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Electron transport in biological processes
Bioelectrochemhy and BioenergtWs. 23 (1990) al-91
A section of J. Electroanal. &~a.. and constituting Vol. 298 (1990) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
Electron transport in biologicd processes Elec~ochemical behaviour of ubiqairroneQJQ adsorbmi on a pyrolytic graphite electrode Ricardo S. Schrebler *, Adriana Arratiaand Susana S$nchez Institute de Quimica, UniversidadCatdlica de Vulparaiso. Cusilia 40%
Valparuiso(Chikj
Marcela Ham Departamento de Bioquimica, lnstituro de Biologio. Universidade Esfoduul de Campirras. CA? 6109. Campinas, CEP 13081. S.P. (Brazil)
Nelson Lauren* fnstituto de Quimica. Biologica/ Chemistry Luboratoq Campinas, CEP 13081, S. P. (Brazil)
UMwrsidude Estadual de Campitms. C. P. 6154,
(Received 17 January 1989: in revised form 12 November 1989)
ABSTRACT The E-pH diagram has enabled us to obtain the acid-base constant of ubiquinonc and the equilibrium potential at pH = 0 for the different couples participating in the process of quinone adsorption on a pyrolytic graphite electrode in aqueous buffered media. The electrochemical behaviour found for ubiquinone may explain in part the in vivo ubiquinone processes. The molecular contour of deoxyribonucleic acid changes the stability of the semiubiquinone intermediate. as shown by chemiluminescence and binding experiments.
INTRODUCTION
Studies on 2,3-dimethoxy-5-methyld-polyisoprenoid-1,4-benzoquinone (CoQ,n, or ubiquinone,n) have shown that it acts as electron carrier from the flavoproteins to the cytochrome in the rnitochondrial respiratory chain [l-3]. Recent studies have shown that the presence of compounds with a quinone group can result in * To whom correspondence should be addressed.
significant death of tumour cells and in the induction of breaks in DNA single and double strands [4-61. It has also been found that interact these molecules strongly with the superoxide anion, O’“-, when generated in cells or in the mitochondria. Furthermore, several reports [5-81 also describe that CoQJO is capable to react in vitro with the superoxide anion generated enzymatically by the xanthine-xanthine oxidase system. The conclusion was that the presence of CoQ,IO in the respiratory chain and the extent of its reduction endows the phospholipid milieu with antioxidant protection, e.g. it has been found that CoQ,lO effectively decreases the lethality of adriamycin [4] and benefits patients treateci with this drug, Thus, it is of capital importance to know the electrochemical characteristics of this quinone and look for an electric technique that enables us to extrapolate the in vitro results to the phenomenological processes that occur in the cell. The most important studies of the electrochemical characteristics of CoQ,n, have been carried out in non-aqueous solvents, due to the low soiubility of this substance in aquous media [9-111. Using polarographic and UV spectroscopic techniques, it was found that CoQ,n shows irreversible electrochemical behaviour in methanol. This behaviour was explained by the formation of dimers or trimers in this medium. In all cases, two-electron processes were found for CoQ,n reduction, which occurs via radical formation. At the moment, little is known about the electrochemical properties of ubiquinone in aqueous solvent [12] or in micelles [13,14]; not even its activity constants are well known. In relation t!lerewith, it is frequently assumed that CoQ,n has a behaviour similar to that of other soluble quinones [IO]. For these. it has been possible to obtain the potential-pH diagram and the acidity constant from the equilibrium potential, the polarographic half-wave potential, the voltammetric peak potential or spectrophotometric data measurements [15]. A technique that has not been utilized yet, is the adsorption of CoQ,n on an appropriate electrode surface. Und,.M these circumstances, a modified electrode is formed. Then it is possible to szudy the electrochemical processes associated with these compounds in any electrolyte in which the quinone is insoluble. The aim of this work is to study the electrochemical behaviour of CoQ,lO (ubiquinone QJO) adsorbed on a pyrolytic graphite electrode in aqueous buffered media over a wide range of pH using the cyclic voltammetric technique, in order to obtain the potential-pH diagram and the acidity constant of this natural quinone. Simultaneously, by means of ESR spectroscopy, an evaluation of the lifetime of semiquinone is carried out and also the interaction of DNA with semiubiquinone is investigated. EXPERIMENTAL
All the voltammetric measurements were carried out with a three-electrode system. The working electrode was a pyrolytic graphite disk (Carbon Lorraine) supported in a PTFE holder [16]. The counter electrode #as a Pt wire maintained in a separate compartment. The potentials were measured through a Luggin capillary
with a saturated calomel electrode (SCE). The electrolytic solutions employed were, for pH 4 2, WC1solutions; for 2 < pH G 10, MacIlvaine buffer prepared from 0.1 M citric acid and 0.1 M disodium hydrogenphosphatc; and for pH > 11, NaOH solution. All these solutions were prepared with triply distilled water and analytical grade reagents {Merck). The ubiquinone used was Cot&10 (Sigma Chem.) from bovine heart. The ionic strength of all solutions was adjusted to 0.5 M with sodium chloride, The potentiostatic and potentiodynamic measurements were carried out under nitrogen saturation at 25°C. These experiments included single (STPS) or repetitive (RTPS) triangular potential sweeps at 0.001 dud 0.4 V/s, run between variable upper (I&) and lower (Es,,) switclling potentials. The graphite disk was wet-ground with an abrasive grade 800 SIC paper, washed with triply distilled water and immersed in the electrolytic cell containing McIlvaine buffer, pH 8.5. Immediately and anodizing pulse of 1.5 V was applied for 5 min, followed by a cathodizing pulse of -1.0 V during 1 min. After the electrode was thus activated, the graphite disk was washed, dried and immersed in a 0.01 M benzene solution of ubiquinone Q,lO. The electrocie was maintained in this solution for an adsorption time of 3 min. Then the ubiquinone Q,lO modified electrode was washed with triply distilled water and placed in the electrolyte. Immediately, a potentios!atic or potentiodynamic program was applied. The measurements were made wit!1 a potentiostat (LADEM 128-B) equipped with a function generator (LADEM 108). The electric responses to the different perturbation programs were recorded on a Rikadenki RW-1OlT recorder. The monovalent ubiquinone Q,lO under study was synthesized chemically in acetonitrile using a superoxide generating system (potassium superoxide in crown ether) as electron source [17]. The existence of the semiubiquinone form was inferred from ESR spectroscopy (not shown). The semiubiquinone remained stable in the absence of oxygen for several minutes. The interaction of semiubiquinone with calf-thymus DNA was followed as described previously [161. Determination of DNA binding was carried out by the method of Witte et a!. [18]. RESULTS AND DISCUSSION
Figure 1 shows the potentiodynamic j-E profile of ubiquinone adsorbed on a pyrolytic graphite electrode, in a buffer so!ution of pH 11. Under these conditions reversible redox processes are found. On the other hand, the anodic to cathodic peak ratio was near unity, and the c unre!l response remained unaltered for several days if the electrode, after finishing the experiments, was kept in the dark. At this pH, the potentiodynamic profiles fitted well the Anson equation 1193for irreversible adsorption with non-ideal behaviour (Fig. 2). However, in this case it was necessary to add a term associated with the ohmic resistance of the electrode. The value of the non-ideality parameter, r, used to calculate the current-potential data was 1.53 X 10’ mol-’ cm*. The fact that the value of t is positive indicates that the processes responsible for the non-idea!ity act by stabilizing the attached reactants, due to the interaction of the isoprenoid group with the electrode surface.
84 I ..........
I
I
I
I.
C AT HODtC
!
E U
t
7 ~ • on;a
:a,
ANO01C I
o
I
I
0.4
I
I
O.B E/V
Fig. l. Potentiodynamic j - E profiles of ubiquinone Q,10 irreversibly adsorbed on a basal-plane pyrolytic graphite electrode ( ). ( - - - - - - ) Graphite electrode. Mcllvaine buffer solution pH 11.0; v = 5 mV/s.
When the pH of the solution was changed systematically from 14 to 1, the characteristics of the profiles were clearly changed. Figure 3 shows the effect of pH on the j-E profiles of ubiquinone adsorbed on the electrode. For 14 > pH >~ 10.5, one anodic and one cathodic peak with reversible character appeared. For 10.5 > pH >/7.5, the ]-E profiles showed two anodic and two cathodic peaks, which can be attributed to the stabilization of semiubiquinone under these conditions. These characteristics were lost in the 7.5 > pH >~4.5 range, where the curves showed only one broad cathodic and one anodic peak, which were difficult to separate in two
~5 i
~
il
i
3O
20
10
I 0.2
I
I
_L_
0.t
0.6
0.~
E/V Fig. 2. Experimental and theoretical cathodic potentiodynamic j - E profiles for 4.4 x 10-to tool cm-2 ubiquinone Q,10 irreversibly adsorbed on a basal-plane pyrolytic graphite electrode. Mcllvaine buffer solution, pH 11, v = 5 mV s-~. ( ) Experimental profile. (e) Points calculated from the Anson equation with non-idcality parameter r = 1.53 × 109 tool - 1 cm2.
contributions. The anodic and cathodic double signal were found again in the pH < 4.5 range. DATA TREATMENT
The potentials used in the E - p H diagrams were obtained as demonstrated in Fig. 4, which shows the three cases found. In eases a and b, where one anodic and one cathodic peak exist, the potential, E', was calculated from:
E'= Ep.a- Ep/2= Ep,o+
(1)
In case c, in which two anodic and two cathodic contributions appeared, the overall curves were divided into single components, using a procedure similar to that applied by other authors [20]. Two potentials E' were then measured according to eqn. (1) and it was assumed that two redox couple participate in this pH range. All the E's were corrected by applying the Nerst equation as follows:
E " = E' - (0.059/n) log(Sc/S ~ )
(2)
86
CAIHODIC
10 pA lcm 2
L
8
?
O.~V
I
E
1.-" t ' t ' "
U
2 3 41
ANOD1C
E/V Fig. 3. Potentiodynamic response of ubiquinone in different phosphate buffer solutions, c = 0.5 M, at 25°C pH: (1) 2.0; (2) 4.3; (3) 5.0; (4) 6.0; (5) "/.2; (6) 9.2; (7) 11.0; (8) 12.1.
where E " is the normalized potential for a charge ratio equal to unity and Sc and S~ are the charges obtained from the cathodic and anodic peaks. The E " - p H diagram of ubiquinone adsorbed on a pyrolytic graphite electrode is shown in Fig. 5. The ranges of stability of six different forms of ubiquinon¢ Q,10 are visible. In this diagram, it is shown that two semiquinone species were found. The first is delimited by lines 1, 2 and 3, corresponding to the HQ" form, which indicates that the overall reduction reaction occurs through: 1
Q+H++e - ~HQ'+H÷+e
2
- ~H2Q
(3)
or, for pH > 3.4: 1
3
Q+H++e - ~ HQ'+e- ~HQ-
(4)
Above pH 4.55, the dismutation reaction of QH' may occur and only one process is observed, Q+H++2e
4
- ~HQ-
(5)
x7 1
I-
,
1
nEp.c/2
Fig. 4. Different cyclic voltammetric curves obtained in the pi-I range studied. (a) 4.5 < pH g 7.5; (b) pH~ll;(c)pH~4Sor7.5cpH~ll.
-0 4.
-0.6-.---l~-~-1-..I_I~ 4 2
6
B
7 S-eY ICI dI ---_I---L-_110_ IO 12
PH
Fig. 5. E-pH diagram for ubiquinone Q.10 adsorbed irrcvcrsibly on a basal-plane pyrolytic graphite
electrodeat 25’ C. which is characterized by (8E”,A?pH) = 30 mV. For pH > 7.5, the semiubiquinone is again stabilized in the form Q’- and the overall process occurs through: Q+e-
2 Q’-+ H++e-
2 HQ-
(6)
Finally, for pH > 11.5 the only process that may occur corresponds to the totally deprotonated species: Q+2e-
&Q’-
(7)
which is characterized by (SE”/6 pH) = 0 mV. The E-pH diagram has enabled use to obtain the different acid-base constants and at the same time, the equilibrium potential at pH 0 for the different participating couples, which are summarized in Tables I and 2. The electrochemical behaviour found for ubiquinone adsorbed on a graphite electrode may explain the in vivo processes of ubiquinone. First of all, the stability of semiubiquinone in the pH range 7.0-11.3 confirms the idea that in the mitochondria the CoQJO participates in one-electron transfer processes. Secondly, the vohammetric results show that no reversible processes are associated with the
89 TABLE
1
Standard redox pokntiuls ! .ines
of the rcdox rcactiors of ubiquintme adsorbed on a graphile clcctrodc
Rmrion
EO/
(SE”/6jlll)/ V dcc-’
Theory Q.lO+H+ -I-e- t= I-IQ:10 HQ;lO+H’+e-+H,Q,IO HQ:lO+eC=HQJO Q,lO+H+2e-=HQ-,lO Q,lO+e- *Q-:10 Q’-,lO+H++e-s l-IQ-.10 Q.10+2 e-t Q2-.lO
- 0.06 - 0.06 0.0 - 0.03 0.0 - 0.06 0.0
V vs. SCE
Exp. - 0.075 - 0.068 0.0 - 0.035 0.0 - cl.062 0.0
0.315 0.220 - 0.035 0.140 -0.170 0.150 - 0.550
TABLE 2 Acid-base
equilibria in the ubiquinone
system
Line
Reaction
PK
9 10 11
HIQ,10 ;t HQ-.lO+H+ HQ-JO ti Q’-JO+ H’ I-IQ;10 G=Q’-.10-l-H*
3.4 11.5 7.5
transfers in this pH range. These facts might explain the reaction schemes in which CoQJO participates, by analogy to that proposed by Slater 1211 (Scheme 1) and other authors 122,231.According to these schemes, the hydroubiquinone is oxidized to semiquinone by cytochrome c s,nd later to quinone by cytochrome bSsH.In these cases, the lifetime OFsemiubiquinone must be sufficiently long t:, permit these two consecutive reactions with two different molecules to occur. The electrochemical bekaviour ?f ubiquincne Q,lO al pH 7.0-11.3 and the stability of the semiubiquinone, estimated to by. several minutes by ESR spectro-
90
scopic measurements, led us to predict a high reactivity toward DNA, as shown previously by the anticancer activity and host toxicity [24,25] of several quinonic drugs. In order to prove this fact, the following experiment was carried out. The reaction of semiubiquinone with hydrogen peroxide exhibited chemiluminescence coming from excited quinone 126,273, in a similar way as that observed in the autooxidation of hydroquinone derivatives [28]. This chemiluminescence is quenched by DNA with a Stern-Volmer constant (KS,) of 660 M-’ (by nucleotides) at the range of 0.07 to 0.14 mg/ml DNA. This quenching is probably due to the binding of semiubiquinone to DNA. The Ksv of = 10” M” for benzoquinone chemiluminescence by ( +)-cyanidazol-3-, a potent free radical quencher [27], indicated, in our case, a low binding value for the semiubiquinone with DNA. This result can be attributed to the fact that the molecular contour of DNA may change the stability of the ubiquinone species, as shown previously by riboflavin, FMN and FAD reduction. In these cases, the stability region of semiquinone changes when cations or proteins also participate in the process [ZO]. ACKNOWLEDGEMENTS
This work has been partially sponsored by the Vicerectoria Academica de la Universidad Catblica de Valparaiso, Research Project No. 125.781-85 and the FAPESP, CNP and FINEP Projects (Brazil). REFERENCES 1 R. Bentley and 1-M. Campbell in S. Patai (Ed.), The Chemistry of the Quinoid Compounds, Part II. Wiley. London, 1974, Ch. 13. p. 683. 2 H.J. Harmon and V.G. Struble, Biochemistry, 22 (1983) 4394. 3 J.M. Anderson and N. Anderson, Trends Biochem. Sci., 7 (1982) 288. 4 G.P. Litarru and S. Lippa. Drugs Exptl. Curr. Res., 7 (1984) 941. 5 W. Bors, M. Saran, E. Lengfeider. R. Spottl and C. Michei, Current Top. Rad. Res. Q., 9 (19741247. 6 J.F. Turrens, A. Alexandre and A.L. Lehninger. Arch, Biochem. Biophys., 237 (1985) 408. 7 J.A. Pcdersen, J. Chem. Sot. Perkin Trans. 2, (1973) 424. 8 H. Nchl, W. Jordan and R.J. Youngman, Advan. Free Radical Biol. Med., 2 (1986) 211. 9 R.C. Prince, P.L. Dutton and J.M. Bruce, FEBS Lett., 160 (1983) 273. 10 J.Q. Chambers in ref. 1, Ch. 14, P. 737. 11 hl. Shaushal and K.H. Hassan. Studia Biophys., 105 (1985) 59. 12 OS. Ksenzhek, S.A. Petrova and M.V. Kolodyazhny, Bioelectrochcm. Bioenerg., 9 (1982) 167. 13 T. Erabi. H. Hiura and M. Tanaka, Bull. Chem. Sac. Jpn., 48 (1975) 1354. 14 T. Erabi. T. Higuti, T. Kakuno. J. Yamashira, M. Tanaka and T. Horio, J. Biochem. (Tokyo). 78 (1975) 795. 15 S.I. Bayley and 1.M. Ritchie, Electrochim. Acta, 30 (1985) 3. 16 R. Schrebler, R. Araya, A. Arratia, D.B. Ciampi, M.E. Hoffmann and N. Duran, Bioelectrochem. Bioenerg.. 17 (1987) 523. 17 H. Nohl and W. Jordan in W. Bors, M. Saran and D. Tail (Eds.), Oxygen Radicals in Chemistry and Biology, Walter de Gruyter. Berlin, 1984, p. 155. 18 1. Witu, U. Juhl and W. Butte, Mutat. Res., 145 (1985) 71.
19 20 21 22 23 24 25 26 27 28
A.P. Brown and F.C. Anson. Anal. Chem., 49 (1977) 1589. OS,, Ksenzhek and S.A. P&ova. Bioelectrochem. Bioenerg.. 11 (1983) 105. E.C. Slater, Trends B&hem. Sic., 8 (1983) 239. B.L. Trumpower. J. Bioecq. Biomembr., 13 (1981) 1. L.I. Hochstein and S.E. Cronin, Can. J. Microbial.. 30 (1984) 572, H. Kappus, B&hem. Pharmacol., 35 (1986) i. A. Begleiter and G.W. 3lair, Cancer Res.. 44 (1984) 78. A. Brunmark and E. Cadenas, Free Radical Biol. Med., 3 (1987) 149, M. Ginsberg and E. Cadenas, Photobiochem. Photobiophys., 9 (1985) 223. M.E. Hoffmann, D.B. Ciampi and N. Durban, Experientia, 43 (1987) 217.
A section of J. Electroanal. &~a.. and constituting Vol. 298 (1990) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
Electron transport in biologicd processes Elec~ochemical behaviour of ubiqairroneQJQ adsorbmi on a pyrolytic graphite electrode Ricardo S. Schrebler *, Adriana Arratiaand Susana S$nchez Institute de Quimica, UniversidadCatdlica de Vulparaiso. Cusilia 40%
Valparuiso(Chikj
Marcela Ham Departamento de Bioquimica, lnstituro de Biologio. Universidade Esfoduul de Campirras. CA? 6109. Campinas, CEP 13081. S.P. (Brazil)
Nelson Lauren* fnstituto de Quimica. Biologica/ Chemistry Luboratoq Campinas, CEP 13081, S. P. (Brazil)
UMwrsidude Estadual de Campitms. C. P. 6154,
(Received 17 January 1989: in revised form 12 November 1989)
ABSTRACT The E-pH diagram has enabled us to obtain the acid-base constant of ubiquinonc and the equilibrium potential at pH = 0 for the different couples participating in the process of quinone adsorption on a pyrolytic graphite electrode in aqueous buffered media. The electrochemical behaviour found for ubiquinone may explain in part the in vivo ubiquinone processes. The molecular contour of deoxyribonucleic acid changes the stability of the semiubiquinone intermediate. as shown by chemiluminescence and binding experiments.
INTRODUCTION
Studies on 2,3-dimethoxy-5-methyld-polyisoprenoid-1,4-benzoquinone (CoQ,n, or ubiquinone,n) have shown that it acts as electron carrier from the flavoproteins to the cytochrome in the rnitochondrial respiratory chain [l-3]. Recent studies have shown that the presence of compounds with a quinone group can result in * To whom correspondence should be addressed.
significant death of tumour cells and in the induction of breaks in DNA single and double strands [4-61. It has also been found that interact these molecules strongly with the superoxide anion, O’“-, when generated in cells or in the mitochondria. Furthermore, several reports [5-81 also describe that CoQJO is capable to react in vitro with the superoxide anion generated enzymatically by the xanthine-xanthine oxidase system. The conclusion was that the presence of CoQ,IO in the respiratory chain and the extent of its reduction endows the phospholipid milieu with antioxidant protection, e.g. it has been found that CoQ,lO effectively decreases the lethality of adriamycin [4] and benefits patients treateci with this drug, Thus, it is of capital importance to know the electrochemical characteristics of this quinone and look for an electric technique that enables us to extrapolate the in vitro results to the phenomenological processes that occur in the cell. The most important studies of the electrochemical characteristics of CoQ,n, have been carried out in non-aqueous solvents, due to the low soiubility of this substance in aquous media [9-111. Using polarographic and UV spectroscopic techniques, it was found that CoQ,n shows irreversible electrochemical behaviour in methanol. This behaviour was explained by the formation of dimers or trimers in this medium. In all cases, two-electron processes were found for CoQ,n reduction, which occurs via radical formation. At the moment, little is known about the electrochemical properties of ubiquinone in aqueous solvent [12] or in micelles [13,14]; not even its activity constants are well known. In relation t!lerewith, it is frequently assumed that CoQ,n has a behaviour similar to that of other soluble quinones [IO]. For these. it has been possible to obtain the potential-pH diagram and the acidity constant from the equilibrium potential, the polarographic half-wave potential, the voltammetric peak potential or spectrophotometric data measurements [15]. A technique that has not been utilized yet, is the adsorption of CoQ,n on an appropriate electrode surface. Und,.M these circumstances, a modified electrode is formed. Then it is possible to szudy the electrochemical processes associated with these compounds in any electrolyte in which the quinone is insoluble. The aim of this work is to study the electrochemical behaviour of CoQ,lO (ubiquinone QJO) adsorbed on a pyrolytic graphite electrode in aqueous buffered media over a wide range of pH using the cyclic voltammetric technique, in order to obtain the potential-pH diagram and the acidity constant of this natural quinone. Simultaneously, by means of ESR spectroscopy, an evaluation of the lifetime of semiquinone is carried out and also the interaction of DNA with semiubiquinone is investigated. EXPERIMENTAL
All the voltammetric measurements were carried out with a three-electrode system. The working electrode was a pyrolytic graphite disk (Carbon Lorraine) supported in a PTFE holder [16]. The counter electrode #as a Pt wire maintained in a separate compartment. The potentials were measured through a Luggin capillary
with a saturated calomel electrode (SCE). The electrolytic solutions employed were, for pH 4 2, WC1solutions; for 2 < pH G 10, MacIlvaine buffer prepared from 0.1 M citric acid and 0.1 M disodium hydrogenphosphatc; and for pH > 11, NaOH solution. All these solutions were prepared with triply distilled water and analytical grade reagents {Merck). The ubiquinone used was Cot&10 (Sigma Chem.) from bovine heart. The ionic strength of all solutions was adjusted to 0.5 M with sodium chloride, The potentiostatic and potentiodynamic measurements were carried out under nitrogen saturation at 25°C. These experiments included single (STPS) or repetitive (RTPS) triangular potential sweeps at 0.001 dud 0.4 V/s, run between variable upper (I&) and lower (Es,,) switclling potentials. The graphite disk was wet-ground with an abrasive grade 800 SIC paper, washed with triply distilled water and immersed in the electrolytic cell containing McIlvaine buffer, pH 8.5. Immediately and anodizing pulse of 1.5 V was applied for 5 min, followed by a cathodizing pulse of -1.0 V during 1 min. After the electrode was thus activated, the graphite disk was washed, dried and immersed in a 0.01 M benzene solution of ubiquinone Q,lO. The electrocie was maintained in this solution for an adsorption time of 3 min. Then the ubiquinone Q,lO modified electrode was washed with triply distilled water and placed in the electrolyte. Immediately, a potentios!atic or potentiodynamic program was applied. The measurements were made wit!1 a potentiostat (LADEM 128-B) equipped with a function generator (LADEM 108). The electric responses to the different perturbation programs were recorded on a Rikadenki RW-1OlT recorder. The monovalent ubiquinone Q,lO under study was synthesized chemically in acetonitrile using a superoxide generating system (potassium superoxide in crown ether) as electron source [17]. The existence of the semiubiquinone form was inferred from ESR spectroscopy (not shown). The semiubiquinone remained stable in the absence of oxygen for several minutes. The interaction of semiubiquinone with calf-thymus DNA was followed as described previously [161. Determination of DNA binding was carried out by the method of Witte et a!. [18]. RESULTS AND DISCUSSION
Figure 1 shows the potentiodynamic j-E profile of ubiquinone adsorbed on a pyrolytic graphite electrode, in a buffer so!ution of pH 11. Under these conditions reversible redox processes are found. On the other hand, the anodic to cathodic peak ratio was near unity, and the c unre!l response remained unaltered for several days if the electrode, after finishing the experiments, was kept in the dark. At this pH, the potentiodynamic profiles fitted well the Anson equation 1193for irreversible adsorption with non-ideal behaviour (Fig. 2). However, in this case it was necessary to add a term associated with the ohmic resistance of the electrode. The value of the non-ideality parameter, r, used to calculate the current-potential data was 1.53 X 10’ mol-’ cm*. The fact that the value of t is positive indicates that the processes responsible for the non-idea!ity act by stabilizing the attached reactants, due to the interaction of the isoprenoid group with the electrode surface.
84 I ..........
I
I
I
I.
C AT HODtC
!
E U
t
7 ~ • on;a
:a,
ANO01C I
o
I
I
0.4
I
I
O.B E/V
Fig. l. Potentiodynamic j - E profiles of ubiquinone Q,10 irreversibly adsorbed on a basal-plane pyrolytic graphite electrode ( ). ( - - - - - - ) Graphite electrode. Mcllvaine buffer solution pH 11.0; v = 5 mV/s.
When the pH of the solution was changed systematically from 14 to 1, the characteristics of the profiles were clearly changed. Figure 3 shows the effect of pH on the j-E profiles of ubiquinone adsorbed on the electrode. For 14 > pH >~ 10.5, one anodic and one cathodic peak with reversible character appeared. For 10.5 > pH >/7.5, the ]-E profiles showed two anodic and two cathodic peaks, which can be attributed to the stabilization of semiubiquinone under these conditions. These characteristics were lost in the 7.5 > pH >~4.5 range, where the curves showed only one broad cathodic and one anodic peak, which were difficult to separate in two
~5 i
~
il
i
3O
20
10
I 0.2
I
I
_L_
0.t
0.6
0.~
E/V Fig. 2. Experimental and theoretical cathodic potentiodynamic j - E profiles for 4.4 x 10-to tool cm-2 ubiquinone Q,10 irreversibly adsorbed on a basal-plane pyrolytic graphite electrode. Mcllvaine buffer solution, pH 11, v = 5 mV s-~. ( ) Experimental profile. (e) Points calculated from the Anson equation with non-idcality parameter r = 1.53 × 109 tool - 1 cm2.
contributions. The anodic and cathodic double signal were found again in the pH < 4.5 range. DATA TREATMENT
The potentials used in the E - p H diagrams were obtained as demonstrated in Fig. 4, which shows the three cases found. In eases a and b, where one anodic and one cathodic peak exist, the potential, E', was calculated from:
E'= Ep.a- Ep/2= Ep,o+
(1)
In case c, in which two anodic and two cathodic contributions appeared, the overall curves were divided into single components, using a procedure similar to that applied by other authors [20]. Two potentials E' were then measured according to eqn. (1) and it was assumed that two redox couple participate in this pH range. All the E's were corrected by applying the Nerst equation as follows:
E " = E' - (0.059/n) log(Sc/S ~ )
(2)
86
CAIHODIC
10 pA lcm 2
L
8
?
O.~V
I
E
1.-" t ' t ' "
U
2 3 41
ANOD1C
E/V Fig. 3. Potentiodynamic response of ubiquinone in different phosphate buffer solutions, c = 0.5 M, at 25°C pH: (1) 2.0; (2) 4.3; (3) 5.0; (4) 6.0; (5) "/.2; (6) 9.2; (7) 11.0; (8) 12.1.
where E " is the normalized potential for a charge ratio equal to unity and Sc and S~ are the charges obtained from the cathodic and anodic peaks. The E " - p H diagram of ubiquinone adsorbed on a pyrolytic graphite electrode is shown in Fig. 5. The ranges of stability of six different forms of ubiquinon¢ Q,10 are visible. In this diagram, it is shown that two semiquinone species were found. The first is delimited by lines 1, 2 and 3, corresponding to the HQ" form, which indicates that the overall reduction reaction occurs through: 1
Q+H++e - ~HQ'+H÷+e
2
- ~H2Q
(3)
or, for pH > 3.4: 1
3
Q+H++e - ~ HQ'+e- ~HQ-
(4)
Above pH 4.55, the dismutation reaction of QH' may occur and only one process is observed, Q+H++2e
4
- ~HQ-
(5)
x7 1
I-
,
1
nEp.c/2
Fig. 4. Different cyclic voltammetric curves obtained in the pi-I range studied. (a) 4.5 < pH g 7.5; (b) pH~ll;(c)pH~4Sor7.5cpH~ll.
-0 4.
-0.6-.---l~-~-1-..I_I~ 4 2
6
B
7 S-eY ICI dI ---_I---L-_110_ IO 12
PH
Fig. 5. E-pH diagram for ubiquinone Q.10 adsorbed irrcvcrsibly on a basal-plane pyrolytic graphite
electrodeat 25’ C. which is characterized by (8E”,A?pH) = 30 mV. For pH > 7.5, the semiubiquinone is again stabilized in the form Q’- and the overall process occurs through: Q+e-
2 Q’-+ H++e-
2 HQ-
(6)
Finally, for pH > 11.5 the only process that may occur corresponds to the totally deprotonated species: Q+2e-
&Q’-
(7)
which is characterized by (SE”/6 pH) = 0 mV. The E-pH diagram has enabled use to obtain the different acid-base constants and at the same time, the equilibrium potential at pH 0 for the different participating couples, which are summarized in Tables I and 2. The electrochemical behaviour found for ubiquinone adsorbed on a graphite electrode may explain the in vivo processes of ubiquinone. First of all, the stability of semiubiquinone in the pH range 7.0-11.3 confirms the idea that in the mitochondria the CoQJO participates in one-electron transfer processes. Secondly, the vohammetric results show that no reversible processes are associated with the
89 TABLE
1
Standard redox pokntiuls ! .ines
of the rcdox rcactiors of ubiquintme adsorbed on a graphile clcctrodc
Rmrion
EO/
(SE”/6jlll)/ V dcc-’
Theory Q.lO+H+ -I-e- t= I-IQ:10 HQ;lO+H’+e-+H,Q,IO HQ:lO+eC=HQJO Q,lO+H+2e-=HQ-,lO Q,lO+e- *Q-:10 Q’-,lO+H++e-s l-IQ-.10 Q.10+2 e-t Q2-.lO
- 0.06 - 0.06 0.0 - 0.03 0.0 - 0.06 0.0
V vs. SCE
Exp. - 0.075 - 0.068 0.0 - 0.035 0.0 - cl.062 0.0
0.315 0.220 - 0.035 0.140 -0.170 0.150 - 0.550
TABLE 2 Acid-base
equilibria in the ubiquinone
system
Line
Reaction
PK
9 10 11
HIQ,10 ;t HQ-.lO+H+ HQ-JO ti Q’-JO+ H’ I-IQ;10 G=Q’-.10-l-H*
3.4 11.5 7.5
transfers in this pH range. These facts might explain the reaction schemes in which CoQJO participates, by analogy to that proposed by Slater 1211 (Scheme 1) and other authors 122,231.According to these schemes, the hydroubiquinone is oxidized to semiquinone by cytochrome c s,nd later to quinone by cytochrome bSsH.In these cases, the lifetime OFsemiubiquinone must be sufficiently long t:, permit these two consecutive reactions with two different molecules to occur. The electrochemical bekaviour ?f ubiquincne Q,lO al pH 7.0-11.3 and the stability of the semiubiquinone, estimated to by. several minutes by ESR spectro-
90
scopic measurements, led us to predict a high reactivity toward DNA, as shown previously by the anticancer activity and host toxicity [24,25] of several quinonic drugs. In order to prove this fact, the following experiment was carried out. The reaction of semiubiquinone with hydrogen peroxide exhibited chemiluminescence coming from excited quinone 126,273, in a similar way as that observed in the autooxidation of hydroquinone derivatives [28]. This chemiluminescence is quenched by DNA with a Stern-Volmer constant (KS,) of 660 M-’ (by nucleotides) at the range of 0.07 to 0.14 mg/ml DNA. This quenching is probably due to the binding of semiubiquinone to DNA. The Ksv of = 10” M” for benzoquinone chemiluminescence by ( +)-cyanidazol-3-, a potent free radical quencher [27], indicated, in our case, a low binding value for the semiubiquinone with DNA. This result can be attributed to the fact that the molecular contour of DNA may change the stability of the ubiquinone species, as shown previously by riboflavin, FMN and FAD reduction. In these cases, the stability region of semiquinone changes when cations or proteins also participate in the process [ZO]. ACKNOWLEDGEMENTS
This work has been partially sponsored by the Vicerectoria Academica de la Universidad Catblica de Valparaiso, Research Project No. 125.781-85 and the FAPESP, CNP and FINEP Projects (Brazil). REFERENCES 1 R. Bentley and 1-M. Campbell in S. Patai (Ed.), The Chemistry of the Quinoid Compounds, Part II. Wiley. London, 1974, Ch. 13. p. 683. 2 H.J. Harmon and V.G. Struble, Biochemistry, 22 (1983) 4394. 3 J.M. Anderson and N. Anderson, Trends Biochem. Sci., 7 (1982) 288. 4 G.P. Litarru and S. Lippa. Drugs Exptl. Curr. Res., 7 (1984) 941. 5 W. Bors, M. Saran, E. Lengfeider. R. Spottl and C. Michei, Current Top. Rad. Res. Q., 9 (19741247. 6 J.F. Turrens, A. Alexandre and A.L. Lehninger. Arch, Biochem. Biophys., 237 (1985) 408. 7 J.A. Pcdersen, J. Chem. Sot. Perkin Trans. 2, (1973) 424. 8 H. Nchl, W. Jordan and R.J. Youngman, Advan. Free Radical Biol. Med., 2 (1986) 211. 9 R.C. Prince, P.L. Dutton and J.M. Bruce, FEBS Lett., 160 (1983) 273. 10 J.Q. Chambers in ref. 1, Ch. 14, P. 737. 11 hl. Shaushal and K.H. Hassan. Studia Biophys., 105 (1985) 59. 12 OS. Ksenzhek, S.A. Petrova and M.V. Kolodyazhny, Bioelectrochcm. Bioenerg., 9 (1982) 167. 13 T. Erabi. H. Hiura and M. Tanaka, Bull. Chem. Sac. Jpn., 48 (1975) 1354. 14 T. Erabi. T. Higuti, T. Kakuno. J. Yamashira, M. Tanaka and T. Horio, J. Biochem. (Tokyo). 78 (1975) 795. 15 S.I. Bayley and 1.M. Ritchie, Electrochim. Acta, 30 (1985) 3. 16 R. Schrebler, R. Araya, A. Arratia, D.B. Ciampi, M.E. Hoffmann and N. Duran, Bioelectrochem. Bioenerg.. 17 (1987) 523. 17 H. Nohl and W. Jordan in W. Bors, M. Saran and D. Tail (Eds.), Oxygen Radicals in Chemistry and Biology, Walter de Gruyter. Berlin, 1984, p. 155. 18 1. Witu, U. Juhl and W. Butte, Mutat. Res., 145 (1985) 71.
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