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Metabolites of carbofuran: Effect on indole-3-acetic acid degradation
PESTICIDE
BIOCHEMISTRY
AND
16, 136-
PHYSIOLOGY
140 (1981)
Metabolites of Carbofuran: Effect Indole-3-acetic Acid Degradation CESAR llnstituto
de Quimica,
FRANCO
AND
NELSON
on
DURAN
Universidade Estadual de Campinas. Campinas, Site Paula. Brazil
C.P.
1170, CEP 13100,
Received April 6, 1981: accepted June 17, 1981 Oxygen uptake, product formation, and photon emission in the indole-3-acetic acid/ peroxidase/O, system were inhibited by carbofuran and its metabolites, the 3-ketocarbofuran phenol and carbofuran phenol. These metabolites acted as competitive inhibitors and concomitantly were degraded. Photon emission, together with uv spectrophotometry and oxygen measurements, shows this to be a rapid and reproducible method to study insecticide interaction with the indole-3-acetic oxidase svstem in \jitro. 3-Ketocarbofuran phenol was metabolized by peroxidase with excited-state production.
Carbofuran insecticides have been found to be degradated rapidly into various metabolites in plants, insects. and mammals (l-5); therefore attention was turned to its metabolites. The effects on plant growth of the interaction, between metabolites of carbofuran and indole-3-acetic acid (IAA)’ were confirmed (3, 6, 7). We want to report the interaction of carbofuran metabolites (Fig. 1) with the IAAi HRP/O, system (8- 10).
conductivity measurement was carried out in a Beckman RC16 B2 conductivity apparatus. HRP-I was prepared by adding to a sample of HRP exhaustively dialyzed in pentadistilled water (resistivity of 1.22 x lo5 R), hydrogen peroxide in a 1.1 to 1.0 proportion (12). HRP-II was prepared from HRP-I by the method of Hewson and Dunford (13). RESULTS
MATERIALS
AND
METHODS
’ Abbreviations horseradish
used: IAA, indole-3-acetic peroxidase;
nylanthracene-2-sulfonate: anthracene-2-sulfonate.
DPAS,
DBAS
acid;
9.10-diphe-
9,10-dibromo136
0048-3575/81/050136-05$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction m any form reserved.
DISCUSSION
Oxygen uptake. Carbofuran phenol delays oxygen uptake by the IAAiHRPi O,/Mn*+ system (Fig. 2). 3-Ketocarbofuran phenol inhibited the oxygen consumption at concentrations above 13 pM (Fig. 3). With increasing 3-ketocarbofuran phenol at concentrations higher than 40 pM an increase of oxygen uptake was observed (results not shown), possibly due to substrate oxidation. The 3-ketocarbofuran phenol oxida-
HRP (Type VI) was obtained from Sigma Chemical Company. IAA was from Merck. Carbofuran phenol and 3-ketocarbofuran phenol were from the U.S. Environmental Protection Agency. Environmental Toxicology Division, Research Triangle Park. Absorption spectra were taken on a Zeiss DMR-21 recording spectrophotometer using l-cm cells. The chemiluminescence was measured in a Beckman LS 100~ liquid scintillation counter or in a Hamamatsu TV Photocounter C-767 (11). Oxygen uptake was determined with a Yellow Springs Instruments Model 53 Oxygen Monitor. The
HRP,
AND
METABOLITES
2
4
MINUTES
6
OF
8
FIG. 2. Inhibition of oxygen uptake in the IAAI HRPIMt?+IO, system by different concentrations oj carbofuran phenol: (Cl) 0.0; (m) I PM; (0) 2 PM; (0) 5 PM. Standard conditions: IAA (0.2 mM). HRP (3.0 nM), Mn2+ (0.1 mM), and p-chlorophenol (0.1 mM) in phosphate buffer (25 mM) at pH 5.9 at 25°C.
137
CABBOFURAN
presence of carbofuran phenol, the enzyme activity was recovered after a lag period with the same rate of product formation as control; the lag time was dependent on the inhibitor concentration. In the case of 3ketocarbofuran phenol the enzyme activity was not recovered. Excited-state generation. It is known that the IAA is generated in its electronically excited state by the IAA/HRP/O, system (9, 14, 15). In this system at pH 5.9 and above, correlation between oxygen uptake, product formation, and photon emission was observed. This means that the IAA degradation produces electronically excited state IAI, presumably through a dioxetane intermediate (16, 17):
IAA
tion catalyzed by peroxidase was confirmed by means of oxygen uptake; photon emission accompanies this oxidation. Product formation. Product formation from IAA was followed at 262 nm in the presence of carbofuran metabolites; results were the same as observed by Lee and Chapman (4). Carbofuran phenol was more inhibitory to enzymatic degradation of IAA than was 3-ketocarbofuran phenol. In the 60c
Figures 4 and 5 show the inhibitory effect of carbofuran phenol on the rate of emission. The presence of an excited species other than IA1 at 200 @4 3-ketocarbofuran phenol is evident (Fig. 6). In fact the 3ketocarbofuran phenol/HRP/O, system was analyzed and a strong emission was observed initially during the reaction.
.
2
4
6
1
8
MINUTES
FIG. 3. Inhibition of oxygen aptake in the IAAI HRPlMn’+IO, system by different concentrations of 3-ketocarbofuran phenol: (0) 0.0; (W) 13 PM: (0) 26 PM; (a) 40 PM; under standard reaction conditions.
MINUTES
FIG. 4. Inhibition of photon emission of the IAAI HRPIMn”l0, system by different concentrations of carbofaran phenol under standard reactions conditions: (0) 0.0: (m) I PM; (0) 2 @I: (0) 5 PM.
138
FRANC0
AND
DURAN
v
1
;
-A-- 1~--5
6
MINUTES
FIG. 5. Changes in lag period of IAA degradation by delayed addition of carbofuran phenol (2 pM) in the IAAIHRPIMnZ+I02 system under standard reaction conditions: (0) control. Carbofuran phenol added after: (W) 60 set; (0) 30 set; and (0) 10 sec.
FIG. 6. Photon emission of IAAIHRPIMnZ+I02 system in the presence of diffeerent concentratiotts of 34etocarbofuran phenol under standard reaction conditions: (0) control; (0) 40 PM: (0) 200 PM; (H) 800 ~.LM. Photon emission by the jr-ketocarbofuran phenol (200 pM)IHRPI02 system (- - - ).
Table 1 shows the enhanced emission when 3-ketocarbofuran phenol was degraded by peroxidase in the presence of DPAS and DBAS which are singlet and triplet excited-state counters, respectively (11). Peroxidase alferations. HRP-I or HRP-II, the intermediate forms of HRP, were transformed almost instantaneously to native peroxidase. in the presence of carbofuran phenol and 3-ketocarbofuran phenol at 10 @f concentration. In contrast, the thiocarbamate herbicides transformed HRP-I into HRP-II (8) and had no effect upon the latter. The formation of HRP-II was followed at 420 nm and that of native peroxidase at 400 nm; the results agreed with those of Lee (6). Our results on oxygen consumption and photon emission together with the product formation in the inhibitor IAA/HRP/O, system showed that carbofuran phenol inhibited the enzymatic degradation of IAA but this was not persistent, indicating that the inhibitor was unstable. Similar results were published with phenolic inhibitors
such as scopolotin and ferulic acid (18, 19). Due to the partially competitive nature of the inhibition by carbofuran phenol the rate of IAA degradation reached the same level as that of the control without the inhibitor after the lag time. Probably, this was due to breakdown of the metabolite. By following spectrophotometrically the reaction, Lee and Chapman (4) concluded that 3-ketocarbofuran phenol was stable under the experimental conditions used. However, our results on oxygen uptake and photon emission showed that this compound consumes oxygen and produces an electronically excited product. Anthracenic acceptors enhanced the emission which lies between 400 and 500 nm. 3-Ketocarbofuran phenol competes with IAA for an HRP active site as was shown by the fact that the emission from IAA oxidation disappears and another one appears in the first minutes of reaction (Fig. 6). This emission in the region 400-500 nm indicates probably a generation of carbonyl excited species via a dioxetane intermediate:
METABOLITES TABLE Enhanced
Emission from PhenollHRPIO,
Acceptor” Control Anthracene-2-sulfonate 9, IO-Diphenylanthracene-2-sulfonate 9, IO-Dibromoanthracene-2-sulfonate
1 Ihe 3-Ketocarbofiran System”
Integrated emission at 60 secC (counts)
Singlet triplet energy (kcal/mol)
20.000 118.000
68
42.5
94.000
65
40.6
95.000
66
40.2
o 3-Ketocarbofuran phenol (200 PM); HRP (3 nM), IAA (0.2 mM), Mn2+ (0.1 mM) in phosphate buffer (25 mA4) pH 5.9 at 25°C. * Acceptor concentration. 5 PM. r Measured in a Hamamatsu TV Photocounter C-767 (10).
139
OF CARBOFURAN
This carbonyl compound in its excited state presumably has (zT,~)* as (n.r)* character, giving enhanced emission with singlet and triplet counter. It is known that the (rr,rr)* character gives more singlet excited carbonyl compounds (22). The products and chemiluminescence of this system are under study. ACKNOWLEDGMENTS The financial support of CNPq (Brasilia), FINEP (Rio de Janeiro), FAPESP (Sao Paulo). CAPES (Brasilia), and UNESCO is appreciated. The authors wish to express their gratitude to Quality Assurance Section, Environmental Toxicology Division, Research Triangle Park, North Carolina, for the gift of pesticidal chemicals and to Dr. G. Cilento for a critical reading of the manuscript as well as for several enlightening discussions.
This is similar to cell-free extract from a Pseudomonas sp. growing on (+)-catechin,
oxidizing dehydrogossypetin by cleaving the A-ring to form oxalacetic and 5-(3,4dihydroxyphenyl)-4-hydroxy-3-oxovaleroSlactone (20). For these processes the formation and cleaving of the dioxetane ring was proposed (21) (Eq. [3]): ?”
1.
2. 3. 4.
REFERENCES E. T. Cherry and C. D. Pless, Effect of carbofuran and disulfoton on parasitism of tobacco budworms and homworms on burley tobacco, J. Econ. Entomol. 64, 187 (1971). J. W. Apple, Response of corn to granular insecticides applied to the row at planting, J. Econ. Enromol. 64, 1208 (1971). T. T. Lee, Insecticide plant interaction: Carbofuran effect on indole-3-acetic acid metabolism and plant growth, Life Sci. 18, 205 (1976). T. T. Lee and R. A. Chapman, Inhibition of en-
0
r
0” 9 ?
zymic oxidation of indole-3-acetic acid by metabolites of the insecticide carbofuran, Phytochemistry 16, 35 (1977). 5. R. L. Metcalf, T. R. Fukuto, C. Collins, K. Borck, S. A. El-Azia, R. Mutioz, and C. C. Cassill, Metabolism of 2,2-dimethyl-2.3dihydrobenzofuranyl-7,N-methylcarbamate (Furadan) in plants, insects and mammals. J. Agric. Food Chem. 16, 300 (1968). 6. T. T. Lee, Role of phenolic inhibitors in peroxidase-mediated degradation of indole-3acetic acid, Plant Physiol. 59, 372 (1977).
140
FRANC0
AND DURAN
7. T. T. Lee, Promotion of plant growth and inhibition of enzymic degradation of indole-3-acetic acid by metabolites of carbofuran, a carbamate insecticide, Canad. /. Bat. 55, 574 (1977). 8. C. Franc0 and N. Durban, Interaction of thiocarbamates with indole-3-acetic acid peroxidase system, Quim Nova 3, 44 (1980). 9. C. C. C. Vidigal, A. Faljoni-Alario, N. Durban. K. Zinner, Y. Shimizu. and G. Cilento, Electronically excited species in the peroxidase catalyzed oxidation of indoleacetic acid: Effect upon DNA and RNA. Photochem. Photobiol. 30, 195 (1979). 10. N.DurBn, K. Zinner, R. C. De Baptista. C. C. C. Vidigal, and G. Cilento, Chemiluminescence from oxidation of auxin derivatives, Photochum. Photobiol. 24, 383 (1976). Il. E. J. H. Bechara, 0. M. M. Faria Oliveira, N. Durban, R. C. De Baptista, and G. Cilento, Peroxidase catalyzed generation of triplet acetone, Photochum. Photobiol. 30, 101 (1979). 12. R. Roman and H. B. Dunford. pH Dependence of the oxidation of iodide by compound I of horseradish peroxidase. Biochemistry 11, 2076 (1972). 13. W. D. Hewson and H. B. Dunford, Stoichiometry of the reaction between horseradish peroxidase and p-cresol, J. Biol. Chem. 251, 6043 (1976). 14. C. C. C. Vidigal, K. Zinner, N. Durban. E. J. H. Bechara. and G. Cilento, Generation of electronic energy in the peroxidase catalyzed oxidation of indole-3-acetic acid, Biochem. Biophys.
Res.
Commun.
65, 138 (1975).
15. M. P. De Mello, S. M. De Toledo, M. Haun, G. Cilento. and N. Durban, Excited indole-3aldehyde from the peroxidase catalyzed aerobic oxidation of indole-3-acetic acid: Reaction with and energy transfer to t-RNA. Biochemistry 19, 5270 (1980). 16. G. Cilento. Generation and transfer of triplet energy in enzymatic systems. Au. Chem. Res. 13, 225 (1980). 17. G. Cilento, Photobiochemistry in the dark, Photothem. Photobiol. Rer*. 5, 199 (1980). 18. J. C. Sirois and R. W. Miller, The mechanism of the scopoletin induced inhibition of the peroxidase-catalyzed degradation by indole-3acetate, Plant Physiol. 49, 1012 (1972). 19. D. A. Gelinas, Proposed model for the peroxidase-catalyzed oxidation of indole-3-acetic acid in the presence of the inhibitor ferulic acid. Plant Physiol. 51, 967 (1973). 20. A. M. Jeffrey, D. M. Jerina, R. Self, and W. C. Evans, The bacterial degradation of flavonoids: Oxidative fission of the A-ring of dehydrogossypetin by a Pseudomona sp.. B&hem. .I. 130, 383 (1972). 21. D. Slawinska, J. Slawinski. K. Polewski, and W. Pukacki, Chemiluminescence in the peroxidation of tannic acid, Photochem. Photobiol. 30, 71 (1979). 22. F. McCapra, I. Beheshti, A. Burford. R. A. Harm. and K. A. Zaklika, Singlet excited states from dioxetane decomposition. J. Chem. Sot. Chem. Cornmltrl.. 944 (1977).
BIOCHEMISTRY
AND
16, 136-
PHYSIOLOGY
140 (1981)
Metabolites of Carbofuran: Effect Indole-3-acetic Acid Degradation CESAR llnstituto
de Quimica,
FRANCO
AND
NELSON
on
DURAN
Universidade Estadual de Campinas. Campinas, Site Paula. Brazil
C.P.
1170, CEP 13100,
Received April 6, 1981: accepted June 17, 1981 Oxygen uptake, product formation, and photon emission in the indole-3-acetic acid/ peroxidase/O, system were inhibited by carbofuran and its metabolites, the 3-ketocarbofuran phenol and carbofuran phenol. These metabolites acted as competitive inhibitors and concomitantly were degraded. Photon emission, together with uv spectrophotometry and oxygen measurements, shows this to be a rapid and reproducible method to study insecticide interaction with the indole-3-acetic oxidase svstem in \jitro. 3-Ketocarbofuran phenol was metabolized by peroxidase with excited-state production.
Carbofuran insecticides have been found to be degradated rapidly into various metabolites in plants, insects. and mammals (l-5); therefore attention was turned to its metabolites. The effects on plant growth of the interaction, between metabolites of carbofuran and indole-3-acetic acid (IAA)’ were confirmed (3, 6, 7). We want to report the interaction of carbofuran metabolites (Fig. 1) with the IAAi HRP/O, system (8- 10).
conductivity measurement was carried out in a Beckman RC16 B2 conductivity apparatus. HRP-I was prepared by adding to a sample of HRP exhaustively dialyzed in pentadistilled water (resistivity of 1.22 x lo5 R), hydrogen peroxide in a 1.1 to 1.0 proportion (12). HRP-II was prepared from HRP-I by the method of Hewson and Dunford (13). RESULTS
MATERIALS
AND
METHODS
’ Abbreviations horseradish
used: IAA, indole-3-acetic peroxidase;
nylanthracene-2-sulfonate: anthracene-2-sulfonate.
DPAS,
DBAS
acid;
9.10-diphe-
9,10-dibromo136
0048-3575/81/050136-05$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction m any form reserved.
DISCUSSION
Oxygen uptake. Carbofuran phenol delays oxygen uptake by the IAAiHRPi O,/Mn*+ system (Fig. 2). 3-Ketocarbofuran phenol inhibited the oxygen consumption at concentrations above 13 pM (Fig. 3). With increasing 3-ketocarbofuran phenol at concentrations higher than 40 pM an increase of oxygen uptake was observed (results not shown), possibly due to substrate oxidation. The 3-ketocarbofuran phenol oxida-
HRP (Type VI) was obtained from Sigma Chemical Company. IAA was from Merck. Carbofuran phenol and 3-ketocarbofuran phenol were from the U.S. Environmental Protection Agency. Environmental Toxicology Division, Research Triangle Park. Absorption spectra were taken on a Zeiss DMR-21 recording spectrophotometer using l-cm cells. The chemiluminescence was measured in a Beckman LS 100~ liquid scintillation counter or in a Hamamatsu TV Photocounter C-767 (11). Oxygen uptake was determined with a Yellow Springs Instruments Model 53 Oxygen Monitor. The
HRP,
AND
METABOLITES
2
4
MINUTES
6
OF
8
FIG. 2. Inhibition of oxygen uptake in the IAAI HRPIMt?+IO, system by different concentrations oj carbofuran phenol: (Cl) 0.0; (m) I PM; (0) 2 PM; (0) 5 PM. Standard conditions: IAA (0.2 mM). HRP (3.0 nM), Mn2+ (0.1 mM), and p-chlorophenol (0.1 mM) in phosphate buffer (25 mM) at pH 5.9 at 25°C.
137
CABBOFURAN
presence of carbofuran phenol, the enzyme activity was recovered after a lag period with the same rate of product formation as control; the lag time was dependent on the inhibitor concentration. In the case of 3ketocarbofuran phenol the enzyme activity was not recovered. Excited-state generation. It is known that the IAA is generated in its electronically excited state by the IAA/HRP/O, system (9, 14, 15). In this system at pH 5.9 and above, correlation between oxygen uptake, product formation, and photon emission was observed. This means that the IAA degradation produces electronically excited state IAI, presumably through a dioxetane intermediate (16, 17):
IAA
tion catalyzed by peroxidase was confirmed by means of oxygen uptake; photon emission accompanies this oxidation. Product formation. Product formation from IAA was followed at 262 nm in the presence of carbofuran metabolites; results were the same as observed by Lee and Chapman (4). Carbofuran phenol was more inhibitory to enzymatic degradation of IAA than was 3-ketocarbofuran phenol. In the 60c
Figures 4 and 5 show the inhibitory effect of carbofuran phenol on the rate of emission. The presence of an excited species other than IA1 at 200 @4 3-ketocarbofuran phenol is evident (Fig. 6). In fact the 3ketocarbofuran phenol/HRP/O, system was analyzed and a strong emission was observed initially during the reaction.
.
2
4
6
1
8
MINUTES
FIG. 3. Inhibition of oxygen aptake in the IAAI HRPlMn’+IO, system by different concentrations of 3-ketocarbofuran phenol: (0) 0.0; (W) 13 PM: (0) 26 PM; (a) 40 PM; under standard reaction conditions.
MINUTES
FIG. 4. Inhibition of photon emission of the IAAI HRPIMn”l0, system by different concentrations of carbofaran phenol under standard reactions conditions: (0) 0.0: (m) I PM; (0) 2 @I: (0) 5 PM.
138
FRANC0
AND
DURAN
v
1
;
-A-- 1~--5
6
MINUTES
FIG. 5. Changes in lag period of IAA degradation by delayed addition of carbofuran phenol (2 pM) in the IAAIHRPIMnZ+I02 system under standard reaction conditions: (0) control. Carbofuran phenol added after: (W) 60 set; (0) 30 set; and (0) 10 sec.
FIG. 6. Photon emission of IAAIHRPIMnZ+I02 system in the presence of diffeerent concentratiotts of 34etocarbofuran phenol under standard reaction conditions: (0) control; (0) 40 PM: (0) 200 PM; (H) 800 ~.LM. Photon emission by the jr-ketocarbofuran phenol (200 pM)IHRPI02 system (- - - ).
Table 1 shows the enhanced emission when 3-ketocarbofuran phenol was degraded by peroxidase in the presence of DPAS and DBAS which are singlet and triplet excited-state counters, respectively (11). Peroxidase alferations. HRP-I or HRP-II, the intermediate forms of HRP, were transformed almost instantaneously to native peroxidase. in the presence of carbofuran phenol and 3-ketocarbofuran phenol at 10 @f concentration. In contrast, the thiocarbamate herbicides transformed HRP-I into HRP-II (8) and had no effect upon the latter. The formation of HRP-II was followed at 420 nm and that of native peroxidase at 400 nm; the results agreed with those of Lee (6). Our results on oxygen consumption and photon emission together with the product formation in the inhibitor IAA/HRP/O, system showed that carbofuran phenol inhibited the enzymatic degradation of IAA but this was not persistent, indicating that the inhibitor was unstable. Similar results were published with phenolic inhibitors
such as scopolotin and ferulic acid (18, 19). Due to the partially competitive nature of the inhibition by carbofuran phenol the rate of IAA degradation reached the same level as that of the control without the inhibitor after the lag time. Probably, this was due to breakdown of the metabolite. By following spectrophotometrically the reaction, Lee and Chapman (4) concluded that 3-ketocarbofuran phenol was stable under the experimental conditions used. However, our results on oxygen uptake and photon emission showed that this compound consumes oxygen and produces an electronically excited product. Anthracenic acceptors enhanced the emission which lies between 400 and 500 nm. 3-Ketocarbofuran phenol competes with IAA for an HRP active site as was shown by the fact that the emission from IAA oxidation disappears and another one appears in the first minutes of reaction (Fig. 6). This emission in the region 400-500 nm indicates probably a generation of carbonyl excited species via a dioxetane intermediate:
METABOLITES TABLE Enhanced
Emission from PhenollHRPIO,
Acceptor” Control Anthracene-2-sulfonate 9, IO-Diphenylanthracene-2-sulfonate 9, IO-Dibromoanthracene-2-sulfonate
1 Ihe 3-Ketocarbofiran System”
Integrated emission at 60 secC (counts)
Singlet triplet energy (kcal/mol)
20.000 118.000
68
42.5
94.000
65
40.6
95.000
66
40.2
o 3-Ketocarbofuran phenol (200 PM); HRP (3 nM), IAA (0.2 mM), Mn2+ (0.1 mM) in phosphate buffer (25 mA4) pH 5.9 at 25°C. * Acceptor concentration. 5 PM. r Measured in a Hamamatsu TV Photocounter C-767 (10).
139
OF CARBOFURAN
This carbonyl compound in its excited state presumably has (zT,~)* as (n.r)* character, giving enhanced emission with singlet and triplet counter. It is known that the (rr,rr)* character gives more singlet excited carbonyl compounds (22). The products and chemiluminescence of this system are under study. ACKNOWLEDGMENTS The financial support of CNPq (Brasilia), FINEP (Rio de Janeiro), FAPESP (Sao Paulo). CAPES (Brasilia), and UNESCO is appreciated. The authors wish to express their gratitude to Quality Assurance Section, Environmental Toxicology Division, Research Triangle Park, North Carolina, for the gift of pesticidal chemicals and to Dr. G. Cilento for a critical reading of the manuscript as well as for several enlightening discussions.
This is similar to cell-free extract from a Pseudomonas sp. growing on (+)-catechin,
oxidizing dehydrogossypetin by cleaving the A-ring to form oxalacetic and 5-(3,4dihydroxyphenyl)-4-hydroxy-3-oxovaleroSlactone (20). For these processes the formation and cleaving of the dioxetane ring was proposed (21) (Eq. [3]): ?”
1.
2. 3. 4.
REFERENCES E. T. Cherry and C. D. Pless, Effect of carbofuran and disulfoton on parasitism of tobacco budworms and homworms on burley tobacco, J. Econ. Entomol. 64, 187 (1971). J. W. Apple, Response of corn to granular insecticides applied to the row at planting, J. Econ. Enromol. 64, 1208 (1971). T. T. Lee, Insecticide plant interaction: Carbofuran effect on indole-3-acetic acid metabolism and plant growth, Life Sci. 18, 205 (1976). T. T. Lee and R. A. Chapman, Inhibition of en-
0
r
0” 9 ?
zymic oxidation of indole-3-acetic acid by metabolites of the insecticide carbofuran, Phytochemistry 16, 35 (1977). 5. R. L. Metcalf, T. R. Fukuto, C. Collins, K. Borck, S. A. El-Azia, R. Mutioz, and C. C. Cassill, Metabolism of 2,2-dimethyl-2.3dihydrobenzofuranyl-7,N-methylcarbamate (Furadan) in plants, insects and mammals. J. Agric. Food Chem. 16, 300 (1968). 6. T. T. Lee, Role of phenolic inhibitors in peroxidase-mediated degradation of indole-3acetic acid, Plant Physiol. 59, 372 (1977).
140
FRANC0
AND DURAN
7. T. T. Lee, Promotion of plant growth and inhibition of enzymic degradation of indole-3-acetic acid by metabolites of carbofuran, a carbamate insecticide, Canad. /. Bat. 55, 574 (1977). 8. C. Franc0 and N. Durban, Interaction of thiocarbamates with indole-3-acetic acid peroxidase system, Quim Nova 3, 44 (1980). 9. C. C. C. Vidigal, A. Faljoni-Alario, N. Durban. K. Zinner, Y. Shimizu. and G. Cilento, Electronically excited species in the peroxidase catalyzed oxidation of indoleacetic acid: Effect upon DNA and RNA. Photochem. Photobiol. 30, 195 (1979). 10. N.DurBn, K. Zinner, R. C. De Baptista. C. C. C. Vidigal, and G. Cilento, Chemiluminescence from oxidation of auxin derivatives, Photochum. Photobiol. 24, 383 (1976). Il. E. J. H. Bechara, 0. M. M. Faria Oliveira, N. Durban, R. C. De Baptista, and G. Cilento, Peroxidase catalyzed generation of triplet acetone, Photochum. Photobiol. 30, 101 (1979). 12. R. Roman and H. B. Dunford. pH Dependence of the oxidation of iodide by compound I of horseradish peroxidase. Biochemistry 11, 2076 (1972). 13. W. D. Hewson and H. B. Dunford, Stoichiometry of the reaction between horseradish peroxidase and p-cresol, J. Biol. Chem. 251, 6043 (1976). 14. C. C. C. Vidigal, K. Zinner, N. Durban. E. J. H. Bechara. and G. Cilento, Generation of electronic energy in the peroxidase catalyzed oxidation of indole-3-acetic acid, Biochem. Biophys.
Res.
Commun.
65, 138 (1975).
15. M. P. De Mello, S. M. De Toledo, M. Haun, G. Cilento. and N. Durban, Excited indole-3aldehyde from the peroxidase catalyzed aerobic oxidation of indole-3-acetic acid: Reaction with and energy transfer to t-RNA. Biochemistry 19, 5270 (1980). 16. G. Cilento. Generation and transfer of triplet energy in enzymatic systems. Au. Chem. Res. 13, 225 (1980). 17. G. Cilento, Photobiochemistry in the dark, Photothem. Photobiol. Rer*. 5, 199 (1980). 18. J. C. Sirois and R. W. Miller, The mechanism of the scopoletin induced inhibition of the peroxidase-catalyzed degradation by indole-3acetate, Plant Physiol. 49, 1012 (1972). 19. D. A. Gelinas, Proposed model for the peroxidase-catalyzed oxidation of indole-3-acetic acid in the presence of the inhibitor ferulic acid. Plant Physiol. 51, 967 (1973). 20. A. M. Jeffrey, D. M. Jerina, R. Self, and W. C. Evans, The bacterial degradation of flavonoids: Oxidative fission of the A-ring of dehydrogossypetin by a Pseudomona sp.. B&hem. .I. 130, 383 (1972). 21. D. Slawinska, J. Slawinski. K. Polewski, and W. Pukacki, Chemiluminescence in the peroxidation of tannic acid, Photochem. Photobiol. 30, 71 (1979). 22. F. McCapra, I. Beheshti, A. Burford. R. A. Harm. and K. A. Zaklika, Singlet excited states from dioxetane decomposition. J. Chem. Sot. Chem. Cornmltrl.. 944 (1977).