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Low-level luminescence from microsomes exposed to enzymatic systems that generate triplet species
ARCHIVES OF BIOCHEMISTRY Vol. 235, No. 2, December,
Low-Level
AND BIOPHYSICS pp. 673-678, 1984
Luminescence from Microsomes Exposed to Enzymatic Systems that Generate Triplet Species ANA
Department
CAMPA
of Biochemistry,
Received
May
GIUSEPPE
AND
CILENTO’
Instituto de Q&mica, Universidode C.P. 20.780, Scio Paulo, Brazil
14, 1984, and in revised
form
August
de Sdo Paulo,
11, 1984
Microsomes exposed to the propanal/horseradish peroxidase/Oz system develop a weak chemiluminescence. The underlying process is distinct from that occurring during lipid peroxidation because the emission intensity peaks at around 560 nm, rather than in the red, and no malonaldehyde is formed. Triplet acetaldehyde appears to be responsible for the induction of the process, which in turn leads to excitation of a component in microsomes, possibly a flavoprotein. o 1984 Academic PRSS, h.
Enzyme-generated triplet species (1) are able to transfer energy to several acceptors, including macromolecules such as phytochrome (2), t-RNAPhe (3,4), DNA (5, 6), and proteins (7). For this reason, energy transfer to an organelle, the chloroplast, was attempted (8,9). The successful results with this system have led us to investigate transfer to other organelles and to preparations obtained from them. In this paper, we report a weak luminescence from microsomes exposed to the peroxidase (HRP)‘-catalyzed oxidation of linear aliphatic aldehydes, a reaction which leads to the lower homolog in the triplet state (9, 10):
H-(CHz),-CHz-C
The choice of the aliphatic aldehyde/ HRP/Oz system was dictated by the fact that it is practically nonemissive, making it highly suitable in cases where one is interested in detecting weak sensitized or induced emissions. MATERIALS
/” H
HRP 3H-(CH2)n-C
No
phosphate buffer
i To whom correspondence should be addressed. a Abbreviations used: Cp, acetaldehyde; Ca, propanaldehyde; C4, butyraldehyde; Cg, valeraldehyde; Cs, caproaldehyde; HRP, horseradish peroxidase; MDA, malonaldehyde; SOD, superoxide dismutase, t-BuOOH, tert-butyl hydroperoxide.
\
H + HCOOH
METHODS
Horseradish peroxidase (type VI), glucose oxidase, catalase, superoxide dismutase, D-glucose, phenobarbital, histidine, thiourea, ascorbic acid, and trypsin were from Sigma Chemical Company. The aldehydes were purchased from Fluka and thiobarbituric acid from Merck. SKF-525 A, from Smith Kline & French Labs., was kindly supplied by Dr. E. Cadenas (University of Dusseldorf). Oxygen uptake was followed on a Yellow Springs Instruments Model 53 Oxygen monitor. Ligh emission was measured in a Hamamatsu TVC-‘767 photon counter provided with a Hamamatsu R-562 photomultiplier tube. In all figures in which emission data are presented, the lower line is the background.
\
+ O2 -
AND
[l] 673
0003-9861/84 $3.00 Copyright 0 1994 by Academic Press. Inc. All rights of reproduction in any form reserved.
674
CAMPA
AND
The microsomal fraction was prepared from livers of male rats (about 180-200 g body wt). Livers were perfused with saline and homogenized with a solution containing 140 mM KC1 and 10 mM KHzPOl at pH 7.0. Homogenates were centrifuged at 10,OOOg for 20 min. The supernatants were recentrifuged at 100,OOOg for 1 h, and then suspended in the same solution. For the phenobarbital treatment, the rats were allowed to consume drinking water containing 0.1% of the drug for 5 days ad libitum Unless otherwise stated, the enzymatic reaction to which the microsomes (8 mg protein) were exposed contained 80 mM propanal, 0.5 M ethanol (to solubilize the aldehyde), 0.5 pM HRP in 0.5 M phosphate140 mM pyrophosphate buffer, pH 7.4. The final volume was 3 ml and the temperature was 37°C. In the experiments with glucose (0.3%)/glucose oxidase (0.4 ~M)/cataktSe (0.04 NM), the scintillation vial was completely filled and sealed with Parafilm to isolate the solution from contact with air. Malonaldehyde formation was measured by previously described methods (11). Protein was estimated by the Biuret method using bovine serum albumin as standard. RESULTS
In the range of pH investigated (6.88.0), a weak emission developed during the HRP-catalyzed aerobic oxidation of propanal when microsomes were present. This emission continued even when Oz had been depleted and was available only by diffusion (Fig. 1). As measured at pH 7.4, microsomes accelerated oxygen consumption (Fig. 2); the same rate of O2 uptake by the Cs/HRP/Oz/microsomes system was observed at pH’s 7.4 and 7.8. Doubling the amount of microsomes did
FIG. 1. Effect of the pH upon the emission from microsomes elicited by the propanal/HRP/Oz system. The vertical arrow indicates oxygen depletion at pH 7.4. No emission was observed upon omitting either the microsomes, propanal, or peroxidase.
CILENTO 1001
201
; _- _ _ -. :’ ,I ;’ I i .!I -I_- I_ -I 5 MINUTES
A 20
FIG. 2. Oxygen uptake by the propanal/HRP/Op system in the absence (- - -) and presence ( __ of microsomes. The lower curve (-*-a -) refers the propanal/microsome system.
) to
not significantly alter the time-emission curve (pH 7.4), but suppressed the accelerating effect. Raising the temperature from 35 to 40°C increased the emission intensity only slightly without altering the shape of the curve (pH 7.4). The timeemission curve was unaffected by increasing the HRP concentration from 0.5 to 2.0 PM. At pH 7.4, optimal emission was observed when the molarity of the buffer was 0.5 M. In Tris buffer, the propanal/ HRP/microsomes system consumed oxygen, but no emission was observed. Reducing the propanal concentration from 80 to 8 mM dramatically reduced the emission from microsomes. The use of low concentrations did, however, permit comparison of propanal with its less-soluble higher homologs (C&J. The results presented in Fig. 3 indicate that the emission intensity increases from C3 to Cs; at these aldehyde concentrations, however, Oz uptake was only barely detectable with our equipment, limiting the amount of additional information which could be obtained. Although the emission was very weak, it was possible to obtain an approximate form from the emission spectrum with a spectral resolution of ca 10 nm (Fig. 4) by using a series of sharp cut-off filters (12).
MICROSOMAL
LUMINESCENCE
MINUTES FIG. 3. Emission HRP (2 pM)-catalyzed aldehydes (8 mM).
from
microsomes induced by the aerobic oxidation of aliphatic
The fluorescence spectrum (X,,, = 400 nm) of our microsome preparation is also shown in this figure. Determination of thiobarbituric acid reactants-an index of lipid peroxidation-gave the same value (5.6 X 10-l’ mol/mg protein of MDA) whether microsomes were kept in buffer or exposed to the propanal/HRP/Oz system for 15 min. It is known that microsomes maintained in phosphate buffer release NADH-cytochrome bs reductase (13). It was therefore important to verify whether the emission arises from some component solubilized
PROMOTED
BY
TRIPLET
SPECIES
675
from the microsomes. In one experiment, the microsomes were kept in buffer for 3 h prior to initiating the aldehyde/HRP/ Oz reaction; the time-emission curve was similar to that obtained with microsomes used immediately. That a solubilized factor was not responsible for the emission was also indicated by the fact that much less emission was elicited from the supernatant than from the pellets obtained from microsomes which had been kept for 30 min in 1 M phosphate buffer, pH 7.4 (not shown). When microsomes were incubated with either propanal or HRP for 10 min prior to adding the other component (HRP or propanal, respectively), the subsequent light emission kinetics were unchanged. Likewise, if the microsomes were added after Oz had been extensively consumed by the CJHRP system, the time-emission curve was not markedly altered. When the system was reoxygenated, the emission persisted unaltered. On the other hand, when the system C3/HRP/microsomes was made essentially anaerobic by addition of glucose/glucose oxidase/catalase, the emission started decreasing (Fig. 5). Active species of oxygen were not involved in the process which ultimately
Lk-ek-+ MINUTES
FIG. 4. Emission spectrum of the microsomal luminescence elicited by -, exposure to the propanal/HRP/Oz system; or - - -, irradiation at 400 nm. These spectra were normalized.
FIG. 5. Effect of oxygen depletion upon the microsomal emission elicited by the propanal/HRP system. The first arrow (9 min) indicates depletion, oxygen becoming available only by diffusion. The second arrow indicates addition, in a duplicate experiment (- - -), of glucose, glucose oxidase, and catalase, the cuvette being completely filled and sealed with Parafilm.
676
CAMPA
AND
gave rise to emission. Thus, the following additions had no significant effect upon the emission at the concentrations employed: 10m3 M histidine, 10-5-10-4 M thiourea, SOD (100 U), lo-” M ascorbate, or 10m7 M catalase. The participation of the HO’ radical was further dismissed by the presence of 0.5 M ethanol in the reaction mixture. To obtain additional evidence against the participation of peroxides, 10-6-10-4 M H202 and 5 InM tert-BuOOH were added to microsomes. None of these additions promoted emission detectable in the equipment used in the present work. Likewise, 0.1 M EDTA had no effect upon the induced emission. The same light kinetics were observed with microsomes which had been prepared from livers of rats that had been treated with phenobarbital, that is, from microsomes containing higher levels of cytochrome P-450. Trypsin treatment of microsomes removed 35% of the total protein; cytochrome b5 and NADPH-cytochrome P-450 reductase were removed to a large extent, while cytochrome P-450 and ATPase were considerably denatured (14). The emission from trypsin-treated microsomes was only moderately reduced, and practically no emission was elicited from the supernatant. Treatment with deoxycholate released 25% of the microsomal proteins, greatly diminishing the IDPase, p-glucuronidase, and esterase activities (14); this treatment moderately increased the emission. Washing microsomes in sucrose solution, a process which reduces the activity of NADPH-cytochrome P-450 reductase (15), slightly increased the emission. SKF-525A inhibits cytochrome P-450 and, therefore, the chemiluminescence associated with microsomal lipid peroxidation (16); only at high concentrations (150 PM) did SKF have any effect and, even so, a modest one upon the sensitized microsomal emission. It was of interest to verify whether the redox state of microsomes may have an influence on the emissive properties of the system. Addition of 10e3 M NADPH, either at the beginning of the reaction or
CILENTO
at a later time, had no effect upon O2 uptake by the C3/HRP/02 system, but markedly reduced the emission in the complete system. However, this effect may be due to quenching of the excited donor; indeed, NADPH decreased the intensity of eosine fluorescence sensitized by energy transfer from the propanal/HRP/Oz system, but had no effect on the fluorescence of optically excited eosine. When microsomes containing bound chlorophyll peroxidized lipids following addition of t-BuOOH, the normal chemiluminescence, if any, was totally masked by chlorophyll fluorescence (17). However, when these chlorophyll-containing microsomes were exposed to the triplet acetaldehyde-generating system, one could infer by the use of filters that the 560-nm chemiluminescence was present in addition to chlorophyll fluorescence. DISCUSSION
Under all experimental conditions, the emission from microsomes exhibits a lag. This indicates that the emission does not arise from a microsomal chromophore directly excited by energy transfer; thus, no lag was observed when the propanal/ HRP/Oz system elicited emission from appropriate acceptors such as xanthene dyes (10, 18), chlorophyll (18), and others (9). The emission must come from an induced process. Provided some oxygen is present (e.g., as a result of diffusion) after the lag period, the microsomal emission develops, becomes maximal and subsequently decay slowly. Emission requires continuous exposure of the microsomes to the propanal/HRP/Oa system. We must infer that it is an intermediate or transient species in this system which promotes the chemiluminescent process in microsomes. The readily conceivable intermediates (19), in particular HRP-I, HRP-II, and the peroxy radical, are excluded by the fact that the microsomes do not inhibit the HRP-promoted oxidation of propanal. Furthermore neither HRP-I (i.e., HRP plus HZOz), t-BuOOH, nor HzO, induce emission from the micro-
MICROSOMAL
LUMINESCENCE
somes detectable with our equipment. Presumably, then, the species which promotes the emissive process is triplet acetaldehyde. This is strongly supported by the fact that NADPH-a quencher of triplet acetaldehyde-quenches the microsomal emission without affecting the propanal/HRP/Oz reaction. In addition, the absence of emission in Tris buffer is in keeping with the quenching effect of the amine form upon triplet acetaldehyde. Although microsomal chemiluminescence under completely anaerobic conditions has been reported (20), we are unable to determine whether the chemiluminescent process in microsomes requires oxygen in our system because oxygen is necessary for the generation of triplet acetaldehyde. Whatever the process induced in microsomes, it does not appear to involve active species of oxygen and is certainly different from common lipid peroxidation process(es). Thus: (i) no malonaldehyde is formed; (ii) SKF-525A has no effect upon the emission; (iii) maximal emission is around 560 nm, not in the red region as expected for bimol singlet oxygen emission (21); and (iv) the emission is still present when chlorophyll-containing microsomes are used. The novel, important features is that the process is subsequent to reaction with-or energy acceptance from-an electronically excited, enzyme-generated triplet species. The emitter is probably a flavoprotein because the emission spectrum resembles that from a variety of cell types which have been ascribed to a flavoprotein (22). Since the optically excited spectrum peaks at somewhat shorter wavelengths, the microsomal process may selectively excite an emitter which is not a major contributor to the optical spectrum. In conclusion, our results show that a chemiluminescent process can be induced in microsomes without formation of malonaldehyde; this nicely explains the fact that induced microsomal chemiluminescence becomes maximal after malonaldehyde is no longer being formed (17). No less important, the stimulus for photoin-
PROMOTED
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6’77
duced chemi- and biochemiluminescence, two well-known phenomena, may be derived from triplet energy sources. The generality of the latter finding is strongly supported by current work with organelles and cells. ACKNOWLEDGMENTS The authors express their gratitude to Dr. Frank Quina for a critical reading of the manuscript. They also thank Dr. E. Cadenas and Dr. 0. August0 for valuable suggestions in the early stages of this work, and Mr. J. F. M. Veiga for technical assistance. This work was supported by grants from the Financiadora de Estudos e Projetos (FINEP), the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), the Volkswagenwerk Stiftung, and the Organization of American States Program (OAS). A.C. is a predoctoral fellow of the FundaCHo de Amparo $ Pesquisa do Estado de SHo Paulo (FAPESP).
REFERENCES 1. CILENTO, G. (1982) in. Chemical and Biological Generation of Excited States (Adam, W., and Cilento, G., eds.), pp. 2’7830’7, Academic Press, New York. 2. AUGUSTO, O., CILENTO, G., JUNG, J., AND SONG, P. S. (1978) B&hem Biophys. Res. Commun, 83, 963-969. 3. DE MELLO, M. P., DE TOLEDO, S. M., HAUN, M., CILENTO, G., AND DURAN, N. (1980) Biochemistry 19, 5270-5275. 4. DE MELLO, M. P., DE TOLEDO, S. M., AOYAMA, H., SARKAR, H. K., CILENTO, G., AND DUR~N, N. (1982) Photo&em. Photobiol. 36, 21-24. 5. FALJONI, A., HAUN, M., HOFFMANN, M. E., MENEGHINI, R., DUR& N., AND CILENTO, G. (1978) B&hem. Biophys. Res. Commun 80,490-495. 6. MENEGHINI, R., HOFFMANN, M. E., DURAN, N., FALJONI, A., AND CILENTO, G. (1978) B&him. Biophgs. Acta 518, 177-180. 7. RIVAS-SUAREZ, E., AND CILENTO, G., Photochem. Photobiol., in press. 8. NASSI, L., AND CILENTO, G. (1983) Photochem Photobiol. 37, 233-237. 9. CAMPA, A., NASSI, L., AND CILENTO, G. (1984) Photochem. Photobiol 40,127-131. 10. HAUN, M., DURAN, N., AUGUSTO, O., AND CILENTO, G. (1980) Arch. Biochem. Biophys. 200, 245252. 11. BUEGE, J. A., AND AUST, S. D. (1978) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 52, pp. 302-310, Academic Press, New York.
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12. INABA, H., SHIMIZU, Y., TSUJI, Y., AND YAMAGISHI, A. (1979) Photochem. PhotobioL 30, 169-1’75. 13. DEY, A. C., RAHAL, S., RIMSAY, R. L., AND SENCIALL, I. R. (1981) Anal B&hem. 110, 373379. 14. DE PIERRE, J. W., AND DALLNER, G. (1975) Biochim Biophys. Acta 415, 411-472. 15. Powrs, G., AND BOOBIS, A. R. (1975) Biochem. PharmacoL 24,1771-1776. 16. CADENAS, E., AND SIES, H. (1982) Eur. J. Biochem 124, 349-356. 17. CADENAS, E., SIES, H., CAMPA, A., AND CILENTO, G. (1984) Photochem PhotobioL, in press.
CILENTO 18. CAMPA, A., AND CILENTO, lication.
G., Submitted
for pub-
19. BECHARA, E. J. H., OLIVEIRA, 0. M. M. F., DURAN, N., DE BAPTISTA, R. C., AND CILENTO, G. (1979) Photochem. PhotobioL 30,101-110. 20. CADENAS, E., SIES, H., GRAF, H ., AND ULLRICH, V. (1983) Eur. J. Biochem. 130, 117-121. 21. CADENAS, E., AND SIES, H. (1983) 2. PhysioL Chem. 364, 519-528.
Hwppe-Seyler’s
22. BENSON, R. C., MEYER, R. A., ZARUBA, M. E., AND MCKHANN, G. M. (1979) J. Histochem. Cyte them. 27, 44-48.
Low-Level
AND BIOPHYSICS pp. 673-678, 1984
Luminescence from Microsomes Exposed to Enzymatic Systems that Generate Triplet Species ANA
Department
CAMPA
of Biochemistry,
Received
May
GIUSEPPE
AND
CILENTO’
Instituto de Q&mica, Universidode C.P. 20.780, Scio Paulo, Brazil
14, 1984, and in revised
form
August
de Sdo Paulo,
11, 1984
Microsomes exposed to the propanal/horseradish peroxidase/Oz system develop a weak chemiluminescence. The underlying process is distinct from that occurring during lipid peroxidation because the emission intensity peaks at around 560 nm, rather than in the red, and no malonaldehyde is formed. Triplet acetaldehyde appears to be responsible for the induction of the process, which in turn leads to excitation of a component in microsomes, possibly a flavoprotein. o 1984 Academic PRSS, h.
Enzyme-generated triplet species (1) are able to transfer energy to several acceptors, including macromolecules such as phytochrome (2), t-RNAPhe (3,4), DNA (5, 6), and proteins (7). For this reason, energy transfer to an organelle, the chloroplast, was attempted (8,9). The successful results with this system have led us to investigate transfer to other organelles and to preparations obtained from them. In this paper, we report a weak luminescence from microsomes exposed to the peroxidase (HRP)‘-catalyzed oxidation of linear aliphatic aldehydes, a reaction which leads to the lower homolog in the triplet state (9, 10):
H-(CHz),-CHz-C
The choice of the aliphatic aldehyde/ HRP/Oz system was dictated by the fact that it is practically nonemissive, making it highly suitable in cases where one is interested in detecting weak sensitized or induced emissions. MATERIALS
/” H
HRP 3H-(CH2)n-C
No
phosphate buffer
i To whom correspondence should be addressed. a Abbreviations used: Cp, acetaldehyde; Ca, propanaldehyde; C4, butyraldehyde; Cg, valeraldehyde; Cs, caproaldehyde; HRP, horseradish peroxidase; MDA, malonaldehyde; SOD, superoxide dismutase, t-BuOOH, tert-butyl hydroperoxide.
\
H + HCOOH
METHODS
Horseradish peroxidase (type VI), glucose oxidase, catalase, superoxide dismutase, D-glucose, phenobarbital, histidine, thiourea, ascorbic acid, and trypsin were from Sigma Chemical Company. The aldehydes were purchased from Fluka and thiobarbituric acid from Merck. SKF-525 A, from Smith Kline & French Labs., was kindly supplied by Dr. E. Cadenas (University of Dusseldorf). Oxygen uptake was followed on a Yellow Springs Instruments Model 53 Oxygen monitor. Ligh emission was measured in a Hamamatsu TVC-‘767 photon counter provided with a Hamamatsu R-562 photomultiplier tube. In all figures in which emission data are presented, the lower line is the background.
\
+ O2 -
AND
[l] 673
0003-9861/84 $3.00 Copyright 0 1994 by Academic Press. Inc. All rights of reproduction in any form reserved.
674
CAMPA
AND
The microsomal fraction was prepared from livers of male rats (about 180-200 g body wt). Livers were perfused with saline and homogenized with a solution containing 140 mM KC1 and 10 mM KHzPOl at pH 7.0. Homogenates were centrifuged at 10,OOOg for 20 min. The supernatants were recentrifuged at 100,OOOg for 1 h, and then suspended in the same solution. For the phenobarbital treatment, the rats were allowed to consume drinking water containing 0.1% of the drug for 5 days ad libitum Unless otherwise stated, the enzymatic reaction to which the microsomes (8 mg protein) were exposed contained 80 mM propanal, 0.5 M ethanol (to solubilize the aldehyde), 0.5 pM HRP in 0.5 M phosphate140 mM pyrophosphate buffer, pH 7.4. The final volume was 3 ml and the temperature was 37°C. In the experiments with glucose (0.3%)/glucose oxidase (0.4 ~M)/cataktSe (0.04 NM), the scintillation vial was completely filled and sealed with Parafilm to isolate the solution from contact with air. Malonaldehyde formation was measured by previously described methods (11). Protein was estimated by the Biuret method using bovine serum albumin as standard. RESULTS
In the range of pH investigated (6.88.0), a weak emission developed during the HRP-catalyzed aerobic oxidation of propanal when microsomes were present. This emission continued even when Oz had been depleted and was available only by diffusion (Fig. 1). As measured at pH 7.4, microsomes accelerated oxygen consumption (Fig. 2); the same rate of O2 uptake by the Cs/HRP/Oz/microsomes system was observed at pH’s 7.4 and 7.8. Doubling the amount of microsomes did
FIG. 1. Effect of the pH upon the emission from microsomes elicited by the propanal/HRP/Oz system. The vertical arrow indicates oxygen depletion at pH 7.4. No emission was observed upon omitting either the microsomes, propanal, or peroxidase.
CILENTO 1001
201
; _- _ _ -. :’ ,I ;’ I i .!I -I_- I_ -I 5 MINUTES
A 20
FIG. 2. Oxygen uptake by the propanal/HRP/Op system in the absence (- - -) and presence ( __ of microsomes. The lower curve (-*-a -) refers the propanal/microsome system.
) to
not significantly alter the time-emission curve (pH 7.4), but suppressed the accelerating effect. Raising the temperature from 35 to 40°C increased the emission intensity only slightly without altering the shape of the curve (pH 7.4). The timeemission curve was unaffected by increasing the HRP concentration from 0.5 to 2.0 PM. At pH 7.4, optimal emission was observed when the molarity of the buffer was 0.5 M. In Tris buffer, the propanal/ HRP/microsomes system consumed oxygen, but no emission was observed. Reducing the propanal concentration from 80 to 8 mM dramatically reduced the emission from microsomes. The use of low concentrations did, however, permit comparison of propanal with its less-soluble higher homologs (C&J. The results presented in Fig. 3 indicate that the emission intensity increases from C3 to Cs; at these aldehyde concentrations, however, Oz uptake was only barely detectable with our equipment, limiting the amount of additional information which could be obtained. Although the emission was very weak, it was possible to obtain an approximate form from the emission spectrum with a spectral resolution of ca 10 nm (Fig. 4) by using a series of sharp cut-off filters (12).
MICROSOMAL
LUMINESCENCE
MINUTES FIG. 3. Emission HRP (2 pM)-catalyzed aldehydes (8 mM).
from
microsomes induced by the aerobic oxidation of aliphatic
The fluorescence spectrum (X,,, = 400 nm) of our microsome preparation is also shown in this figure. Determination of thiobarbituric acid reactants-an index of lipid peroxidation-gave the same value (5.6 X 10-l’ mol/mg protein of MDA) whether microsomes were kept in buffer or exposed to the propanal/HRP/Oz system for 15 min. It is known that microsomes maintained in phosphate buffer release NADH-cytochrome bs reductase (13). It was therefore important to verify whether the emission arises from some component solubilized
PROMOTED
BY
TRIPLET
SPECIES
675
from the microsomes. In one experiment, the microsomes were kept in buffer for 3 h prior to initiating the aldehyde/HRP/ Oz reaction; the time-emission curve was similar to that obtained with microsomes used immediately. That a solubilized factor was not responsible for the emission was also indicated by the fact that much less emission was elicited from the supernatant than from the pellets obtained from microsomes which had been kept for 30 min in 1 M phosphate buffer, pH 7.4 (not shown). When microsomes were incubated with either propanal or HRP for 10 min prior to adding the other component (HRP or propanal, respectively), the subsequent light emission kinetics were unchanged. Likewise, if the microsomes were added after Oz had been extensively consumed by the CJHRP system, the time-emission curve was not markedly altered. When the system was reoxygenated, the emission persisted unaltered. On the other hand, when the system C3/HRP/microsomes was made essentially anaerobic by addition of glucose/glucose oxidase/catalase, the emission started decreasing (Fig. 5). Active species of oxygen were not involved in the process which ultimately
Lk-ek-+ MINUTES
FIG. 4. Emission spectrum of the microsomal luminescence elicited by -, exposure to the propanal/HRP/Oz system; or - - -, irradiation at 400 nm. These spectra were normalized.
FIG. 5. Effect of oxygen depletion upon the microsomal emission elicited by the propanal/HRP system. The first arrow (9 min) indicates depletion, oxygen becoming available only by diffusion. The second arrow indicates addition, in a duplicate experiment (- - -), of glucose, glucose oxidase, and catalase, the cuvette being completely filled and sealed with Parafilm.
676
CAMPA
AND
gave rise to emission. Thus, the following additions had no significant effect upon the emission at the concentrations employed: 10m3 M histidine, 10-5-10-4 M thiourea, SOD (100 U), lo-” M ascorbate, or 10m7 M catalase. The participation of the HO’ radical was further dismissed by the presence of 0.5 M ethanol in the reaction mixture. To obtain additional evidence against the participation of peroxides, 10-6-10-4 M H202 and 5 InM tert-BuOOH were added to microsomes. None of these additions promoted emission detectable in the equipment used in the present work. Likewise, 0.1 M EDTA had no effect upon the induced emission. The same light kinetics were observed with microsomes which had been prepared from livers of rats that had been treated with phenobarbital, that is, from microsomes containing higher levels of cytochrome P-450. Trypsin treatment of microsomes removed 35% of the total protein; cytochrome b5 and NADPH-cytochrome P-450 reductase were removed to a large extent, while cytochrome P-450 and ATPase were considerably denatured (14). The emission from trypsin-treated microsomes was only moderately reduced, and practically no emission was elicited from the supernatant. Treatment with deoxycholate released 25% of the microsomal proteins, greatly diminishing the IDPase, p-glucuronidase, and esterase activities (14); this treatment moderately increased the emission. Washing microsomes in sucrose solution, a process which reduces the activity of NADPH-cytochrome P-450 reductase (15), slightly increased the emission. SKF-525A inhibits cytochrome P-450 and, therefore, the chemiluminescence associated with microsomal lipid peroxidation (16); only at high concentrations (150 PM) did SKF have any effect and, even so, a modest one upon the sensitized microsomal emission. It was of interest to verify whether the redox state of microsomes may have an influence on the emissive properties of the system. Addition of 10e3 M NADPH, either at the beginning of the reaction or
CILENTO
at a later time, had no effect upon O2 uptake by the C3/HRP/02 system, but markedly reduced the emission in the complete system. However, this effect may be due to quenching of the excited donor; indeed, NADPH decreased the intensity of eosine fluorescence sensitized by energy transfer from the propanal/HRP/Oz system, but had no effect on the fluorescence of optically excited eosine. When microsomes containing bound chlorophyll peroxidized lipids following addition of t-BuOOH, the normal chemiluminescence, if any, was totally masked by chlorophyll fluorescence (17). However, when these chlorophyll-containing microsomes were exposed to the triplet acetaldehyde-generating system, one could infer by the use of filters that the 560-nm chemiluminescence was present in addition to chlorophyll fluorescence. DISCUSSION
Under all experimental conditions, the emission from microsomes exhibits a lag. This indicates that the emission does not arise from a microsomal chromophore directly excited by energy transfer; thus, no lag was observed when the propanal/ HRP/Oz system elicited emission from appropriate acceptors such as xanthene dyes (10, 18), chlorophyll (18), and others (9). The emission must come from an induced process. Provided some oxygen is present (e.g., as a result of diffusion) after the lag period, the microsomal emission develops, becomes maximal and subsequently decay slowly. Emission requires continuous exposure of the microsomes to the propanal/HRP/Oa system. We must infer that it is an intermediate or transient species in this system which promotes the chemiluminescent process in microsomes. The readily conceivable intermediates (19), in particular HRP-I, HRP-II, and the peroxy radical, are excluded by the fact that the microsomes do not inhibit the HRP-promoted oxidation of propanal. Furthermore neither HRP-I (i.e., HRP plus HZOz), t-BuOOH, nor HzO, induce emission from the micro-
MICROSOMAL
LUMINESCENCE
somes detectable with our equipment. Presumably, then, the species which promotes the emissive process is triplet acetaldehyde. This is strongly supported by the fact that NADPH-a quencher of triplet acetaldehyde-quenches the microsomal emission without affecting the propanal/HRP/Oz reaction. In addition, the absence of emission in Tris buffer is in keeping with the quenching effect of the amine form upon triplet acetaldehyde. Although microsomal chemiluminescence under completely anaerobic conditions has been reported (20), we are unable to determine whether the chemiluminescent process in microsomes requires oxygen in our system because oxygen is necessary for the generation of triplet acetaldehyde. Whatever the process induced in microsomes, it does not appear to involve active species of oxygen and is certainly different from common lipid peroxidation process(es). Thus: (i) no malonaldehyde is formed; (ii) SKF-525A has no effect upon the emission; (iii) maximal emission is around 560 nm, not in the red region as expected for bimol singlet oxygen emission (21); and (iv) the emission is still present when chlorophyll-containing microsomes are used. The novel, important features is that the process is subsequent to reaction with-or energy acceptance from-an electronically excited, enzyme-generated triplet species. The emitter is probably a flavoprotein because the emission spectrum resembles that from a variety of cell types which have been ascribed to a flavoprotein (22). Since the optically excited spectrum peaks at somewhat shorter wavelengths, the microsomal process may selectively excite an emitter which is not a major contributor to the optical spectrum. In conclusion, our results show that a chemiluminescent process can be induced in microsomes without formation of malonaldehyde; this nicely explains the fact that induced microsomal chemiluminescence becomes maximal after malonaldehyde is no longer being formed (17). No less important, the stimulus for photoin-
PROMOTED
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SPECIES
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duced chemi- and biochemiluminescence, two well-known phenomena, may be derived from triplet energy sources. The generality of the latter finding is strongly supported by current work with organelles and cells. ACKNOWLEDGMENTS The authors express their gratitude to Dr. Frank Quina for a critical reading of the manuscript. They also thank Dr. E. Cadenas and Dr. 0. August0 for valuable suggestions in the early stages of this work, and Mr. J. F. M. Veiga for technical assistance. This work was supported by grants from the Financiadora de Estudos e Projetos (FINEP), the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), the Volkswagenwerk Stiftung, and the Organization of American States Program (OAS). A.C. is a predoctoral fellow of the FundaCHo de Amparo $ Pesquisa do Estado de SHo Paulo (FAPESP).
REFERENCES 1. CILENTO, G. (1982) in. Chemical and Biological Generation of Excited States (Adam, W., and Cilento, G., eds.), pp. 2’7830’7, Academic Press, New York. 2. AUGUSTO, O., CILENTO, G., JUNG, J., AND SONG, P. S. (1978) B&hem Biophys. Res. Commun, 83, 963-969. 3. DE MELLO, M. P., DE TOLEDO, S. M., HAUN, M., CILENTO, G., AND DURAN, N. (1980) Biochemistry 19, 5270-5275. 4. DE MELLO, M. P., DE TOLEDO, S. M., AOYAMA, H., SARKAR, H. K., CILENTO, G., AND DUR~N, N. (1982) Photo&em. Photobiol. 36, 21-24. 5. FALJONI, A., HAUN, M., HOFFMANN, M. E., MENEGHINI, R., DUR& N., AND CILENTO, G. (1978) B&hem. Biophys. Res. Commun 80,490-495. 6. MENEGHINI, R., HOFFMANN, M. E., DURAN, N., FALJONI, A., AND CILENTO, G. (1978) B&him. Biophgs. Acta 518, 177-180. 7. RIVAS-SUAREZ, E., AND CILENTO, G., Photochem. Photobiol., in press. 8. NASSI, L., AND CILENTO, G. (1983) Photochem Photobiol. 37, 233-237. 9. CAMPA, A., NASSI, L., AND CILENTO, G. (1984) Photochem. Photobiol 40,127-131. 10. HAUN, M., DURAN, N., AUGUSTO, O., AND CILENTO, G. (1980) Arch. Biochem. Biophys. 200, 245252. 11. BUEGE, J. A., AND AUST, S. D. (1978) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 52, pp. 302-310, Academic Press, New York.
678
CAMPA
AND
12. INABA, H., SHIMIZU, Y., TSUJI, Y., AND YAMAGISHI, A. (1979) Photochem. PhotobioL 30, 169-1’75. 13. DEY, A. C., RAHAL, S., RIMSAY, R. L., AND SENCIALL, I. R. (1981) Anal B&hem. 110, 373379. 14. DE PIERRE, J. W., AND DALLNER, G. (1975) Biochim Biophys. Acta 415, 411-472. 15. Powrs, G., AND BOOBIS, A. R. (1975) Biochem. PharmacoL 24,1771-1776. 16. CADENAS, E., AND SIES, H. (1982) Eur. J. Biochem 124, 349-356. 17. CADENAS, E., SIES, H., CAMPA, A., AND CILENTO, G. (1984) Photochem PhotobioL, in press.
CILENTO 18. CAMPA, A., AND CILENTO, lication.
G., Submitted
for pub-
19. BECHARA, E. J. H., OLIVEIRA, 0. M. M. F., DURAN, N., DE BAPTISTA, R. C., AND CILENTO, G. (1979) Photochem. PhotobioL 30,101-110. 20. CADENAS, E., SIES, H., GRAF, H ., AND ULLRICH, V. (1983) Eur. J. Biochem. 130, 117-121. 21. CADENAS, E., AND SIES, H. (1983) 2. PhysioL Chem. 364, 519-528.
Hwppe-Seyler’s
22. BENSON, R. C., MEYER, R. A., ZARUBA, M. E., AND MCKHANN, G. M. (1979) J. Histochem. Cyte them. 27, 44-48.