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Copper-catalyzed oxidation of thiomalic acid
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Copper-Catalyzed
144, 496-502 (1971)
Oxidation
of Thiomalic
Acid
CARLO DE MARCO, SILVESTRO DUPRR, CARLO CRIFO, ROTILIO, AND DORIANO CAVALLINI
GIUSEPPE
Institute of BioZogicaE Chemistry, University of Cagliari, Cagliari, Italy and Institute of Biological Chemistry, University of Rome and Cenler of Molecular Biology, National Research Council, Rome, Italy Received
November
11, 1970; accepted
February
8, 1971
The copper-catalyzed alkaline oxidation of thiomalic acid (TMA) to the corresponding disulfide occurs with the metal firmly bound in a stable TMA-Cntr complex, which is characterized by a sharp absorption peak with maximum at 340 nm. In this complex the TMA to copper ratio is 2 : 1. The complex remains unchanged during TMA oxidation by molecular oxygen, and quickly disappears at the end of the reaction. In t,he absence of oxygen the TMA-01” complex slowly disappears, Cur’ being reduced by the excess of TMA. In t.he course of TMA oxidation an intermediate compound, showing a characteristic absorption band centered at 300 am, accumulates in the solut,ion, and quickly disappears at the end of the reaction, in coincidence with the complete oxidation of TMA. It has been demonstrated that the amount of the 300-nm absorbing compound is proportional to the initial TMA concentration and to the oxygen concentration, while is independent of the concentration of TMA-Cu” complex. It may possibly represent an intermediate between TMA and the disulfide in which oxygen is involved. Hz02 is produced during TMA oxidation, accumulates in the reaction mixture in amounts proportional to the oxygen concentration, and is quickly destroyed at the end of the reaction, when CuT1 is set free from the TMA-CuIr complex.
In previous studies on t.he copper-catalyzed oxidation of cy&eine and other thiols (1, 2) to disulfides, it has been demonstrated that a copper’I-cysteine complex, in which the cysteine to copper ratio is 211, is the main intermediate in the reaction, and represents t,he catalyst of the oxidation. In extending the study of copper-catalyzed oxidations to thiomalic acid (TMA), t,he formation of a similar TMA-Cu” complex has been demonst,rat.ed in alkaline medium. Moreover, spectral evidence has been obtained for another different intermediate of the react,ion, on which we will report here. The metal-ion catalyzed oxidation of TMA had been extensively studied, but under different conditions, by Hill and McAuley (3-6). These authors followed the oxidation catalyzed by ceriumIv, cobalP, and vanadiumV at acidic pH. They were able to detect the t,ransient formation of 496
metal ion-TNA complexes with half-Iives of formation of the order of milliseconds. These complexes probably may be compared to the copper-TMA one, which under the conditions of the present study is much more stable, allowing a more detailed analysis of the int’ermediat)es. MATERIALS
AND
METHODS
Thiomalic acid (purum) was a product from Fluka, Switzerland. It was used without further purification. All other reagents were analytical grade products. The standard reaction mixture contained 1OP M TMA and 10-h in CuCl2 in 0.1 N NaOH. The reaction was started by the addition of copper chloride, and was usually allowed to occur in a stoppered quartz cuvette in the spectrophotometer (l-cm light path, 3-ml volume). Spectra were recorded in a Beckman DB-G spectrophotometer equipped with a Sargent SRL-G recorder. Variable oxygen concentrations in the reaction mix-
OXIDATION
OF THIOMALIC
ture were obtained by adding different volumes of helium-saturated solution of CuCls to oxygensaturated TMA in NaOH. Experiments in oxygenfree solutions were carried out in a Thunberg tube sealed to the upper part of a quartz Beckmau cuvette. Wheu larger volumes were necessary to perform chemical analyses at various times, the reaction was allowed to occur in an all-glass syringe, from which the desired amounts of the reaction mixture were directly injected in graduated test tubes containing the reagent. In this way contact with air was avoided during the reaction and when withdrawing the samples. SII and SS groups were detected by the Folin-Marenzi reaction (7). H*O:! was determined calorimetrically with titanous chloride according to Egertou et ul. (8). Lumiuol chemiluminescence was detected as previously described (1). Electrou paramagnetic resonance (EPR) analyses were carried out at 123°K in a Varian V 4502-14 spectrometer, equipped with a loo-kc field modulation uuit and with the Varian variable temperature accessory. Microwave frequency was 9.16 kc, aud microwave power was 6.4 mW.
197
ACID
1.5
O.D.
1.0
xlO*M
Cu'*
5
RESULTS
The TMA-Cu”
Complex
When an excess of TMA is added to an alkaline solution of copper’I, a sharp absorption peak centered at 340 nm immediately appears. The peak extinction is a function of the copper concentration (Fig. 1). By varying the TXIA to copper ratio it has been demonstrated that the maximal absorbancy at 340 nm is obtained when the rat,io is 2 (CuII concentration ranging from 2.5 X 10-j to 2 X lop4 M), suggesting that the stoichiometry for the formation of the compound absorbing at 340 nm is 2 moles of TMA for 1 mole of copper (Fig. 2). In oxygen-saturated standard reaction mixture the 340-nm peak remains unchanged for about 12 min, and then quickly disappears (see inset of Fig. 6). During this time T&IA is slowly and completely oxidized, as shown by analyses for SH groups. In analogy lvith the results obtained with cysteine (2) it has been supposed that t,he 340-nm peak is due to a complex between T&IA and CuII, which remains unchanged until an excess of oxygen is present in solution and disappears when copper is reduced by t,he thiol. This suggestion was confirmed by correlating absorption at 340
l-
I
I
’
300 nm LOo FIG. 1. Optical spectra recorded immediately after mixing thiomalic acid aud copper in alkaline solution. TMA 10-a -“I; CuCls 0.25, 0.5, 0.75, 1, and 1.5 X 10e4 M (from bot,tom to top), ill 0.1 N NaOH. Light path: 1 cm. In t,he iuset: absorbancy at 340 nm as a function of CuCl:! concentration under the same conditions.
nm with the presence of CuII in solutlion, as detected by EPR analyses. Figure 3 shows t,he EPR spectra of an oxygen-sat,urated standard reaction mixture at various stages of t,he reaction. A characteristic EPR spectrum att,ributable to a distinct complex between TMA and Cu’I is present as long as the 340 nm peak is detectable. In fact, even though the hyperfine structure is not resolved enough to permit a detailed EPR analysis of the spectrum in terms of g and A values, the signal is shifted to magnetic field values which are comparable to those of the cysteine-Cu complex under the same
49%
DE MARCO
1
i
FIG. 2. Absorbancy at 340 nm as a function of the TMA to copper ratio. OD recorded immediately after mixing. Solutions in 0.1 N NaOH. A = CuClz 5 X IO-& M; B = CuClz IO+ M.
MAGNETIC
FIELD
(bauss)
FIG. 3. EPR spectra in oxygen-saturated standard reaction mixture (TMA 10-Z M, CuCl? lo+ M in 0.1 N NaOH). a, CuCln; b, after addition of TMA, as long as the 340-nm peak is present; c, after disappearance of the 340 nm peak.
(2) and indicate the formation of a compfex with lower g values than the hydroxycuproate complex. The latter feature can be related to a high degree of covalency
conditions
ET AL.
(9) which is in keeping with the participation of sulfur in the complex (10). When the absorption at 340 nm disappears, the EPR spectrum shows t#hepresence of free copperI in the solution. Therefore, it may be concluded that, as in t’he case of cysteine (2), the absorbancy at 340 nm is directly correlated with the presence in solution of a complex between TMA and copperII. Moreover, double integration of t#he EPR spectra shows that almost all the copper is complexed with TMA. Experiments performed in the presence of neocuproine (2,9 dimethyl-l , 10 phenanthroline, 3 X 1O-4 M in standard oxygen-saturated reaction mixture) demonstrated that no free copper’ is present in the solution during the course of TMA oxidation.’ When the oxidat.ion of TMA was completed, the t,ypical spectrum of the neocuproine-Cu’ complex suddenly appeared, concomitant with the disappearance of the 340-nm peak. These results and those obtained by EPR analyses indicate that during the course of the TMA oxidation almost all t,he copper is present as copperII. Just at the end of the reaction the copper is fully reduced: it may be recovered as copper1 if an approprate chelating agent is present,, as free copperI in the absence of it. In this respect TMA behaves like cysteine (cf. Fig. 4 of Ref. 2). In oxygen-free standard reaction mixtures, the absorbancy at 340 nm shows a continuous slow decrease (Fig. 4) and disappears in about 60 min. The decrease of the 340-nm peak is attribut’able to the reduction of copper wit’h the consequent’ disappearance of the TMA-CuI’ complex. This has been confirmed by EPR analyses (Fig. 5) which indicate that in the absence of oxygen the CuII signal progressively decreases in intensity, paralleling the decrease in absorbancy at 340 nm. If oxygen is admitted to these solut,ions, the copper is immediately reoxidized, the absorption at 340 nm reaches again the initial values, and then remains unchanged until all the TMA is oxidized. 1 Cur tightly bound to TMA or it.s oxidation product(s) cannot be excluded by neocuproine tests; however, the presence of Cur in more than traces is ruled out by the EPR results.
OXIDATION
OF THIOMALIC
499
ACID
1.1
lI.l. lo
d d
min.
3o
1.2
1. an .0 4
8
12
.6
.1
.2 3m
4uu
“ml
FIG. 4. Spectral changes in oxygen-free stand. ard react.ion mixture, (TMA 10-a M; CuCl2 lo+ M in NaOH 0.1 N). The figures on the curves indicate the t.ime in minutes after the addition of C!uC12 fo TMA. In the inset: decrease with time of log ODaao , for the first 30 min.
a
H-, b c FIQ. 5. EPR spectra of oxygen-free reaction mixture containing 2.10+ M TMA and 2.1W4 M CuClz in 0.1 NaOH. a, spectrum recorded after 1 min (ODW, = 1.76); b, spectrum recorded after 5 min (OD3a = 1.39) ; c, spectrum recorded aft.er 10 min. (ODsdO = 0.78).
The resu1t.s obtained in oxygen-free solut,ions are, therefore, anot,her clear indication that the 340-nm peak is due to a eomplex between TlUA and copperI’.
2 Fro. G. Spectral changes in oxygen-saturated standard reaction mixture, (TMA 1OW M, CuClz 1OV M in 0.1 N NaOH). From bottom t,o top, curves recorded 1, 4, 7, and 11 min after mixing. Inset: time course of the OD variations at 300 nm (full line) and at 340 nm (broken line).
The 300-nm Contpound In addition to the immediate formation of the TMA-Cul’ complex absorbing at 340 nm, other spectral changes are observable at shorter wavelengths. In fact another absorption peak with maximum at 300 nm is slowly formed during the oxidation of TMA, and disappears abruptly at the end of the reaction. Figure 6 shows the optical spect,ra recorded at various times in an oxygen-saturated standard react,ion mixture. In the inset the time course of the absorption peaks at 340 and 300 nm is recorded. There is no isosbest’ic point for the curves recorded at various times, and this indicates that t,he two peaks are due to different, compounds.
500
DE MARCO
-0-s .x2
.66
.
M
FIG. 7. Maximal absorbancies attained at 300 nm (full line) and at 340 nm (broken line) in oxygeil-saturated reaction mixtures (0.1 N NaOH) as function of TMA concentration (abscissa). Open circles: 5.10V5 M CuClz . Full circles: 1P M CuCla. The small increase in absorbancy at 340 nm is ascribable to the residual absorption at this wavelength of the 300-nm peak (cf. Fig. 6).
Data were obtained indicating that the 300-nm peak: (a) is independent of copper concentration; (b) is directly correlated to TMA concentration; (c) is correlated to oxygen concentration. Figure 7 shows the maximum absorption values reached at 300 and 340 nm in oxygensaturated react’ion mixtures at different concentrations of TMA and of CuClz . It is evident that the extinction at 300 nm is directly proportional to SH concentration, and independent of copper concentration, whereas the 340-nm absorption is independent of thiol concentration. Figure 8 shows the effects of varying the oxygen concentration on the reaction rate and on the absorbancies at 300 and 340 nm. It is evident that a primary effect of the increasing oxygen concentration is to increase the reaction rate: the oxidation of TMA which is completed in 12 min in oxygen-saturated solution, requires about 20 min at 25% oxygen saturation. Aloreover, while the absorption at 340 nm is practically unaffected, the ext,inction at
ET AL.
FIG. 8. Effect of varying oxygen concentratiou on the reaction time, and on the absorbancy at 340 nm (full lines) and 300 nm (broken lines) in a standard reaction mixture. Figures on the curves indicate the percentage oxygen saturation. In the inset: maximal absorbancy attained at 300 nm as a function of the oxygen concentration.
300 nm reaches values that are directly correlated with the oxygen concentration. In oxygen-free reaction mixtures, the peak at 300 nm is completely absent (Fig. 4). All these observat’ions indicat,e that the 300-nm peak may represent a transient intermediate in the oxidation of TMA to the disulfide, and oxygen seems to be involved in t’his intermediate. Reaction Products During the reaction TMA is oxidized to the corresponding disulfide. This has been qualitatively checked at the end of the reaction in the following way. The standard reaction mixture was acidified with concentrated HCl and repeatedly extracted with ether; the ether was taken to dryness and the residue subjected to paper chromatography in water-saturated phenol, using as control nearly equal amount of the disulfide of TMA prepared according t’o Hill and McAuley by oxidation with cerium sulfate (4). The chromatograms were developed with the Folin-Marenzi reagent (9). In this solvent the RF for TMA was 0.4 and for the disulfide 0.1.
OXIDATION
OF THIOMALIC
TMA disulfide was also det,ected by gaschromatography. A standard reaction mixture was acidified at the end of the reaction and repeatedly extracted with ether. The ethereal ext,ract was taken t,o dryness and t,he residue then subjected to met’hylatjon in met,hanol-HiSO under reflux for 6 hr. Gas-chromatographic analyses (WE gas chromatograph; 1.5 m X 0.4-cm column of 20 5%DEGS on Anachrom A, 70-80 mesh; carrier: iU2 40 ml/min; programmed temperatures increase: 3”/min from 110 t’o 175” C) shoTI-ed only one peak (elution t’ime: 7-S min) in t,he same position as that of TfilA disulfide met’hyl est’er prepared as above from TMA disulfide, and well separated from TIBIA methyl ester (elution t’ime: 20 minj. Quantitatively, the formation of the disulfide was checked by performing at the end of t,he reaction the Folin-RIarenzi react.ion in the presence of bisulfite (for detecting SS groups), using as standard the TRfA disulfide. The results obtained indicate that almost8 all TXA may be recovered as disulfide. During t#hereaction H202 is also produced and accumulates in the reaction mixture. It is destroyed suddenly and concomit,antly \vith the disappearance of the 340- and 300-nm peaks, at the end of t’he reaction. This behavior is exactly the same as that observed \vith cyst,eine (2) : until t’he CulI is complexed with the thiol H302 accumulates; when the thiol is completely oxidized, the Cu’I is set free in solution and catalyzes the immediate decomposition of Hz02. Figure 9 shows the time course of Hz02 production in standard reaction mixtures at different oxygen saturation. At’ lower oxygen concentration a lower amount of Hz02 is produced. A good parallelism is evident bet,ween the amount’ of HsOr, t,hat of the 300-nm absorbing compound and the oxygen concentration. H,O? production and decomposition has also been invest’igated by det’ecting the chemiluminescence excited on luminol (5 amino-?, X-dihydro-1 ,4-phtalazinedione). When t,he oxidation of TJIA is allowed to occur in the presence of luminol (10e3 M), a light, flash appears at the end of the reaction.
301
ACID
FIG. 9. Time course of 11202 production in standard reaction mixtures at different, oxygen concentratioll. Figures on the curves indicate the percentage of oxygen saturation. In the inset: maximal IIIOz product,ion as a function of the oxygen concentration.
Since luminol chemiluminescence is due to the decomposition of HzOp catalyzed by free CulI (1). t)he production of light just at the end of the reaction indicates t,hat (i) H202 is formed and accumulates during the reaction; (ii) HZO, is not, decomposed during t,he reaction, as no free Cul’ is present; and (iii) H& is xuddently decomposed at’ the end of t’he reaction, since when all the T&IA is oxidized, t)he CuT1 is set, free in solution. Moreover, if H202 is added to a standard reaction mixture in the presence of luminol, no light emission is observed until the end of the reaction. This is another indication that t’he Cu” is st)rongly complexed by TMA, and even an excess of H?O, cannot be destroyed. DISCUSSION
E’rom the results described above, the following conclusions may be drawn. In alkaline solutions TMA binds to Cu” in a stable complex whose st,oichiomet,ry is 2 moles of TRZA for 1 mole of copper. Almost all the copper is complexed. The TXL-CU’~ com-
502
DE MARCO
plex is detected spectrophot’ometrically by a sharp absorption peak centered at 340 nm. TMA is oxidized to the corresponding disulfide with production of Hz02 . If oxygen is present the TMA-Cu’I complex is stable until the thiol oxidation is complete, and Hz02 accumulates in the solution. In the absence of oxygen the copper of the complex is slowly reduced. The fact that when a large excess of oxygen is present more HzOZ accumulates may suggest that TMA, or the T&IA-C@ complex, is better oxidized by molecular oxygen than by HzOz. However, when oxygen is present in limited amount, the oxidation of TMA may proceed at the expense of H202, which is, therefore, recovered in smaller amounts. The oxidation of TMA is accompanied by the formation of an intermediate detected spectrophotometrically by its absortion at 300 nm. It has been excluded that this absorption could be due to the products of the reaction, since neither the disulfide of TMA nor Hz02 shows absorption at this wavelength. Furthermore, the behavior of the 300-nm peak is clearly that of an intermediate, since it increases during the reaction but completely disappears at the end. Its concentration increases almost linearly with time, paralleling the production of the disulfide and of HtOz, and finally disappears suddenly. In the presence of limited amounts of molecular oxygen, the concentration of the intermediate reaches smaller values, as does also the production of HsOz. The concentration of the 300-nm compound is a function of the time of the reaction, of the thiol concentration, and of the oxygen concentration, while it is independent of the amount of copper. This seems to
ET AL.
exclude possible complexes of the type TMA-Cu-02 or TMA-Cu-H202, even if these have not been really ruled out. An addition compound between TMA (TMA radical?) and oxygen may be possibly taken into consideration. In conclusion, the results obtained with TMA indicate that the copper-catalyzed oxidation of this thiol occurs virtually with the same general features as in the case of cysteine and other thiols, that is, with the metal firmly bound in a stable complex, which possibly represents the electron donor to oxygen (2). The main difference appears to be the formation, or perhaps the easier detection, of an intermediate absorbing at 300 nm, the structure of which at present remains undefined. REFERENCES 1. C~VALLINI,
2.
3. 4. 5.
6. 7. 8.
9. 10. 11.
D., DE MARCO, C., AND DUPRI?,, S., Arch. Biochem. Biophys. 134, 18 (1968). CBVALLINI, D., DEMARCO, C., DUPR&, S., END ROTILIO, G., Arch. Biochem. Biophys. 130,354 (1969). HILL, J., AND MCAULEY, A., J. Chem. Sot. A, 2405 (1968). HILL, J., AND MCAULEY, A., J. Chem. Sot. A, 156 (1968). PICKERING, W. F., BND MCAULEY, A., J. Chem. Sot. A, 1173 (1968). HILL, J., MCAULEY, A., AND PICKERING, W. F., Chem. Commun. 573 (1967). FoLIN,O.,~~NDM.~RENZI, A.D., J.Biol. Chem. 33,109 (1929). EGERTON, A. C., EVERETT, A. J., MINKOFF, G. J., RUDRAK~NCHANA, S.,SALOOJA, K. C., Anal. Chim. Acta 10,422 (1954). KIVELSON, D., AND NEIMAN, R., J. Chem. Phys. 36, 149 (1961). ROTILIO, G., AND CALABRESE, L., Arch. Biochem. Biophys., in press. MONDOV?, B., MODIANO, G., AND DE MARCO, C., G. Biochim. 4, 324 (1955).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Copper-Catalyzed
144, 496-502 (1971)
Oxidation
of Thiomalic
Acid
CARLO DE MARCO, SILVESTRO DUPRR, CARLO CRIFO, ROTILIO, AND DORIANO CAVALLINI
GIUSEPPE
Institute of BioZogicaE Chemistry, University of Cagliari, Cagliari, Italy and Institute of Biological Chemistry, University of Rome and Cenler of Molecular Biology, National Research Council, Rome, Italy Received
November
11, 1970; accepted
February
8, 1971
The copper-catalyzed alkaline oxidation of thiomalic acid (TMA) to the corresponding disulfide occurs with the metal firmly bound in a stable TMA-Cntr complex, which is characterized by a sharp absorption peak with maximum at 340 nm. In this complex the TMA to copper ratio is 2 : 1. The complex remains unchanged during TMA oxidation by molecular oxygen, and quickly disappears at the end of the reaction. In t,he absence of oxygen the TMA-01” complex slowly disappears, Cur’ being reduced by the excess of TMA. In t.he course of TMA oxidation an intermediate compound, showing a characteristic absorption band centered at 300 am, accumulates in the solut,ion, and quickly disappears at the end of the reaction, in coincidence with the complete oxidation of TMA. It has been demonstrated that the amount of the 300-nm absorbing compound is proportional to the initial TMA concentration and to the oxygen concentration, while is independent of the concentration of TMA-Cu” complex. It may possibly represent an intermediate between TMA and the disulfide in which oxygen is involved. Hz02 is produced during TMA oxidation, accumulates in the reaction mixture in amounts proportional to the oxygen concentration, and is quickly destroyed at the end of the reaction, when CuT1 is set free from the TMA-CuIr complex.
In previous studies on t.he copper-catalyzed oxidation of cy&eine and other thiols (1, 2) to disulfides, it has been demonstrated that a copper’I-cysteine complex, in which the cysteine to copper ratio is 211, is the main intermediate in the reaction, and represents t,he catalyst of the oxidation. In extending the study of copper-catalyzed oxidations to thiomalic acid (TMA), t,he formation of a similar TMA-Cu” complex has been demonst,rat.ed in alkaline medium. Moreover, spectral evidence has been obtained for another different intermediate of the react,ion, on which we will report here. The metal-ion catalyzed oxidation of TMA had been extensively studied, but under different conditions, by Hill and McAuley (3-6). These authors followed the oxidation catalyzed by ceriumIv, cobalP, and vanadiumV at acidic pH. They were able to detect the t,ransient formation of 496
metal ion-TNA complexes with half-Iives of formation of the order of milliseconds. These complexes probably may be compared to the copper-TMA one, which under the conditions of the present study is much more stable, allowing a more detailed analysis of the int’ermediat)es. MATERIALS
AND
METHODS
Thiomalic acid (purum) was a product from Fluka, Switzerland. It was used without further purification. All other reagents were analytical grade products. The standard reaction mixture contained 1OP M TMA and 10-h in CuCl2 in 0.1 N NaOH. The reaction was started by the addition of copper chloride, and was usually allowed to occur in a stoppered quartz cuvette in the spectrophotometer (l-cm light path, 3-ml volume). Spectra were recorded in a Beckman DB-G spectrophotometer equipped with a Sargent SRL-G recorder. Variable oxygen concentrations in the reaction mix-
OXIDATION
OF THIOMALIC
ture were obtained by adding different volumes of helium-saturated solution of CuCls to oxygensaturated TMA in NaOH. Experiments in oxygenfree solutions were carried out in a Thunberg tube sealed to the upper part of a quartz Beckmau cuvette. Wheu larger volumes were necessary to perform chemical analyses at various times, the reaction was allowed to occur in an all-glass syringe, from which the desired amounts of the reaction mixture were directly injected in graduated test tubes containing the reagent. In this way contact with air was avoided during the reaction and when withdrawing the samples. SII and SS groups were detected by the Folin-Marenzi reaction (7). H*O:! was determined calorimetrically with titanous chloride according to Egertou et ul. (8). Lumiuol chemiluminescence was detected as previously described (1). Electrou paramagnetic resonance (EPR) analyses were carried out at 123°K in a Varian V 4502-14 spectrometer, equipped with a loo-kc field modulation uuit and with the Varian variable temperature accessory. Microwave frequency was 9.16 kc, aud microwave power was 6.4 mW.
197
ACID
1.5
O.D.
1.0
xlO*M
Cu'*
5
RESULTS
The TMA-Cu”
Complex
When an excess of TMA is added to an alkaline solution of copper’I, a sharp absorption peak centered at 340 nm immediately appears. The peak extinction is a function of the copper concentration (Fig. 1). By varying the TXIA to copper ratio it has been demonstrated that the maximal absorbancy at 340 nm is obtained when the rat,io is 2 (CuII concentration ranging from 2.5 X 10-j to 2 X lop4 M), suggesting that the stoichiometry for the formation of the compound absorbing at 340 nm is 2 moles of TMA for 1 mole of copper (Fig. 2). In oxygen-saturated standard reaction mixture the 340-nm peak remains unchanged for about 12 min, and then quickly disappears (see inset of Fig. 6). During this time T&IA is slowly and completely oxidized, as shown by analyses for SH groups. In analogy lvith the results obtained with cysteine (2) it has been supposed that t,he 340-nm peak is due to a complex between T&IA and CuII, which remains unchanged until an excess of oxygen is present in solution and disappears when copper is reduced by t,he thiol. This suggestion was confirmed by correlating absorption at 340
l-
I
I
’
300 nm LOo FIG. 1. Optical spectra recorded immediately after mixing thiomalic acid aud copper in alkaline solution. TMA 10-a -“I; CuCls 0.25, 0.5, 0.75, 1, and 1.5 X 10e4 M (from bot,tom to top), ill 0.1 N NaOH. Light path: 1 cm. In t,he iuset: absorbancy at 340 nm as a function of CuCl:! concentration under the same conditions.
nm with the presence of CuII in solutlion, as detected by EPR analyses. Figure 3 shows t,he EPR spectra of an oxygen-sat,urated standard reaction mixture at various stages of t,he reaction. A characteristic EPR spectrum att,ributable to a distinct complex between TMA and Cu’I is present as long as the 340 nm peak is detectable. In fact, even though the hyperfine structure is not resolved enough to permit a detailed EPR analysis of the spectrum in terms of g and A values, the signal is shifted to magnetic field values which are comparable to those of the cysteine-Cu complex under the same
49%
DE MARCO
1
i
FIG. 2. Absorbancy at 340 nm as a function of the TMA to copper ratio. OD recorded immediately after mixing. Solutions in 0.1 N NaOH. A = CuClz 5 X IO-& M; B = CuClz IO+ M.
MAGNETIC
FIELD
(bauss)
FIG. 3. EPR spectra in oxygen-saturated standard reaction mixture (TMA 10-Z M, CuCl? lo+ M in 0.1 N NaOH). a, CuCln; b, after addition of TMA, as long as the 340-nm peak is present; c, after disappearance of the 340 nm peak.
(2) and indicate the formation of a compfex with lower g values than the hydroxycuproate complex. The latter feature can be related to a high degree of covalency
conditions
ET AL.
(9) which is in keeping with the participation of sulfur in the complex (10). When the absorption at 340 nm disappears, the EPR spectrum shows t#hepresence of free copperI in the solution. Therefore, it may be concluded that, as in t’he case of cysteine (2), the absorbancy at 340 nm is directly correlated with the presence in solution of a complex between TMA and copperII. Moreover, double integration of t#he EPR spectra shows that almost all the copper is complexed with TMA. Experiments performed in the presence of neocuproine (2,9 dimethyl-l , 10 phenanthroline, 3 X 1O-4 M in standard oxygen-saturated reaction mixture) demonstrated that no free copper’ is present in the solution during the course of TMA oxidation.’ When the oxidat.ion of TMA was completed, the t,ypical spectrum of the neocuproine-Cu’ complex suddenly appeared, concomitant with the disappearance of the 340-nm peak. These results and those obtained by EPR analyses indicate that during the course of the TMA oxidation almost all t,he copper is present as copperII. Just at the end of the reaction the copper is fully reduced: it may be recovered as copper1 if an approprate chelating agent is present,, as free copperI in the absence of it. In this respect TMA behaves like cysteine (cf. Fig. 4 of Ref. 2). In oxygen-free standard reaction mixtures, the absorbancy at 340 nm shows a continuous slow decrease (Fig. 4) and disappears in about 60 min. The decrease of the 340-nm peak is attribut’able to the reduction of copper wit’h the consequent’ disappearance of the TMA-CuI’ complex. This has been confirmed by EPR analyses (Fig. 5) which indicate that in the absence of oxygen the CuII signal progressively decreases in intensity, paralleling the decrease in absorbancy at 340 nm. If oxygen is admitted to these solut,ions, the copper is immediately reoxidized, the absorption at 340 nm reaches again the initial values, and then remains unchanged until all the TMA is oxidized. 1 Cur tightly bound to TMA or it.s oxidation product(s) cannot be excluded by neocuproine tests; however, the presence of Cur in more than traces is ruled out by the EPR results.
OXIDATION
OF THIOMALIC
499
ACID
1.1
lI.l. lo
d d
min.
3o
1.2
1. an .0 4
8
12
.6
.1
.2 3m
4uu
“ml
FIG. 4. Spectral changes in oxygen-free stand. ard react.ion mixture, (TMA 10-a M; CuCl2 lo+ M in NaOH 0.1 N). The figures on the curves indicate the t.ime in minutes after the addition of C!uC12 fo TMA. In the inset: decrease with time of log ODaao , for the first 30 min.
a
H-, b c FIQ. 5. EPR spectra of oxygen-free reaction mixture containing 2.10+ M TMA and 2.1W4 M CuClz in 0.1 NaOH. a, spectrum recorded after 1 min (ODW, = 1.76); b, spectrum recorded after 5 min (OD3a = 1.39) ; c, spectrum recorded aft.er 10 min. (ODsdO = 0.78).
The resu1t.s obtained in oxygen-free solut,ions are, therefore, anot,her clear indication that the 340-nm peak is due to a eomplex between TlUA and copperI’.
2 Fro. G. Spectral changes in oxygen-saturated standard reaction mixture, (TMA 1OW M, CuClz 1OV M in 0.1 N NaOH). From bottom t,o top, curves recorded 1, 4, 7, and 11 min after mixing. Inset: time course of the OD variations at 300 nm (full line) and at 340 nm (broken line).
The 300-nm Contpound In addition to the immediate formation of the TMA-Cul’ complex absorbing at 340 nm, other spectral changes are observable at shorter wavelengths. In fact another absorption peak with maximum at 300 nm is slowly formed during the oxidation of TMA, and disappears abruptly at the end of the reaction. Figure 6 shows the optical spect,ra recorded at various times in an oxygen-saturated standard react,ion mixture. In the inset the time course of the absorption peaks at 340 and 300 nm is recorded. There is no isosbest’ic point for the curves recorded at various times, and this indicates that t,he two peaks are due to different, compounds.
500
DE MARCO
-0-s .x2
.66
.
M
FIG. 7. Maximal absorbancies attained at 300 nm (full line) and at 340 nm (broken line) in oxygeil-saturated reaction mixtures (0.1 N NaOH) as function of TMA concentration (abscissa). Open circles: 5.10V5 M CuClz . Full circles: 1P M CuCla. The small increase in absorbancy at 340 nm is ascribable to the residual absorption at this wavelength of the 300-nm peak (cf. Fig. 6).
Data were obtained indicating that the 300-nm peak: (a) is independent of copper concentration; (b) is directly correlated to TMA concentration; (c) is correlated to oxygen concentration. Figure 7 shows the maximum absorption values reached at 300 and 340 nm in oxygensaturated react’ion mixtures at different concentrations of TMA and of CuClz . It is evident that the extinction at 300 nm is directly proportional to SH concentration, and independent of copper concentration, whereas the 340-nm absorption is independent of thiol concentration. Figure 8 shows the effects of varying the oxygen concentration on the reaction rate and on the absorbancies at 300 and 340 nm. It is evident that a primary effect of the increasing oxygen concentration is to increase the reaction rate: the oxidation of TMA which is completed in 12 min in oxygen-saturated solution, requires about 20 min at 25% oxygen saturation. Aloreover, while the absorption at 340 nm is practically unaffected, the ext,inction at
ET AL.
FIG. 8. Effect of varying oxygen concentratiou on the reaction time, and on the absorbancy at 340 nm (full lines) and 300 nm (broken lines) in a standard reaction mixture. Figures on the curves indicate the percentage oxygen saturation. In the inset: maximal absorbancy attained at 300 nm as a function of the oxygen concentration.
300 nm reaches values that are directly correlated with the oxygen concentration. In oxygen-free reaction mixtures, the peak at 300 nm is completely absent (Fig. 4). All these observat’ions indicat,e that the 300-nm peak may represent a transient intermediate in the oxidation of TMA to the disulfide, and oxygen seems to be involved in t’his intermediate. Reaction Products During the reaction TMA is oxidized to the corresponding disulfide. This has been qualitatively checked at the end of the reaction in the following way. The standard reaction mixture was acidified with concentrated HCl and repeatedly extracted with ether; the ether was taken to dryness and the residue subjected to paper chromatography in water-saturated phenol, using as control nearly equal amount of the disulfide of TMA prepared according t’o Hill and McAuley by oxidation with cerium sulfate (4). The chromatograms were developed with the Folin-Marenzi reagent (9). In this solvent the RF for TMA was 0.4 and for the disulfide 0.1.
OXIDATION
OF THIOMALIC
TMA disulfide was also det,ected by gaschromatography. A standard reaction mixture was acidified at the end of the reaction and repeatedly extracted with ether. The ethereal ext,ract was taken t,o dryness and t,he residue then subjected to met’hylatjon in met,hanol-HiSO under reflux for 6 hr. Gas-chromatographic analyses (WE gas chromatograph; 1.5 m X 0.4-cm column of 20 5%DEGS on Anachrom A, 70-80 mesh; carrier: iU2 40 ml/min; programmed temperatures increase: 3”/min from 110 t’o 175” C) shoTI-ed only one peak (elution t’ime: 7-S min) in t,he same position as that of TfilA disulfide met’hyl est’er prepared as above from TMA disulfide, and well separated from TIBIA methyl ester (elution t’ime: 20 minj. Quantitatively, the formation of the disulfide was checked by performing at the end of t,he reaction the Folin-RIarenzi react.ion in the presence of bisulfite (for detecting SS groups), using as standard the TRfA disulfide. The results obtained indicate that almost8 all TXA may be recovered as disulfide. During t#hereaction H202 is also produced and accumulates in the reaction mixture. It is destroyed suddenly and concomit,antly \vith the disappearance of the 340- and 300-nm peaks, at the end of t’he reaction. This behavior is exactly the same as that observed \vith cyst,eine (2) : until t’he CulI is complexed with the thiol H302 accumulates; when the thiol is completely oxidized, the Cu’I is set free in solution and catalyzes the immediate decomposition of Hz02. Figure 9 shows the time course of Hz02 production in standard reaction mixtures at different oxygen saturation. At’ lower oxygen concentration a lower amount of Hz02 is produced. A good parallelism is evident bet,ween the amount’ of HsOr, t,hat of the 300-nm absorbing compound and the oxygen concentration. H,O? production and decomposition has also been invest’igated by det’ecting the chemiluminescence excited on luminol (5 amino-?, X-dihydro-1 ,4-phtalazinedione). When t,he oxidation of TJIA is allowed to occur in the presence of luminol (10e3 M), a light, flash appears at the end of the reaction.
301
ACID
FIG. 9. Time course of 11202 production in standard reaction mixtures at different, oxygen concentratioll. Figures on the curves indicate the percentage of oxygen saturation. In the inset: maximal IIIOz product,ion as a function of the oxygen concentration.
Since luminol chemiluminescence is due to the decomposition of HzOp catalyzed by free CulI (1). t)he production of light just at the end of the reaction indicates t,hat (i) H202 is formed and accumulates during the reaction; (ii) HZO, is not, decomposed during t,he reaction, as no free Cul’ is present; and (iii) H& is xuddently decomposed at’ the end of t’he reaction, since when all the T&IA is oxidized, t)he CuT1 is set, free in solution. Moreover, if H202 is added to a standard reaction mixture in the presence of luminol, no light emission is observed until the end of the reaction. This is another indication that t’he Cu” is st)rongly complexed by TMA, and even an excess of H?O, cannot be destroyed. DISCUSSION
E’rom the results described above, the following conclusions may be drawn. In alkaline solutions TMA binds to Cu” in a stable complex whose st,oichiomet,ry is 2 moles of TRZA for 1 mole of copper. Almost all the copper is complexed. The TXL-CU’~ com-
502
DE MARCO
plex is detected spectrophot’ometrically by a sharp absorption peak centered at 340 nm. TMA is oxidized to the corresponding disulfide with production of Hz02 . If oxygen is present the TMA-Cu’I complex is stable until the thiol oxidation is complete, and Hz02 accumulates in the solution. In the absence of oxygen the copper of the complex is slowly reduced. The fact that when a large excess of oxygen is present more HzOZ accumulates may suggest that TMA, or the T&IA-C@ complex, is better oxidized by molecular oxygen than by HzOz. However, when oxygen is present in limited amount, the oxidation of TMA may proceed at the expense of H202, which is, therefore, recovered in smaller amounts. The oxidation of TMA is accompanied by the formation of an intermediate detected spectrophotometrically by its absortion at 300 nm. It has been excluded that this absorption could be due to the products of the reaction, since neither the disulfide of TMA nor Hz02 shows absorption at this wavelength. Furthermore, the behavior of the 300-nm peak is clearly that of an intermediate, since it increases during the reaction but completely disappears at the end. Its concentration increases almost linearly with time, paralleling the production of the disulfide and of HtOz, and finally disappears suddenly. In the presence of limited amounts of molecular oxygen, the concentration of the intermediate reaches smaller values, as does also the production of HsOz. The concentration of the 300-nm compound is a function of the time of the reaction, of the thiol concentration, and of the oxygen concentration, while it is independent of the amount of copper. This seems to
ET AL.
exclude possible complexes of the type TMA-Cu-02 or TMA-Cu-H202, even if these have not been really ruled out. An addition compound between TMA (TMA radical?) and oxygen may be possibly taken into consideration. In conclusion, the results obtained with TMA indicate that the copper-catalyzed oxidation of this thiol occurs virtually with the same general features as in the case of cysteine and other thiols, that is, with the metal firmly bound in a stable complex, which possibly represents the electron donor to oxygen (2). The main difference appears to be the formation, or perhaps the easier detection, of an intermediate absorbing at 300 nm, the structure of which at present remains undefined. REFERENCES 1. C~VALLINI,
2.
3. 4. 5.
6. 7. 8.
9. 10. 11.
D., DE MARCO, C., AND DUPRI?,, S., Arch. Biochem. Biophys. 134, 18 (1968). CBVALLINI, D., DEMARCO, C., DUPR&, S., END ROTILIO, G., Arch. Biochem. Biophys. 130,354 (1969). HILL, J., AND MCAULEY, A., J. Chem. Sot. A, 2405 (1968). HILL, J., AND MCAULEY, A., J. Chem. Sot. A, 156 (1968). PICKERING, W. F., BND MCAULEY, A., J. Chem. Sot. A, 1173 (1968). HILL, J., MCAULEY, A., AND PICKERING, W. F., Chem. Commun. 573 (1967). FoLIN,O.,~~NDM.~RENZI, A.D., J.Biol. Chem. 33,109 (1929). EGERTON, A. C., EVERETT, A. J., MINKOFF, G. J., RUDRAK~NCHANA, S.,SALOOJA, K. C., Anal. Chim. Acta 10,422 (1954). KIVELSON, D., AND NEIMAN, R., J. Chem. Phys. 36, 149 (1961). ROTILIO, G., AND CALABRESE, L., Arch. Biochem. Biophys., in press. MONDOV?, B., MODIANO, G., AND DE MARCO, C., G. Biochim. 4, 324 (1955).