly through Reactions 1 and 6. 13. Agner, K. (1958) Acta Chem. Scand. 12, 89-94 This true catalase activity probably caused the 14. Nelson, D.P. & Kiesow, L.A. (1972) Anal. Biochem. abnormal stoichiometry of the reaction between 49,474-478 MPO and H2O2 and a part of the instability of 15. Winterbourn, C.C., Garcia, R.C., & Segal, A.W.

compound I. Since it was difficult to capture the (1985) Biochem. J. 228, 583-592

spectrum of MPO compound I, as in the case of 16. Odajima, T. & Yamazaki, 1. (1970) Biochim. Bio catalase (24), the rate of Reaction 6 was estimated phys. Acta 206, 71-77

to be faster than that of Reaction 1, the rate con 17. Bolscher, B.G.J.N. & Wever, R. (1984) Biochim. Biovhvs. Acta 788. 1-10 stant of the latter being reported to be 2.3•E107 18. Jones, P. & Dunford, H.B. (1977) J. Theor. Biol. M-1•Es-1 (17). Nevertheless, we could not deter 69,457-470 mine the exact rate constant of Reaction 6 be 19. Wilson, I., Bretscher, K.R., Chea, C.K., & Kelly, cause the 02 evolution rates observed with an H.C. (1983) J. Inorg. Biochem. 19, 345-357 oxygen electrode are limited by the diffusion rate 20. Matheson, N.R., Wong, P.S., & Travis, J. (1981) of 02 molecules through the Teflon membrane 20, 325-330 covering the cathode. When more than several 21. Baron, D.N. & Ahmed, S.A. (1969) Clin. Sci. 37, fold excess of H2O2 was added, most of the ferric 205-219

enzyme was converted to compound II, which is 22. Bakkenist, A.R.J., De Boer, J.E.G., Plat, H., &

an inactive form as regards the true catalase ac Wever, R. (1980) Biochim. Biophys. Acta 613, 337-348 tivity, and relatively slow 02 evolution was ob 23. Ohlsson, P.-I. (1986) Acta Chem. Scand. B40 served. The mechanism of this pseudocatalatic , 358 -362 degradation of H2O2 to 02 has not been elucidated 24. Chance, B. (1947) Acta Chem. Scand. 1, 236-267 yet; this 02 evolution probably proceeded by the same mechanism as that of HRP.

J. Biochem.