Anal. Chem. 1995,67, 283-287
Electron Transfer from a Polythiophene Derivative to Compounds I and II of Peroxidases Tetsu Tatsuma, Kaoru Ariyama, and Noboru Oyama*
Department of Applied Chemistry, Faculty of Technolow, Tokyo University of Agriculture and Technology, Naka-cho, Koganei, Tokyo 184, Japan
Electron transfer from poly(3-(3'-thienyl)propanesulfonic acid) (P'TS) to compounds I and I1 of horseradish peroxidase (=), microbial peroxidase (ARP),and lactoperoxidase (LPO)and to compound I of catalase (CAT) is studied. PTS polymerized chemically and p d e d to exclude the monomer and oligomersis dissohed in water together with an enzyme and cast on an Sn02 electrode. Cathodic current increase upon H202 addition is measured in 0.05 M BaClz aqueous solution, in which the enzyme/PTS film is insoluble. The electron transfer reactions are observed at t-200to 1000 mV vs1" for the HRF', ARP, and LPO systems and at +200 to +500 mV for the CAT system. HRP, ARP,and LPO may have similar redox potentials of compound Vcompound I1 and compound II/ferric enzyme. The apparent rate of the charge transfer from PTS to compounds I and I1 of the enzymes in conjunction with the proton uptake process is in the order ARP > HRF' > LPO > CAT.
+
Since 1986, many researchers have addressed themselves to observing charge transfer between redox enzymes and conducting po1ymers.'-l0 In most cases, however, the observed electron transfer was mediated by a small redox-active Even in the other cases in which direct transfer is ~laimed,4-~ the observed current was not necessarily large. Recently, one authorlo employed horseradish peroxidase (HRP), which is a relatively small molecule (MW = 40 000) and a highly active enzyme, and observed a fairly large response to HzOz (0.2-0.3 A cm-2 M-' in the linear region) at a HRP-incorporated polypyrrole (PPy) electrode. However, PPy was electrochemically polymerized from a pyrrole solution containing HRP so that the HRP-incorporated PPy film necessarily contained small redox-active molecules such ~~~
(1) Foulds, N. C.; Lowe, C. R J. Chem. SOC.,Faraday Trans. 1 1986,82,12591264. (2)Umaria, M.; Waller, J. Anal. Chem. 1986,58,2979-2983. (3) Bartlett, P.N.; Cooper, J. M. J. Electroanal. Chem. 1993,362,1-12. (4)Yabuki, S.;Shinohara, H.; Aizawa, M. J. Chem. SOC.,Chem. Commun. 1989, 945-946. (5)du Poet, P. D. T.; Miyamoto, S.; Murakami, T.; Kimura, J.; Karube, I. Anal. Chim. Acta 1990,235,255-263. (6)Wollenberger, U.; Bogdanovskaya, V.; Bobrin, S.; Scheller, F.; Tarasevich, M. Anal. Lett. 1990,23,1795-1808. (7) (a) Koopal, C. G. J.; de Ruiter, B.; Nolte, R J. M. /. Chem. SOC.,Chem. Commun. 1991,1691-1692. @) Koopal, C. G. J.; Feiters, M. C.; Nolte, R J. M.; de Ruiter, B.; Schasfoort, R B. M. Biosens. Bioelectron. 1992,7,461. (8)Matsue, T.; Kasai, N.; Narumi, M.; Nishizawa, M.; Yamada, H.; Uchida, Lj. Electroanal. Chem. 1991,300,111-118. (9)Khan, G.F.; Kobatake, E.; Shmohara, H.; Ikariyama, Y.; Aizawa, M. Anal. Chem. 1992,64,1254-1258. (10) (a) Tatsuma, T.; Watanabe, T. Anal. Chem. 1992, 64, 1183-1187. (b) Tatsuma, T.; Watanabe, T.; Tatsuma, S.; Watanabe, T. Anal. Chem. 1994, 66, 290-294. 0003-2700/95/0367-0283$9.00/0 0 1995 American Chemical Society
- e-
7
+ e-
Figure 1. Molecular structure of PTS.
as pyrrole monomer, oligomers, and other oxidized pyrroles which might act as mediators. Actually, it was revealed that water-soluble species of air-oxidized1Oaand electrochemically oxidizedll pyrrole can mediate electron transfer between HRP and electrode, while pyrrole monomer cannot. Thus, direct electron transfer from PPy to HRP has not yet been evidenced. Further, small redox-active species contained in electropolymerized conducting polymers may work as mediators for other redox enzymes. In the present work, we employed a water-soluble conducting polymer, poly(3-(3'-thienyl)propanesulfonic acid) (€93, Figure l), which had been polymerized chemically and puriiied to exclude the monomer and oligomers.12 It is known that a water-soluble redox polymer can be a good polymer mediator for water-soluble enzymes13J4because the polymer and enzyme can be mixed well in an aqueous cast solution, though the polymer should be insolubilized by ~ross-linking~~ or another method14 before measurements. PTS is soluble in water and water containing a monovalent cation but is only slightly soluble or insoluble in water containing a multivalent cation. PTS is therefore dissolved in water together with a peroxidase and then cast on the electrode surface, and electrochemical measurements are performed in a BaClz aqueous solution, in which PTS is insoluble. This is the first report on electrochemistry of redox enzymes in a watersoluble conducting polymer, to the best of our knowledge. An SnOacoated glass plate is used as a substrate electrode because the catalytic activity of SnO2 as well as PTS for oxidation and reduction of HzOz is so low that direct redox currents do not interfere with the current from PTS to a peroxidase. As the enzyme, we use not only HRP but also other peroxidases: microbial peroxidase from Arthromyces rumosus (ARP),15 lactoperoxidase &PO) , and catalase (CAT). All the enzymes used in this work have heme as an active center and show similar catalytic reactions, as follows:15-17 (11)Tatsuma, T.; Watanabe, T., unpublished results (1992). (12)Ikenoue, Y.; Saida, Y.; Kira, M.; Tomozawa, H.; Yashima, H.; Kobayashi, M. J. Chem. Soc., Chem. Commun. 1990,1694-1965. (13)Heller, A J. Phys. Chem. 1992,96,3579-3587. (14)Tatsuma, T.; Saito, IC; Oyama, N. Anal. Chem. 1994,66,1002-1006. (15) (a) Shinmen, Y.; Asami, S.; Amachi, T.;Shimizu, S.; Yamada, H. Agric. Biol. Chem. 1986, 50, 247-249. @) Akimoto, IC; Shinmen, Y.; Sumida, M.; Asami, S.; Amachi, T.; Yoshizumi, H.; Saeki, Y.; Shimizu, S.; Yamada, H. Anal. Biochem. 1990,189,182-185.
Analytical Chemistry, Vol. 67, No. 2, January 15, 1995 283
I + H' + e- I1 + H+ + e- - ferric
ferric peroxidase
+ H,O,
compound I
k2
compound compound
kl
k3
+ H,O
compound I1
peroxidase
+ H20
(1)
(2) (3)
Ferric peroxidase has heme with Fe(I1I) ([Fe(III)], where the brackets represent protoporphyrin E).Compound I has a mation radical of heme with F e O to which an oxygen atom is coordinated ([Fe0=OI*+). Compound I1 has heme with F e O to which OH is coordinated ([FeOOHl). The catalytic activity dictated by eqs 1-3 is called peroxidase activity. Although CAT exhibits catalase activity, in which compound I is reduced directly to ferric peroxidase by HzOz, CAT also has peroxidase activity. Although direct electron transfers from carbon electrodes to compounds I and I1 of HRP,6J8-23ARF',24 and LP021bas well as oxidized cytochrome c p e r o x i d a ~ ehave ~ ~ ~been ~ ~ reported, it is not clear whether transfer from conducting polymers is possible, as mentioned above. Further, a direct oxidation current of HzOz was signiiicant in those systems, and so electron transfer to HRP compounds has not yet been observed at a more positive potential than +600 mV vs Ag/AgCl, though reported redox potentials of compound 1/11 and compound II/femc enzyme for HRP are about +770 and +790 mV vs Ag/AgCl, respecti~ely.~~ Molecular weights of the peroxidases and catalase used here are as follows: ca. 40 000 for HRP,17ca. 41 000 for ARP,15 ca. 82 000 for LP0,17 and ca. 250 000 for CAT17 (one CAT molecule has four subunits, each of which has one heme). Therefore, here we can discuss a correlation between size of enzyme molecules and the electron transfer rate, which has never been studied.
Procedures. An SnOsoated glass plate (1.0 cmz) was pretreated with sulfuric acid that had been heated by dilution (1: 1) for a few minutes. Enzyme (1 mg) was dissolved in a 1 wt % PTSaqueous solution (10 pL), and the enzyme/PTS solution was cast on the SnOz electrode surface (1pL cm-2, unless otherwise noted). Thus obtained enzyme/PTS electrode was immersed in a BaClrsaturated aqueous solution for about 1 min before the electrochemical measurements. Electrochemical measurements were performed in an airsaturated 0.05 M BaClz aqueous solution (PH 6.0 f 0.2) at room temperature. Potential of an enzyme/€% electrode was controlled with a potentiostat (LC-4B, BAS). Reference and counter electrodes were Ag/AgCl/NaCl (saturated) and a platinum wire, respectively. The BaClz solution was stirred gently in the course of a measurement. H202 standard solutions were added stepwise, and subsequent changes in the current were observed. THEORY
As will be shown below, a cathodic current vs HzOz concentration curve for a peroxidase electrode can be divided into two regions: the linear region and the saturated region. In the liner region, HzOz concentration is so low that reaction 1 determines the overall reaction rate and the cathodic current is proportional to the HzOz concentration. In the saturated region, H202 concentration is so high that reaction 1 no longer determines the overall reaction rate and the current is independent of the HZOZ concentration. The current in the saturated region is determined by reaction 2 and/or 3. Steady-state enzymatic reaction rate can be obtained by solving the following differential equations together with the boundary conditions:10b,14,28
(4)
EXPERIMENTAL SECTION
Materials. PTS (n > 500), which had been purified by gel permeation chromatography and ultraiiltration,12was a gift from Dr. Ikenoue, Showa Denko Co. (Japan). Horseradish peroxidase (grade 11, Boehringer, Germany), microbial peroxidase (from Arthromyces ramosus, Suntory, Japan), lactoperoxidase (from bovine milk, Sigma), and catalase (from bovine liver, Sigma) were used as obtained. ?"moxide (9000 A thick, F-doped) coated glass plates as the base electrode were obtained from Nippon Sheet Glass (Japan). (16) Yamada, H.; Yamazaki, I. Arch. Biochem. Biophys. 1974, 165, 728-738. (17) Barman, T.E. Enzyme Handbook;Springer-Verlag: Berlin, 1969; Vol. 1, pp 232-235. (18) Iwai, H.; Akihama, S. Chem. Phann. Bull. 1986, 34,3471-3474. (19) Kulys, J.; Bilitewski, U.; Schmid, R D. Bioelectrochem. Bioenerg. 1 9 9 1 , 2 6 , 277-286. (20) Wollenberger, U.; Wang, J.; Ozsoz, M.; Gonzalez-Romero, E.; Sheller, F. Bioelectrochem. Bioenerg. 1991, 26, 287-296. (21) (a) Jonsson, G.; Gorton, L. Electroanalysis 1989, 1,465-468. (b) Gorton, L.; Bremle, G.; Csoregi, E.;Jonsson-Pettersson, G.; Persson, B. Anal. Chim. Acta 1991, 249, 43-54. (c) Gorton, L.; Jonsson-Pettersson, G.; Csoregi, E.; Johansson, IC; Dominguez, E.; Marko-Varga, G. Analyst 1992, 117, 1235-1241. (22) Ho, W. 0.;Athey, D.; McNeil, C. J.; Hager, H. J.; Evans, G. P.; Mullen, W. H.J. Electroanal. Chem. 1 9 9 3 , 3 5 1 , 185-197. (23) Armstrong, F. A; Bond, A M.; Buchi, F. N.; Hamnett, A; Hill, H. A 0.; Lannon, A M.; Lettington, 0. C.; Zoski, C. G. Analyst 1993,118,973-978. (24) Kulys, J.; Schmidt, R D. Bioelectrochem. Bioenerg. 1990,24, 305-311. (25) Armstrong, F. A; Lannon, A M. J. Am. Chem. SOC.1987,109,7211-7212. (26) (a) Scott, D.L.; Paddock, R M.; Bowden, E. F. J. Electroanal. Chem. 1992, 341,307-321. (b)Scott, D. L.; Bowden, E. F. Anal. Chem. 1 9 9 4 , 6 1 2 1 7 1223. (27) Hayashi, Y.; Yamazaki, I. J. Biol. Chem. 1 9 7 9 , 2 5 4 , 9101-9106.
204 Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
(5)
where DSand Ds*are diffusion coefficients of a substrate in the film and in the solution, respectively, CS and CS* are substrate concentrations in the film and in the solution bulk, respectively, CE is the enzyme concentration in the film, KS is the partition coefficient of substrate for the film, k~ is the reaction rate constant, x is the distance from the electrode surface, and d and 1 are thicknesses of the diffusion layer and the film, respectively. In the linear region, reaction 1 is the ratedetermining step, so the substrate is HzOz and k~ = k l . In the saturated region, reaction 2 and/or 3 determines the rate, so the substrate is H+ and kE = h k 3 / (kZ
+ k3)
Thus, the steady-state current density is given by
2F€Cs*
-
ed
d
Ds
+
+ e-d
(7)
(ed - e-d)&(kECED&1'2
where E is the enzyme-electrode charge transfer efficiency, which (28) Tatsuma, T.;Watanabe, T.Anal. Chem. 1993, 65, 3129-3133.
Table 1. Comparison of Apparent Reaction Rate Constants
electrode
& (A cm-2)O CE (mol ~ m - ~ kE,app ) (cm3mol-’ s-l)* 6x 2
HRP/PTS AFWPTS
A
A
CAT
LPO/PTS
1
1
CAT/PTS
6 x 103 > 3 x 104
1.7 x
3.5
10-6
10-7
1.2 1.1 x
6x
10-5 10-5
2 x 102 9 x 10-1 e
Saturated cathodic current. * Apparent kE, which was estimated on the assumption that KH+and DH+are assumed to be 1 and cm2s-l, res ectively. kE = k&3(kz + k3) for HRP, ARP,and LPO, and kE = klk&Aklk2 + klk3 + k3k4) for CAT. e Values for the CAT subunit. (1
IO-’ I 10-8
10.7o 10-6
10-5o 1 0 - 4
10.3
L
IH2021 (M)
Figure 2. Cathodic current increases for the HRP/PTS, ARP/PTS, LPOIPTS, and CAT/PTS electrodes upon addition of HZOZinto a 0.05 M BaC12 aqueous solution at +400 mV vs Ag/AgCI.
is unity in the absence of mediators other than PTS. Constant a is given by
If the film is infinitely thin, eq 7 can be rewritten as
2FdS* a = d/Ds*
+ 1/&k&
On the other hand, if the film is infinitely thick, eq 7 can be rewritten as
The use of a dfierent enzyme may give different kE, CE,Ds, and Ks, and thereby different i. RESULTS AND DISCUSSION
Horseradish Peroxidase. A cathodic current increase was observed at the HRP/PTS film-coated electrode upon addition of HzOz into the electrolyte solution. This reflects that electron transfer from PTSto HRP occurs. In the presence of 5 pM HzOz, at least 5 mC of cathodic charge was passed through the HRP/ PTS electrode at +400 mV vs Ag/AgC1; at least 5 x mol of electrons was consumed. Since the total amount of HRP in the HRP/PTS film is about 2.5 x mol, HRP must have been turned over at least 10 times, and electrons must be transferred to both compound I (eq 2) and compound I1 (eq 3). Incidentally, the passed charge should not exceed 2.5 x mol of electrons if the electron is transferred only to compound I. Figure 2 shows the current-concentration profile obtained in 0.05 M BaClz aqueous solution at f 4 0 0 mV vs Ag/AgCl. Linear region, in which reaction 1is the rate-determining step and thus the current is proportional to the HzOz concentration,extends up to 0.5 pM HzOz. The cathodic current was almost saturated at above 5 pM HzOz. Since the cathodic current increase was independent of the film thickness 1, which was controlled by cast amount, it is given by eq 10. Further, the current increase depended upon diffusion layer thickness d, which was controlled by the stirring rate, in the linear region, while it was independent of d in the saturated region. That is, ~ / D H ~isonot ~ *negligible before ~ / K H(klC&Hzo2) ~ o ~ lIz, and d/DH+* is negligible before &+(C&+k&?3/(k~ k3))1/2. Therefore,the apparent value of k&3/ (kz k3) can be estimated from the saturated current on the
+
+
50
4
i
201
-101
0
1
I
400
200
I
600
I
800
1000
I
1200
E (V vs.AgiAgCl) Figure 3. Potential dependence of the cathodic current increase for the HRPlPTS electrode upon addition of HZOZ(0.5 pM) into a 0.05 M BaClp aqueous solution.
assumption that KH+and &+ are 1and cmzs-l, respectively. The estimated value is shown in Table 1. Since the diffusion coefficient of proton in the film must be smaller than that in the solution, the true value of kzk3/(kz k3) may be larger than the apparent value. Correlation between the cathodic current increase upon addition of 0.5 pM HzOz (final concentration) and the potential of the HRP/PTS electrode was depicted in Figure 3. AU the data points were acquired with independently prepared HRP/PTS electrodes; scatter of the data is due chiefly to this. The cathodic current increase was rather small and less reproducible at f200 to f 3 0 0 mV, because the PTS is reduced and almost insulating at that potential. PTS electrode containing no enzyme exhibited cathodic current increase lower than 0.3 nA cm-2 upon addition of 0.5 pM HzOz (final concentration) at +400 mV. This cathodic reaction is as follows:
+
H,O,
+ 2Hf + 2e-
-
2H,O
(11)
At +800 mV, on the contrary, an anodic current increase was observed for the PTS electrode upon addition of HzOz. This anodic current was ascribed to the following reaction:
H,O,
- 0, +
2H’
+ 2e-
(12)
Since this reaction proceeds very slowly at the PTScoated SnOz electrode (fortunately), the cathodic current was not canceled at a potential below f l O O O mV. Nonetheless, it is noteworthy that, at +800 to +lo00 mV, the HRF’/PTS electrode showed anodic current responses to 5 pM HzOz, while it showed cathodic current responses to 0.5 pM HzO2. This is because the reaction rate of HRP was saturated above 0.5 pM, while the rate of reaction 12 was not saturated. These results support that the cathodic Analytical Chemisfry, Vol. 67, No. 2, January 15, 1995
285
120 I I 1 currents observed even at +800 to +lo00 mV are ascribed to the reactions catalyzed by HRP. 100 Hayashi and YamazakiZ7reported that the redox potentials of compound I/compound I1 and compound II/ferric enzyme couples for HRP are about +770 and +790 mV vs Ag/AgCl, respectively, at pH 6.06. However, they evaluated the redox potentials by indirect means, so some error may be involved. Indeed, they did not measure the redox potential of IrCl,jz-/3-, which was used as an oxidant/reductant for the enzyme in their own system. Taking this into account, the direct observation of the cathodic currents 0 200 400 600 800 1000 1200 at f l O O O mV in this work does not necessarily contradict the E (V vs. AglAgCI) reported potentials, because the redox potential is a potential at Figure 4. Potential dependence of the cathodic current increase which the activity ratio of an oxidized form to a reduced form is for the ARP/PTS electrode upon addition of H202 (0.5 pM) into a 0.05 unity. That is, in the present case, the oxidized form is produced M BaCh aqueous solution. continuously via reactions 1and 2; hence, the current is observed at a potential even above the redox potential. A similar case has a redox potential. On the basis of the results obtained in this been reported by Aizawa et al.9 for a polypyrrole-fructose work, namely that a direct oxidation current is more significant dehydrogenase system. than a cathodic current via enzyme at a high HZOZconcentration, Microbial Peroxidase. The ARP/PTS electrode also exhib we infer that a higher HZOZconcentration lowers the open circuit ited a cathodic current increase upon addition of HzOz into 0.05 potential. M BaClz aqueous solution. It was also verified coulometrically Lactoperoxidase. The LPO/PTS electrode also showed a that electrons are transferred from PTS to both compound I (eq cathodic current increase upon addition of HzOz. It was also 2) and compound I1 (eq 3) of ARP (examined at +400 mV vs Ag/ verified coulometrically that electrons are transferred from PTS AgCl, 2.5 pM HzOz). to both compound I (eq 2) and compound I1 (eq 3) of LPO Figure 2 shows the current increase as a function of the HzOz (examined at +400 mV vs Ag/AgCl, 10 pM HzOz). concentration observed at +400 mV vs Ag/AgCl. The current As can be seen in Figure 2, showing correlation between the increases agree with those observed for the HRP/PTS electrode cathodic current increase and the HzO2 concentration, the curve up to 0.5 pM (i.e., in the linear region). On the other hand, the does not show a distinct saturated region. This is probably saturated current of the ARP/PTS electrode was about 3-fold because the direct reduction current of HZOZ(eq 11) is not higher than that of the HRP/PTS electrode. Over the concentraneghgible at high concentrations. Subtracting the direct reduction tion range examined, the current increase was independent of the current (observed at a PTS electrode without enzyme) from the film thickness 1 and depended upon the diffusion layer thickness observed cathodic current (at 5 x W 5 - 5 x low4M HzOz) gave d. Therefore,d / h ; is not negligible before l/KH&(klc&#$l/z, the net saturated current of about 100 nA cm-2. That is, the and d/DH+* is not negligible before ~/KH+(CEDH+~Z~~/(~Z + cathodic current increases are smaller than those for the ARP/ k3))lIZ (see eq 10). Namely, K H + ( C Q H + ~ Z ~ ~ k/ 3( )~) lZI 2 for PTS electrode by about an order of magnitude over the concentrathe ARP/PTSelectrode was at least 3-fold larger than that for the tion range examined. Since the current of the LPO/PTS electrode HRP/PTS electrode. This difference may be explained in terms was independent on the film thickness 1 in both linear and of larger KH+and/or &+ for the ARP/PTS electrode because ARP saturated regions, the current increase is given by eq 10 again. is an acidic enzyme, the isoelectric point of which is 3.4,15 and Further, the current increase was independent of the diffusion HRP is a mixture of acidic and basic isozymes.29The other cause layer thickness d. Therefore, K H ~ ~ ~ ( ~ ~ 1C 2’ E D and H~OJ for the difference is larger kz and k3 of ARP than those of HRP. KH+(C&+k&3/(kZ + k3))ll2values for the LPO/PTS electrode are The apparent value of k&3/(kz k3), estimated on the assumption smaller than those for the ARP/PTS and HRP/PTS electrodes. that KH+and DH+are common between the ARP/PTS and HRP/ This is owing to smaller KH+, DH+,and/or kZk3/(kZ + k3). PTS electrodes, is at least Sfold larger than that of the HRP/PTS The apparent value of kZk3/(kz k3) for the LPO/PTS system system (Table 1). Although the kinetics of ARP has not been is also estimated on the assumption that KH+and DH+are 1 and fully studied, ARP is known to be much more active than HRP.15 cm2s-l, respectively, and is listed in Table 1. The hue value Therefore, it is not surprising that the reaction of ARP with PTS may be larger because DH+should be smaller than cm2 s-l, and proton was faster than that of HRP. as mentioned above. In either event, the rate constant for the Figure 4 shows the correlation between the cathodic current LPO/PTS system may be smaller than those for the HRP/PTS increase upon addition of 0.5 pM HzOz (final concentration) and and ARP/PTS systems. This is reasonable in light of the molecule the potential of the ARP/PTS electrode. The plot is similar to size; the distance between an active center and PTS would be that for HRP/PTS electrode. Therefore, the redox potentials of larger for a larger enzyme. Afsnity of PTS for the enzyme may compound I/compound I1 and compound II/fenic enzyme for also be responsible for the difference, though it is not yet known. ARP are probably close to those for HRP. Although Kulys and Figure 5 shows the potential dependence of the current Schmidtz4demonstrated that the open circuit potential of an ARPincrease upon addition of 0.5 pM HzOz. The curve is similar in modified graphite electrode in the presence of a “high concentrashape to those of the HRP/PTS and ARP/PTS electrodes, tion” of HzOz was 732 mV vs Ag/AgCl and suggested that it is suggesting that the thermodynamics of LPO compounds I and I1 the redox potential of ARP compounds, Gorton et al?lc stated that are similar to those of HRF’ and ARP compounds. an open circuit potential in the presence of HzOz is not necessarily Catalase. The CAT/PTS electrode also exhibited a cathodic current increase upon addition of HzOz, though the current was (29) Shannon, L. M.; Kay, E.; Lew, J. Y.J. B i d . Chem. 1966,241, 2166-2172.
t
I
I
L”
+
+
+
286 Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
f
3 -0
':cI
i
2 t
6
4
,
2
0 0
A 1
I
I
I
I
200
400
600
800
1000
J 1200
-2 0
200
,
m
400
600
,
1 800
E (V vs. Ag/AgCl)
E (V vs. AgIAgCI)
Figure 5. Potential dependence of the cathodic current increase for the LPO/PTS electrode upon addition of He02 (0.5pM) into a 0.05 M Back aqueous solution.
Flgure 6. Potential dependence of the cathodic current increase for the CAT/PTS electrode upon addition of H202 (0.5 pM) into a 0.05 M BaC12 aqueous solution.
DH+must be smaller than the assumed value. In either event, substantially lower. The current is too low to examine coulcmetrically whether PTS donates electrons to not only compound I but also compound 11. The current increase as a function of the HzOz concentration is shown in Figure 2 . In the case of the CAT/PTS electrode, response analysis is more complicated due to catalase activity. As mentioned above, compound I of CAT can be directly reduced to ferric CAT by HzOz (eq 13).17 When the HzOz concentration
compound I
+ H,O,
-ferric CAT + H,O + k4
0, (13)
is infinitely small, the rate of this reaction (eq 13) is much slower than reactions 2 and 3, so it need not be considered. At an infinitely large HzOz concentration,the overall rate of reactions 2 and 3 is 2C&~+k&3/(kz k3) in the absence of the reaction 13. On the other hand, in the presence of reaction 13, the rate is 2C~C~+klk2k3/(kik2klk3 + k3k4). Although the current increases for the CAT/PTS electrode in the linear region (> 10 mA cm-2 M-l) were larger than those for the PTS electrode without enzyme (0.6 mA cm-2 M-l), the saturated current for the CAT/PTS electrode (ca. 5 nA cm-2) was smaller than that for the PTSelectrode. This is probably because the HzOz concentration at the h/solution interface is much lower than the bulk concentration,even in the saturated region, due to the fast and diffusion-controlled catalase reaction (eqs 1and 13). This HzOTpurging effect has been observed for the HRPcontaining polypyrrole electrode covered with a CAT film.'Ob Due to this effect, the contribution of the direct reduction current (eq 11) to the observed current is difficult to estimate. However, the current is saturated at 5-50 pM HzOz, and a further increase in the current was not observed; hence the contribution of the direct current may be negligible. Since the current increase was almost independent of the film thickness I, it is as given by eq 10. Thus, the apparent value of klkzk3/(klk~ klk3 k3k4) can be estimated from the saturated current on the assumption that KH+and DH+are 1 and cmz s-', respectively (Table 1). The apparent value listed in the table is for the CAT subunit. Since the values of kl and kq are reported to be 6 x lo9 and 1.5 x 1Olo cm3 mol-' s-l, re~pectively,'~ kz = 4 cm3 mol-' s-l assuming that kz = k3. The true value of klk~k3/ (klkz + klk3 + k3k4) is larger than the above-shown value because
+
+
+
+
the electron transfer reaction from PTS to compound I (and ID of CAT is much slower than those for HRP, ARP, and LPO. Although the molecular weight of the subunit of CAT (60 00065 000) is smaller than that of LPO,CAT exists as an aggregate consisting of four identical subunits; hence, heme in a subunit may be difficult to access for PTS. Figure 6 depicts the potential dependence of the current increase upon addition of 0.5 pM HzOz. As can be seen, the catalytic current drops at $600 to $700 mV vs Ag/AgCl. In this case, however, the cathodic current is so small that the direct oxidation current of HzOz (eq 12) may not be negligible at a potential positive of +600 mV, unlike the other systems of HRP, ARP,and LPO. The maximum current was obtained at around +400 mV, similarly to the other three systems. Concluding Remarks. PTS was confirmed to donate electrons to compounds I and I1 of HRP, ARP,and LPO and at least compound I of CAT. This electron transfer can be observed at t 2 0 0 to flOOO mV vs Ag/AgCl for the HRP,ARP, and LPO systems and at +200 to +500 mV for the CAT system. Compounds I and I1 of HRP, ARP, and LPO exhibit similar thermodynamics. The apparent rate of the electron transfer reaction in conjunction with the proton uptake process was in the order ARP > HRP > LPO > CAT. The small rates for the LPO and CAT systems can be explained in terms of large molecule size and dacult accessibility for PTS to the enzyme active center heme. Further explanation for the difference in the kinetics should follow the elucidation of the molecular structure of the enzymes, though this is beyond the scope of the present work. ACKNOWLEDGMENT
We are grateful to Dr. Y. Ikenoue (Showa Denko Co.) for the supply of PTS.This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 05750731 for T.T. and No. 05235211 for N.O.). Received for review September September 22, 1994.@
20, 1994.
Accepted
AC9409391 @Abstractpublished in Advance ACS Abstracts, November 15, 1994.
Analytical Chemistry, Vol. 67,No. 2, January 15, 1995
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