Oxidation of isonicotinic acid hydrazide by the peroxidase system

ARCHIVES

OF

Oxidation

BIOCHEMISTRY

AND

BIOPHYSICS

of lsonicotinic

180,

452-458

(1977)

Acid Hydrazide

The Formation

by the Peroxidase

of an Excited

Product

KLAUS ZINNER, CARMEN C. C. VIDIGAL, NELSON DURAN, GIUSEPPE CILENTO Department

of Biochemistry,

Znstituto

de Quimica,

Received

September

System

Universidade

AND

de Sbo Paula, Brazil

16, 1976

The tuberculostatic and carcinogenic drug isonicotinic acid hydrazide (“isoniazid”) is oxidized to pyridine-4-carboxaldehyde by the horseradish peroxidase/Mn2+/0, system. Eosin and related sensitizers greatly accelerate the reaction and generate light detectable with the liquid scintillation counter or with the photon counter. If the isoniazid concentration is raised, the rate and extent of O2 uptake are increased, but above a certain concentration of isoniazid, emission is reduced and even suppressed. The strong quencher of triplet eosin, potassium ferricyanide, abolished both effects of eosin, that is, catalysis and light emission. Superoxide dismutase at high concentrations partially suppressed the emission and almost totally removed the catalytic effect. It is tentatively proposed that the isoniazid/peroxidase/MnZ+/02 system efficiently produces the aldehyde in the triplet state, which in turn transfers energy to eosin. Because of the presence of oxygen, only a small yield of triplet eosin is obtained and only a small fraction of these triplet eosin molecules is able to react with isoniazid. Nevertheless, it will contribute efficiently to a cyclic process of oxidation of the isoniazid.

The generation of nonemissive, or only very weakly emissive, excited electronic states in biochemical systems is under active investigation in this laboratory (l-9). The main conclusion that is emerging is that the most likely systems to generate such excited states are peroxidase-catalyzed reactions, a finding which is of considerable interest because luciferases may act as peroxidases (10). It was suggested in an earlier paper from this laboratory (11) that the tuberculostatic drug INH,’ a linear hydrazide, might generate excited species in uiuo because luminol, which is a cyclic hydrazide, yields light in the presence of microsomes (12, 13). Two facts have since reinforced this suggestion. First, linear hydrazides also yield light in chemical systems (14); second, luminol also undergoes a chemiluminescent oxidation in the peroxidase-0, system (13). It was therefore important to investi1 Abbreviations used: INH, drazide; uv, ultraviolet.

isonicotinic

acid hy-

gate whether or not INH is acted upon by the peroxidase-0, system, and if so, to look for generation of electronically excited states. A further very important reason for such a study is the fact that the tuberculostatic activity of INH is related to peroxidase activity; thus, INH-resistant strains of Mycobacteria have low or no peroxidase activity (15, 16). Peroxidase, acting peroxidatically upon INH, produces pyridine-4-carboxylic acid (16). It will be reported here that INH is oxidized by the peroxidase/Mn2+/0, system and that sensitizers such as eosin and fluorescein catalyze the reaction with concomitant light emission.2 ’ A referee has pointed out the possibility of a low-level nonspecific chemiluminescence involving eosin, especially since peroxidase systems can “leak” minute quantities of peroxides and also because catalase reduces the light emission appreciably at a concentration of 10m6 M. We comment as follows: (0 The above explanation would dissociate the two concomitant effects of eosin, namely, catalysis and emission. It would then be difficult to ex452

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN

0003-9861

EXCITED MATERIALS

AND

PRODUCT

FROM ISONICOTINIC

ACID HYDRAZIDE

453

METHODS

INH was from Ely Lilly; horseradish peroxidase (type VI), methylene blue, and *H,O (99.8%) were from Sigma. Eosin, riboflavin, p-benzoquinone, and pyronine G were from Fisher; sodium fluorescein was from BDH and potassium ferricyanide was from Baker. Superoxide dismutase (sp act 2500 units/mg of protein) was prepared from bovine red blood cells by the method of McCord and Fridovich (17). Catalase was from General Biochemicals. The standard reaction mixture consisted of 3.2 x IO-” M horseradish peroxidase, 7.2 x 10e5 M Mn*+, 5.8 x 10e5 M INH, and 2.5 x 10e5 M eosin. The buffer was 0.2 M phosphate, pH 7.5. In all cases the reaction was initiated by the addition of INH. Oxygen consumption was determined with a Yellow Springs Instruments Model 53 oxygen monitor. Absorption spectra were taken on a Zeiss DMR-10 recording spectrophotometer using l-cm cells. Analysis of the final reaction mixture was carried out by paper (Whatman No. 3) and thin-layer chromatography (Silica gel, Merck) using ethanol-ammonium hydroxide-water (6:3:1) as eluent (18, 19). Pyridine-4-carboxaldehyde (R, 0.72 and 0.76 in paper and thin-layer, chromatography, respectively) was isolated and characterized by comparing the R, values and the uv spectra with those of an authentic sample. The yield of aldehyde was calculated from eggoH = 2270. Repeated attempts to detect pyridine4-carboxylic acid (R, = 0.64 and 0.70 in paper and thin-layer chromatography (20)) were negative. The hydrazone of pyridine-4-carboxyaldehyde was detected spectrophotometrically (21). To measure the very weak emission from the eosin-catalyzed reaction, a Beckman Model LS-250 liquid scintillation counter, with the coincidence circuit turned off, was used. A Hamamatsu TV C767 photon counter (22) was used to measure some of the weak emissions. Measurements were made at room temperature; this accounts for the small differences occasionally observed. RESULTS

Peroxidase, provided it is type VI, promotes a slow oxidation of INH which is markedly accelerated by Mn2+ ions; however, a considerable part of this increased rate is due to a nonenzymic oxidation of INH (Fig. 1). In the binary systems, INHI plain the catalytic effect. (ii) There is only an insignificant emission from the system peroxidase/Mi?+/ eosin/O, plus 1 x 1O-6 M H,O,. (iii) The catalase effect upon the emission appears to be a quenching effect, in the same way that raising the peroxidase concentration decreases the emission. Accordingly, catalase does not influence the rate of reaction.

minutes

FIG. 1. Oxygen uptake by the systems, INH/peroxidase (lower curve), INH/Mn2+ (middle), and INH/peroxidase/Mr?+ (upper), under standard conditions.

Mn2+ and INH/peroxidase, and also in the ternary system, INH/Mr?+/peroxidase, the product formed is pyridine-4-carboxaldehyde isolated in yields of 70-75%. By spectral and chromatographic criteria, pyridine-4-carboxaldehyde and its hydrazone (low yield) were the only products present in the final reaction mixture. Accordingly, 0, consumption was stoichiometric, or very slightly below, for aldehyde formation. Thus, the reaction which occurs is O=C-NH-NHZ O=C-” 15. + Hz0+ N2 i^’ NJ + 1’zo2 Since isonicotinic acid may be formed when peroxidase acts peroxidatically upon INH (16) and since the chemical oxidation of linear acylhydrazides leads to the luminescent acid (141, we carefully searched for minute amounts of the acid. However, results were negative. The addition of eosin had little or no effect upon the rate of O2 uptake by the INH/Mn2+ and INH/peroxidase systems; however, a striking effect was observed in the INH/peroxidase/Mr?+ system (Fig. 2). With respect to product formation, the three systems behave as in the absence of eosin. The reaction in the INH/peroxidase/ Mn*+/eosin system was accompanied by light emission detectable with the liquid scintillation counter or with the photon counter (Fig. 3); further addition of INH at the end of reaction results in another burst, from which it is inferred that neither peroxidase nor eosin are altered during the process. Manganese ions could not be replaced by Ca2+ or Sr2+ ions. The total

454

ZINNER ET AL.

minutes

FIG. 2. The effect of 2.5 x 10m5M eosin upon the rate of 0, uptake by the INH/peroxidase/MrP+/O~ system. Lower curve: in the absence of eosin.

1

minubr

FIG. 3. The effect of 2.5 x 10m5M eosin upon the rate of photon emission as a function of time by the INH/peroxidase/Mn*+/O~ system. The reaction was initiated by the addition of INH. The arrow denotes addition of substrate to the final reaction mixture.

light emission curve did not correlate with 0, uptake (data not shown). The reaction can be followed spectrophotometrically. With the system, INH/peroxidase/MrP+/O,, the Soret band of the enzyme is bathochromically shifted (Fig. 4): When the reaction is complete the Soret band returns to the original position. If eosin is present in the system, the return is markedly faster. At the end of reaction, the differential visible spectrum is zero, or almost so, throughout the region investigated, indicating that neither the enzyme nor eosin are altered by the process. Effect of the concentration of the participants. Increasing the concentration of

FIG. 4. The Soret band of peroxidase before C---j, during (-.-I, and after (- - -1 the peroxidase/ eosin catalyzed aerobic oxidation of INH. Similar behavior was observed in the absence of eosin, except for the slower rate of reaction. The Soret band before reaction was obtained in the absence of either O2 or INH.

eosin increases the rate of the reaction and also the emission. Increasing the substrate concentration causes the emission to increase up to a maximum value, after which it decreases until at high INH concentration no emission is observed. The rate of O2 uptake increased in the range of INH concentrations investigated, 5.8 x lo-' t0 1.2 X lo-3 M. The effect of Mn*+ ions could be detected at levels as low as 1 x 10e6 M. At this concentration, Mn*+ ions increased the rate of O2 uptake by the INH/peroxidase system only slightly; yet under these conditions eosin exerted a pronounced catalytic effect and yielded significant emission. Increasing the Mn2+ ion concentration up to 7.2 x 10-j M resulted in increased initial rates of photon emission. Total light emission appears to increase with decreasing peroxidase concentration. This quenching effect of peroxidase was not surprising (9). Boiling the enzyme for a few minutes greatly reduced light emission. Effects of other factors. The emission greatly increases as the pH is raised from 7.0 to 7.5 and 8.0. When the molarity of the buffer was varied from 0.1 to 0.4 at 7.5, a small decrease in emission was observed. At pH 7.1 the rate of the noncatalyzed (eosin absent) reaction in H,O is reduced by a factor of 8 relative to pH 7.5, whereas in the presence of eosin the same high rate

EXCITED PRODUCT FROM ISONICOTINIC

of O2 uptake is observed as at pH 7.5. Therefore, the catalytic effect of eosin at pH 7.1 is dramatic (x 30). The same behavior was observed in 2H,0 buffer (pD 7.1) except that the emission was stronger than that in H,O (Fig. 5). Note that these results are derived on the basis of pH = pD, whereas according to some authors (23, 24), pH = pD + 0.4. In 2H20 the halflife of ‘Ag lo, is 10 times longer than in water (25). Effects of other sensitizers. Eosin could efficiently be replaced by Rose Bengal and fluorescein. Riboflavin (3 x 1O-4 M) was only slightly active in inducing emission and in speeding up the rate of 0, uptake. Pyronine G (2.5 x 1O-5 MI was essentially inactive. Methylene blue (2.5 x lop5 M) had an inhibitory effect upon the rate of O2 uptake and failed to induce significant emission. With riboflavin, pyronine G, and especially fluorescein, a slightly larger 0, consumption was observed. Effect of cat&se. Catalase, 1.6 x 10e6 M or 3.2 x lO-‘j M, reduced the maximum rate of emission by 50%. However, it did not seem to affect the rate of reaction. Effect of superoxide dismutase. Because the large increase in the rate of 0, uptake in the presence of eosin might be due to a cyclic oxidation of INH in which 02- would participate, we have assayed the effect of

FIG. 5. The effect of replacing H,O by ‘Hz0 upon the rate of photon emission as a function of time by the INH/peroxidase/Mn2+/eosin/02 system in 0.1 M phosphate buffer. Upper curve, pH = 7.5; middle, pD = 7.1; lower, pH = 7.1.

ACID HYDRAZIDE

455

superoxide dismutase upon the rate of 0, consumption and also upon the emission (26). Superoxide dismutase showed an inhibitory effect at high concentrations (Table I). Effects of quenchers of triplet eosin. In connection with possible mechanisms of eosin catalysis and light induction, it was of interest to test the effect of quenchers of triplet eosin upon both O2 consumption and photon emission. Since quenchers acting through electron transfer would be expected to be oxidized by the peroxidase system, and thus, to complicate the system, we selected two powerful triplet eosin quenchers which are not donors, namely, potassium ferricyanide and p-benzoquinone (27). Ferricyanide, 1 x 10e4 M, completely suppressed both eosin effects, namely, catalysis and emission, without appreciably affecting the uncatalyzed 0, uptake (Table I). p-Benzoquinone was too insoluble in water to be employed as triplet quencher. However, because it interferes in radical processes (28), it was tested at saturating concentrations. p-Benzoquinone, 2.5 x lO-‘j M, considerably reduced the rate of 0, uptake; when 2.5 x 10e5 M eosin was present, there was little, if any, increase in the rate of 0, consumption and no emission was observed (Table I). The eosin spectrum was not altered in the final reaction mixtures. Interaction of INH with eosin. No interaction was observed between INH and eosin; thus, the absorption spectrum of the binary system corresponded to the sum of the spectra of the components and did not change with time. As expected, INH did not interact with excited singlet eosin at the usual concentrations of the experiments (5.8 x 1O-5 M INH and 2.5 x 10m5M eosin); the fluorescence band of eosin was not changed at all. Irradiation of the INH-eosin system with visible light resulted in INH oxidation. Neither INH nor eosin alone consumed 0, in control experiments. Interaction between ferricyanide and excited singlet eosin. When potassium ferricyanide at the concentration used in the inhibition experiments (1 x 10e4 M) was tested upon the fluorescence of 2.5 x 1O-5

ZINNER ET AL.

456

TABLE I EFFECT OF DIFFERENT INHIBITORS UPON THE INITIAL RATE OF 0, UPTAKE BY THE INH/PERoXIDAsE/Mn*+ AND INH/PERoxIDAsE/Mn*+/EoSIN SYSTEMS AND UPON THE EMISSION BY THE LATTER SYSTEM" Emission O2 uptake (pl/min)

0.6 pM superoxide dismutase 2.4 pM superoxide dismutase 100 pM ferricyanide 2.5 PM p-benzoquinone

No eosin

With eosin

0.63

2.63 2.10 0.96 0.60 0.38

0.30 0.53 0.30

Taken as reference Moderate reduction Marked reduction Suppressed suppressed

a Standard reaction mixture. M eosin, a 20% quenching effect was observed. Binding of eosin to peroxidase. Interaction between eosin (ground state) and peroxidase was investigated spectrophotometrically as in the case of the lysozyme-eosin complex (29). However, the 518~nm absorption band of eosin was not affected by the presence of peroxidase, even at a concentration of 6.4 x 1O-5M. Neither was the eosin band altered in the reacting system. Conversely, eosin did not affect the Soret band of the resting enzyme, nor, at least apparently, that of the reacting enzyme.

The possibility that eosin exerts its effect through an excited state formed by energy transfer from an electronically excited product of the reaction, presumably pyridine-4-carboxaldehyde, is analyzed here. Since a diazene is likely to be an intermediate in the oxidation (30) it is pertinent that energy bond considerations indicate that there is enough energy (ca. 60 kcal) in the step

DISCUSSION

The first conclusion from this work is that INH is oxidized in the peroxidase/ Mn2+/02 system; this is important in view of the correlation between the tuberculostatic action of INH and peroxidase activity (15, 16). The reaction bears some analogy to the recently reported oxidation of phenylhydrazine to benzene by both oxyand methemoglobin functioning as a peroxidase (30). However, whereas catalase inhibited the latter reaction, it did not seem to slow down the oxidation of isoniazid. Also important is the observation that eosin and structurally similar sensitizers greatly increase the rate of 0, consumption. The dye effect must be exerted either through the enzyme or through the substrate (INH). No evidence for the formation of an enzyme-eosin complex has been found; however, the presence of small amounts of a very active complex cannot be excluded. On the other hand, no interaction occurs between ground-state eosin and INH.

to produce, in cooperation with the activation energy, the excited aldehyde (ET - 70 kcal) . The formation of an electronically excited product is supported by light emission concomitant with the reaction. The very weak emission is not detectable with conventional spectrofluorometers. A contributing, though minor, reason is that the substrate itself acts as quencher as shown by the decreasing emission at high INH concentration. Certainly, a more important factor is that the excited product would be quenched by oxygen if it is in the triplet state. If an excited state of eosin is involved in the catalytic process it is not the singlet state because INH at the concentration of the experiments could not, and indeed did not, affect the fluorescence spectrum of the dye; therefore, the excited state which participates in the reaction would be triplet eosin. Quenching of this species by several reductants, including phenylhydrazine, is well known (27) and quenching by INH

EXCITED

PRODUCT

FROM ISONICOTINIC

would not be surprising. It is possible that the quenching action of INH is connected to the catalytic effect of the dye. Thus, the quenching may be exerted through electron donation from the substrate to triplet eosin. A plausible mechanism would be peroxidase 3P* Mn2+ 3P* + eosin -+ P + 3eosin*

INH + 0,

INH + 3eosin* --+ R ’ + eosin H

457

ACID HYDRAZIDE

emitting species. One possibility is that it is triplet eosin. Emission from triplet eosin is consistent with the fact that phosphorescence can be observed in fluid solution, provided of course, that 0, is excluded (32). The presence of oxygen in our systems as a participant of the reaction would explain why the emission is only detected with very sensitive equipment. Very weak phosphorescence spectra in the presence of oxygen have been reported (33). The following scheme appears likely:

INH+ 0, INH ii P + heat

P + 0, + heat

where P is pyridine-4-carboxaldehyde. The semireduced eosin would be regenerated to eosin by oxygen: eosin H + 0, + eosin + HO, * and the radicals R * and HO, ‘/O,- would initiate a cyclic process: R’+02++P+02INH + O,- + R ’ + H,O, (fate unknown). The superoxide ion/perhydroxyl would disappear by dismutation: 02- + o,- - H+

radical

02 + H,O,.

The participation of triplet eosin is supported by the results with the efficient quencher ferricyanide. It inhibited the two effects of eosin, namely, the increase in the rate of 0, uptake and light emission. The observed inhibitions with ferricyanide are in part due to the following processes (31): “eosin + Fe(CN)Z- + eosin + Fe(CN&t

3eosin + Fe(CN),3- + X + Fe(CN),4-. The product of the enzymic reaction, pyridine-4-carboxaldehyde, must be formed in, or rapidly converted into, the triplet state; thus, no emission whatsoever is observed in the absence of sensitizers. Without knowing the emission spectrum, one cannot identify which is the

OL

The enzymic system should generate the excited product in substantial yield; otherwise it would be difficult to explain the effect of eosin at all. Indeed, this sensitizer at the 2.5 x 10e5 M level must compete with 2 x 1O-4M oxygen for triplet pyridine4-carboxaldehyde in order for its triplet state to be populated. Whatever triplet eosin is formed, only a fraction will survive to react with INH. Yet this fraction will very efficiently contribute to a cyclic process of INH oxidation. It is for these reasons that the “photochemistry without light” effect (34) is detectable. The lack of correlation between light emission and 0, uptake may be explained as follows. At the beginning of the reaction, the emission appears relatively weak due to the quenching effect of INH. In the final stages it becomes relatively strong because the decomposition of the intermediate (a “stabilized” diazene?) is slower than its formation. The rate of emission as a function of time (Fig. 2) is consistent with this view. ACKNOWLEDGMENTS The authors were greatly aided by grants from the “Fundacao de Amparo a Pesquisa do Estado de S&o Paulo” (BIOQ/FAPESP programme). Dr. Nelson Duran (Universidad Catolica de Valparaiso, Chile) is a FAPESP Visiting Professor. Miss Carmen C. C. Vidigal is a predoctoral fellow of FAPESP. The authors thank Dr. F. Quina for enlightening discussions.

ZINNER REFERENCES 1. CILENTO, G., (1973) Quart. Rev. Biophys. 6,485501. 2. CILENTO, G., NAKANO, M., FUKUYAMA, H., SUWA, K., AND KAMIYA, I. (1974) Biochem. Biophys. Res. Commun. 58, 296-300. 3. FARIA OLIVEIRA, 0. M. M., SANIOTO, D. L., AND CILENTO, G. (1974) Biochem. Biophys. Res. Commun. 56, 391-396. 4. ZINNER, K., CASADEI DE BAPTISTA, R., AND CILENTO, G. (1974) Biochem. Biophys. Res. Commun. 61, 889-898. 5. CILENTO, G. (1975)5. Theor. Biol. 52,255-257. 6. VIDIGAL, C. C. C., AND CILENTO, G. (1975) Biothem. Biophys. Res. Commun. 62, 184-190. 7. CILENTO, G. (1975)5. Theor. Biol. 55,471-479. 8. VIDIGAL, C. C. C., ZINNER, K., DURAN, N., BECHARA, E. J. H., AND CILENTO, G. (1975) Biothem. Biophys. Res. Commun. 65, 138-145. 9. ZINNER, K., DUR~N, N., VIDIGAL, C. C. C., SHIMIZU, Y., AND CILENTO, G. (1976) Arch. Biochem. Biophys. 173, 58-69. 10. CORMIER, M. J., AND TOTTER, J. R. (1964) Ann. Rev. Biochem. 33, 431-458. 11. CILENTO, G. (1965) Photochem. Photobiol. 4, 1243-1247. 12. NILSSON, R., ORRENIUS, S., AND ERNSTER, L. (1964) Biochem. Biophys. Res. Commun. 17, 303-309. 13. NILSSON, R. (1969) Biochim. Biophys. A& 184, 237-251. 14. RAPAPORT, E., CASS, M. W., AND WHITE, E. H. (1972) J. Amer. Chem. Sot. 94, 3153-3159. 15. GAYATHRI DEVI, B., SHAILA, M. S., RAMAKRISHNAN, T., AND GOPINATHAN, K. P. (1975) Biothem. J. 149, 187-197. 16. SEYDEL, J. K., SCHAPER, K-J., WEMPE, E., AND CORDES, H. P. (1976) J. Med. Chem. 19, 483492.

ET AL. 17. MCCORD, J. M., AND FRIDOVICH, I. (1969)5. Biol. Chem. 244, 6049-6055. 18. KUFFNER, F., AND FADERL, N. (1955) Mh. Chem. 86, 995-1003. 19. KODICEK, E., AND REDDI, K. K. (1951) Nature (London) 168, 475-477. 20. H~~BNER, C. F. (195l)Nuture (London) 167, 119120. 21. INOUE, S., OGINO, A., AND ONO, Y. (1966) Yakuzaiguku 26, 302-307; Chem Abstr. 69, 99472 q. 22. INABA, H., SHIMIZU, Y., AND TSUJI, Y. (1975) Japan. J. Appl. Phys. 14, 23-32. 23. GLASOE, P. K., AND LONG, F. A. (1960) J. Phys. Chem. 64, 188-190. 24. SRERE, P. A., KOSICKI, G. W., AND LUMRY, R. (1961) Biochim. Biophys. Acta 50, 184-185. 25. MERKEL, P. B., NILSSON, R., AND KEARNS, D. R. (1972) J. Amer. Chem. Sot. 94, 1030-1031. 26. HODGSON, E. K., AND FRIDOVICH, I. (1973) Photothem. Photobiol. 18, 451-455. 27. RIZZUTO, F., AND SPIKES, J. D. (1975) Rad. Environ. Biophys. 12, 217-232. 28. SIMIC, M., AND HAYON, E. (1973) Biochem. Biophys. Res. Commun. 50, 364-369. 29. BAUGHER, J. F., GROSSWEINER, L. I., AND LEWIS, C. (1974) J. Chem. Sot. Faraday Trans. II 70, 1389-1398. 30. G~LBERG, B., STERN, A., AND PEISACH, J. (1976) J. Biol. Chem. 251, 3045-3051. 31. KEPKA, A. G., AND GROSSWEINER, L. I. (1971) Photochem. Photobiol. 14, 621-639. 32. PARKER, C. A., AND HATCHARD, C. G. (1962) J. Phys. Chem. 66, 2506-2511. 33. STAUFF, J., AND BARTOLMES, P. (1970) Angew. Chem. (Znt.) 9, 307-308. 34. WHITE, E. H., MIANO, J. D., WATKINS, C. J., ANDBREAUX, E. J. (1974)Angew. Chem. (Int.) 13, 229-243.