Anal. Chem. 1998, 70, 4690-4695
Mediator-Assisted Continuous-Flow Column Electrolytic Spectroelectrochemical Technique for the Measurement of Protein Redox Potentials. Application to Peroxidase Masaki Torimura,† Manabu Mochizuki,† Kenji Kano,*,† Tokuji Ikeda,*,† and Teruhisa Ueda‡
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, and Chromatographic Instruments Division, Shimadzu Corporation, Nakagyo-ku, Kyoto 604-8511, Japan
A mediator-assisted continuous-flow column electrolytic spectroelectrochemical method was designed to study redox properties of proteins. This method is based on a flow-through column electrolytic control of a redox buffer containing relatively high concentrations of mediators and an accelerated reaction of a protein with the mediators to reach equilibrium within the electrolysis time. A small amount of a protein sample is introduced into the electrochemically regulated redox buffer in the mode of flow injection analysis (FIA). The equilibrated redox state of the protein is evaluated by a highly sensitive flow-through photodiode array detector. Reproducible and stable background spectra and the employment of the FIA mode allow precise background subtraction in spectral analysis. This method was successfully applied to determining the redox potentials of horseradish peroxidase (HRP) using potassium hexachloroiridate as a mediator. The absorbance versus the electrode potential curves for the redox reactions among the ferric form, compound II, and compound I of HRP were well interpreted by a Nernstian equation based on the two-step one-electron-transfer model. The advantage and the kinetic aspects of this method are discussed in detailed.
There are substantial demands for knowledge of redox potentials (E°′) of proteins, since E°′ values are one of the most important physicochemical parameters for better understanding of physiological redox processes and also for further developments of biosensors, bioelectrocatalytic reactors, and biofuel cells. Cyclic voltammetry might be applied to the direct determination of the E°′ of proteins at suitable electrodes.1-4 However, direct electrode reactions of redox proteins are often irreversible or undetectable, * Corresponding authors: (fax) +81-75-753-6128; (e-mail) (K.K.) kkano@ kais.kyoto-u.ac.jp and (T.I.)
[email protected]. † Kyoto University. ‡ Shimadzu Co. (1) Frew, J. E.; Hill, H. A. O. Eur. J. Biochem. 1988, 172, 261. (2) Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21, 407. (3) Armstrong, F. A. Adv. Inorg. Chem. 1992, 38, 117. (4) Hawkridge F. W.; Taniguchi, I. Comments Inorg. Chem. 1995, 17, 163.
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since the redox-active centers of proteins are usually bound strongly within deep inside of the polypeptide chains and/or the strong adsorption properties of proteins lead to denaturation on electrode surfaces. Thus, potentiometric titration or that coupled with spectroscopic detection (potentiometric-spectroscopic titration) using some redox mediators becomes a routine technique for the determination of the E°′ of redox proteins.5,6 However, the potentiometric technique is often complicated by slow potential responses at (platinum) indicator electrodes or spectral overlapping of mediators and proteins. Optically transparent thinlayer cells provide in situ UV-visible absorption spectroelectrochemistry.7,8 Since proteins can undergo indirect electrontransfer processes in the presence of mediators or promoters, the optically transparent thin-layer spectroelectrochemical technique (OTTLSET)9,10 or other optically transparent spectroelectrochemical techniques11 can be used as the alternative for determining thermodynamic parameters of redox proteins. Numerous literature reports emphasize the use of the OTTLSET for studying the redox reaction of proteins.12 The disadvantages of the OTTLSET might be that it also takes a long time to reach the equilibrium state after applying the potential to the cell and that the spectra become complicated when the absorption spectra of mediators and proteins are overlapped. On the other hand, continuous-flow column electrolysis is a novel method for rapid and quantitative electrolysis.13-17 It is well(5) Wilson, G. S. In Methods in Enzymology; Fleischer, S., Packer, L., Eds.; Academic Press: New York, 1978; Vol. 54, pp 396-410. (6) Dutton, P. L. In Methods in Enzymology; Fleischer, S., Packer, L., Eds.; Academic Press: New York, 1978; Vol. 54, pp 411-435. (7) Petek, M.; Neal, T. E. Murray, R. W. Anal. Chem. 1971, 43, 1069. (8) Heineman, W. R.; Hawkridge, F. M., Blount, H. N. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13, pp 1-113. (9) Heineman, W. R.; Norris, B. J.; Goelz, J. F. Anal. Chem. 1975, 47, 1975. (10) Anderson, C. W.; Halsall, H. B.; Heineman, W. R. Anal. Biochem. 1979, 93, 366. (11) Hawkridge, F. M.; Kuwana, T. Anal. Chem. 1973, 45, 1021. (12) Dong, S.; Niu, J.; Cotton, T. M. In Methods in Enzymology; Sauer, K., Ed.; Academic Press: New York, 1995; Vol. 246, pp 701-732. (13) Fujinaga, F. Pure Appl. Chem. 1971, 25, 709. (14) Okazaki, S. Rev. Polarogr. 1968, 15, 154. (15) Shioda, R. E. Electrochim. Acta 1968, 13, 375. (16) Kihara, S. J. Electroanal. Chem. 1973, 45, 31. (17) Bard, A. J.; Faulkner, L. R. In Electrochemical Methods. Fundamentals and Applications; John Wily: New York, 1980; pp 398-405. 10.1021/ac980621z CCC: $15.00
© 1998 American Chemical Society Published on Web 10/15/1998
known that this method has a great advantage over other electrochemical techniques in view of the electrolysis efficiency. This would result from an extremely large electrode surface area compared to an electrolysis cell volume as well as a hydrodynamic effect. Consequently, complete electrolysis to the equilibrium state can be achieved quickly under suitable conditions. Furthermore, this method can be easily coupled with various spectroscopic devices (e.g., UV-visible photodiode array, electron spin resonance, or Raman spectroscopic detectors) to obtain electrochemical and spectroscopic signals simultaneously.18-20 The continuous-flow column electrolytic spectroelectrochemical technique (CFCESET) allows direct and reversible redox reactions of redox proteins such as a single-hemoprotein horse heart cytochrome c (12 kDa)21 and quinohemoprotein alcohol dehydrogenase from Gluconobacter suboxidans containing one pyrroloquinoline quinone and four hemes c (140 kDa).22 However, some proteins including horseradish peroxidase (HRP) exhibited only ill-defined irreversible characteristics in the direct column electrolysis. Although HRP shows five redox states at one porphyrincentered active-site, the ferric (native) form, compound II, and compound I are important redox states in peroxidase reaction and the three redox states are reported to be reversible in principle as written by eq 1.23 step I -e-
step II -e-
ferric form {\ } compound II {\ } compound I (1) E°′ E°′ 1
2
In the HRP reaction, the ferric form as the resting state is oxidized by H2O2 to form a two-electron oxidized state known as compound I containing oxyferrylheme (Fe4+dO) and a porphyrin radical. The porphyrin radical in compound I abstracts one electron from a substrate to generate compound II, which is subsequently reduced back to the ferric form by taking one additional electron from a substrate. The two redox potentials E°′1 and E°′2 were evaluated by means of potentiometric-spectroscopic titration using the K2IrCl6-K3IrCl6 redox system as a mediator.23 Recently, the OTTLSET has been also applied to the determination of the redox potentials using the K2IrCl6-K3IrCl6 couple, although 1-1.5 h was required for equilibration after each potential step.24 The electron-transfer processes between proteins and electrodes would be facilitated by the use of increased concentrations of redox mediators, and the redox state of the mediators can be easily controlled by column electrolysis. Thus, our attempt in this work is to couple the CFCESET with flow injection analysis (FIA) for the assessment of E°′of proteins, in which a protein sample will be introduced into a continuous flow of a redox buffer of mediators controlled by column electrolysis and the redox state of the protein will be monitored spectroscopically. In this work, (18) Oyama, M.; Nozaki, K.; Okazaki, S. Anal. Chem. 1991, 63, 1387. (19) Fujinaga, T.; Okazaki, O.; Nagaoka, T. Bull. Chem. Soc. Jpn. 1980, 53, 2241. (20) Yamanuki, M.; Oyama, M.; Okazaki, S. Vibr. Spectrosc. 1997, 13, 205. (21) Oyama, M.; Okada, M.; Okazaki, S. In Redox Mechanisms and Interfacial Properties of Molecules of Biological Importance; Schultz, F. A., Taniguchi, I., Eds.; The Electrochem. Soc.: Pennington, NJ, 1993; p 343. (22) Torimura, M.; Kano, K.; Ikeda, I.; Ueda, T. Chem. Lett. 1997, 525. (23) Hayashi, Y.; Yamazaki, I. J. Biol. Chem. 1979, 254, 9101. (24) Farhangrazi, Z. S.; Fossett, M. E.; Powers, L. S.; Ellis, W. R., Jr. Biochemistry 1995, 34, 2866.
Figure 1. Schematic diagram of a CFCESET-FIA system. A, reservoir of a mobile phase (0.2 M phosphate buffer of pH 7.0 with ionic strength adjusted 1.0 with KCl); B, reservoir of a mediator solution (usually 6.8 × 10-4 M K2IrCl6); C, HPLC pump; D, injector; E, T-mixing; F, column electrolysis cell; G, potentiostat; H, photodiode array detector; I, waste.
HRP and the K2IrCl6-K3IrCl6 couple were used as a model redox protein and as a redox mediator, respectively. EXPERIMENTAL SECTION Reagents. Horseradish peroxidase C (EC1.11.1.7) is an isoenzyme of HRP and was purchased from Toyobo (Osaka, Japan). Potassium hexachloroiridate(III) and -(IV) (K3IrCl6 and K2IrCl6) were purchased from Wako (Kyoto, Japan) and used without further purification. Hexachloroiridate solutions were prepared with distilled water just before use. All other chemicals were of analytical-reagent grade and used as received. Apparatus. Column electrolysis was performed using a Hokuto Denko (Kanagawa, Japan) HX-110 cell consisting of a carbon wool working electrode with a fibrous structure packed tightly in a Bicole glass tube (i.d. 8 mm, length 50 mm), an Ag/ AgCl (saturated KCl) reference electrode, and a platinum coiled counter electrode. All potentials were referred to the Ag/AgCl reference electrode, unless otherwise stated. The column electrode potential was changed in a staircase mode with a Yanaco (Kyoto, Japan) P-1000 potentiostat and a Hokuto Denko HB-104 function generator. The continuous-flow system used in the present study is illustrated in Figure 1. A mobile phase (0.2 M phosphate buffer, pH 7.0) in reservoir A and a mediator (usually K2IrCl6) solution in reservoir B flowed continuously at 0.25 mL min-1 with Shimadzu LC-6A HPLC pumps and mixed with a T-piece before introduction to the column electrode, the total flow rate at the column electrode being 0.5 mL min-1 unless otherwise stated. The electrolyzed solution was spectroscopically monitored with a Shimadzu SPD-M10Avp photodiode array detector in the wavelength range from 300 to 700 nm at a data acquisition interval of 2 s. Stable stationary absorption spectra of the mediator in the electrolyzed solution (background spectra) were obtained within ∼1 min after applying a given potential to the column electrode. After stabilization of the background spectra, a 10-µL portion of an HRP solution was injected on the mobile-phase flow with a Rheodyne injector 7725 in an FIA mode. The FIA peak was monitored at a suitable wavelength. A potential step interval of 10 min was enough to take spectroscopic data for each injection of protein samples and then the volume of the mediator solution in reservoir B required for 15 successive injections was ∼40 mL. All experiments were performed at a room temperature controlled at 25 ( 3 °C. Data Treatment. The absorption spectrum at the top of an HRP peak in the FIA (peak-top total spectrum) was taken as the sum of the spectra of HRP and the mediator. The background spectrum ascribed to the mediator alone was subtracted from the peak-top total spectrum to extract the spectral information ascribed Analytical Chemistry, Vol. 70, No. 22, November 15, 1998
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to HRP alone (background-corrected spectrum) at each applied potential. Absorbance (A) of the background-corrected spectrum of HRP at a given wavelength (λ) was plotted against the electrode potential (E). A two-step one-electron Nernstian equation (eq 12) was fitted to the A-E curve to determine E°′1 and E°′2 by means of a nonlinear least-squares analysis.
ln
[
ηP
(ηP + 1)θ - 1
kf
(2)
b
where ox and red denote the oxidized and reduced forms, respectively, and kf and kb are the bimolecular rate constants of the corresponding processes. The rate equation of reaction 2 is given by
ν)
-d[Pred] ) kf[Pred][Mox] - kb[Pox][Mred] dt
(3)
τ1/2 )
[Mox]e
) exp
[RTF (E - E°′ )] ≡ η M
(4)
M
[
ln
(kfηM + kb)[Pred] kfηM[P]o
-
] (
)
kb kfηM + kb )[M]ot (5) kfηM ηM + 1
where [M]o is defined as [M]o ) [Mox] + [Mred] ([Mred]e ) [M]o/ (1 + ηM)). When reaction 2 is in equilibrium (v ) 0 in eq 3), the equilibrium concentrations of Pox and Pred ([Pox]e and [Pred]e) are expressed as a function of E,
[Pox]e [Pred]e
) exp
[RTF (E - E°′ )] ≡ η P
P
(6)
and the equilibrium constant K in reaction 2 is expressed using thermodynamic and kinetic parameters by
K≡
[Pox]e[Mred]e [Pred]e[Mox]e
)
ηP kf ) ηM kb
(7)
where E°′P is the formal redox potential of P. Substitution of eqs 6 and 7 into eq 5 yields 4692 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998
(8)
(ηM + 1)ηP kf[M]o(ηP + 1)ηM
ln 2
(9)
Aλ ) {ox,λ[Pox]e + red,λ[Pred]e}l )
(ox,ληP + red,λ) [P]ol ηP + 1
(10)
Equation 10 can be rewritten as
[
ln where E°′M is the formal redox potential of M. Since [Mox]e and [Mred]e are constant in the column electrolysis cell, reaction 2 can be considered as a pseudo-first-order reaction and then eq 3 can be solved as follows under the initial condition of [Pred]t)o ) [P]o(≡[Pox] + [Pred]).
(ηM + 1)ηP
In the column electrolytic FIA, the reaction time t may be considered as the retention time during which protein molecules pass through the column. When t is sufficiently larger than τ1/2, reaction 2 can reach the equilibrium state. Such situations would be realized by elongation of the column length, decrease in the flow rate to increase t, and/or increase in [M]o (or kf[M]o) to decrease τ1/2. In the CFCESET, the background spectrum ascribed to the mediator is very stable; then it is easy to correct the large background spectrum precisely at increased [M]o. Under the equilibrium conditions, E dependence of the backgroundcorrected spectrum can be expressed by
In continuous-flow column electrolysis, the equilibrium state of the mediator with reversible redox characteristics is quickly achieved just after application of a potential to an appropriate column electrode. The equilibrium concentrations of Mox and Mred ([Mox]e and [Mred]e) are expressed as a function of E by
[Mred]e
kf[M]o(ηP + 1)ηMt
with θ ≡ [Pred]/[P]o. At the half-life time τ1/2, θ ) (1 + [Pred]e/ [P]o)/2, then τ1/2 is given by
PRINCIPLES Let us consider a simple bimolecular one-electron reaction between a redox protein (P) and a mediator (M),
Pred + Mox {\ } Pox + Mred k
]
)
]
Aλ - red[P]ol ox[P]ol - Aλ
)
F(E - E°′P) RT
(11)
Since ox,λ[P]ol and red,λ[P]ol can be evaluated experimentally at E . E°′P and E , E°′P, respectively, Aλ vs E curves can be analyzed based on eq 10 or 11 to evaluate E°′P. The above argument can be extended to the two-step oneelectron-transfer reaction such as reaction 1, though kinetic treatments will be somewhat complicated due to a potent comproportionation reaction between the ferric form and compound I to generate compound II. For reaction 1 in equilibrium, eq 10 can be replaced by25
Aλ ) {F,λ[ferric]e + CII,λ[compound II]e + CI,λ[compound I]e}l )
[P]ol ( + CII,λη1 + CI,λη1η2) η1η2 + η1 + 1 F,λ
(12)
where F,λ, CII,λ, and CI,λ are the absorption coefficients of the ferric form, compound II, and compound I, respectively, η1 ) exp[F(E - E°′1)/RT], and η2 ) exp[F(E - E°′2)/RT]. Since F,λ[P]ol and CII,λ[P]ol can be evaluated experimentally, Aλ vs E curves can be analyzed based on eq 12 using E°′1, E°′2, and CII,λ (or CII,λ[P]ol) as adjustable parameters. (25) Torimura, M.; Mochizuki, M.; Kano, K.; Ikeda, T.; Ueda, T. J. Electroanal. Chem. 1998, 451, 229.
RESULTS AND DISCUSSION Redox Behavior of the IrCl62-/3- Couple. The CFCESET was applied to examine the redox behavior of the K3IrCl6 or K2IrCl6 aqueous solution at pH 7.0. The E dependence of A488 values of the background spectra well satisfied a one-electron Nernstian equation to yield a redox potential of 0.756 ( 0.001 V (see eq 10, in which [P]o may be replaced by [M]o). This value is in agreement with a reported one (0.74 V at pH 0-9 26). This means attainment of the redox equilibrium of the IrCl62-/3- couple in the column. It is well-known that IrCl62-/3- ions are reduced and hydrolyzed spontaneously above neutral pH as follows:26,27
IrCl62- + Cl- f IrCl63- + 1/2Cl2
(13)
IrCl63- + H2O f Ir(OH2)Cl52- + Cl-
(14)
In a pH 9.0 buffer, K2IrCl6 gave a new redox wave at ∼0.5 V and the wave grew with time. The new wave seems to be assigned to the Ir(OH2)Cl5-/2- couple. A similar but moderate reaction was observed even at pH 7.0. Although the Ir(OH2)Cl5-/2- couple might serve as a mediator, the hydrolysis and the subsequent redox reaction would cause time dependence of the background spectra and then complicate the background subtraction after the long reaction time for equilibration with HRP. Since aqueous solutions of K2IrCl6 (and K3IrCl6) were stable for several hours, the K2IrCl6 solution for reservoir B was prepared with distilled water in this work. Compared with the OTTLSET, the reaction time could be remarkably shortened and the side reactions of IrCl62-/3- during the electrolysis can be ignored in the CFCESET. Mediator-Assisted Redox Reaction of HRP. At a total flow rate of 0.5 mL min-1, the peak of HRP was detected ∼4.5 min after the injection. The FIA peak shape of HRP and the time from the injection to the peak top were practically independent of E, suggesting no adsorption of HRP on the column electrode. The standard deviation of the peak height was 3.1% for five successive injections of the HRP solution at a given E. Judging from the peak height, the HRP sample solution (3.2 × 10-4 M) was diluted ∼200-fold at the peak top (1.6 × 10-6 M), while the total concentration of the IrCl62-/3- ions in the column was 3.4 × 10-4 M. The background and peak-top total spectra are given in the inset of Figure 2. Although the difference between the spectra was relatively small due to an large concentration ratio of IrCl62-/3against HRP () 212), both of the absorption spectra were reproducible and then a simple subtraction treatment was acceptable to get the background-corrected spectra of HRP. Some examples of the background-corrected 3-D spectra are depicted in Figure 2. Determination of E°′ of HRP. Figure 3 shows the E dependence of A402 of the background-corrected spectra of HRP. The curve was practically independent of the total flow rate up to 1.0 mL min-1 as well as the direction and the width of the potential step. These results strongly support the electrochemical reversibility of HRP or the redox equilibrium between HRP and IrCl62-/3-. (26) Fergusson, R. R. J. Am. Chem. Soc. 1956, 78, 741. (27) Kukuskin, Y. N.; Soboleva, M. S. Russ. J. Inorg. Chem. 1972, 17, 619.
Figure 2. Typical background-corrected 3-D spectra of FIA peaks of HRP at E ) 0.550, 0.715, and 0.850 V vs Ag/AgCl. HRP, 3.2 × 10-4 M × 10 µL; K2IrCl6 in reservoir B, 6.8 × 10-4 M; total flow rate, 0.5 mL min-1. The inset shows (A) a peak-top total spectrum and (B) a background spectrum at E ) -0.850 V, where the difference between the peak-top total spectrum and the background spectrum was very small. Even in such cases, reproducible backgroundcorrected spectra were obtained.
Figure 3. A402-E curve of HRP in pH 7.0 phosphate buffer: (b) experimental data; (s) fitting curve based on a two-step electrontransfer mechanism (eq 12).
The A402-E plot is composed of one sigmoidal part with two plateaus. Absorption spectra at three values of E are depicted in Figure 4. At E ) 0.550 V (a potential on the plateau of the negative potential side), HRP is in the ferric state as evidenced by the spectrum identical with that of the native one. On the other hand, at E ) 0.850 V (a potential on the plateau of the positive potential side), a decrease and a blue shift of the Soret band were observed. The spectrum is reasonably assigned to compound I.24,28 During the potential change from E ) 0.550 V to E ) 0.900 V, no clear isosbestic point was observed and a red shift of the Soret band was observed on the partial oxidation prior to the blue shift on (28) George, P. Science 1953, 117, 220.
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Figure 4. Absorption spectra of HRP at E ) (A) 0.550, (B) 0.715, and (C) 0.850 V vs Ag/AgCl. Spectra A and C correspond to the ferric form and compound I, respectively, at [P]o ≈ 1.6 × 10-6 M. The inset is a calculated spectrum of compound II normalized to be [P]o, which is extracted from spectrum B as described in the text.
Kinetic Aspects. The complete redox equilibrium between HRP and IrCl62-/3- may be also supported from the kinetic point of view. As expected from eq 9, τ1/2 exhibits a sigmoidal dependence on E with two limiting values of K ln 2/(kf[M]o) and ln 2/(kf[M]o). Using the E°′ values of the mediator and HRP estimated here, K can be calculated based on eq 7 as 4.1 and 2.3 for steps I and II in reaction 1, respectively. On the other hand, the kf values for the oxidation of HRP with IrCl62- were reported as 2.5 × 102 and 1.0 × 104 M-1 s-1 for steps I and II, respectively.23 Thus, the maximum value of τ1/2 can be assessed as 48 and 4 s, for steps I and II, respectively, for our experimental conditions. These values would be acceptably smaller than the electrolysis time (∼4.5 min). This argument supports the achievement of the redox equilibrium during the mediator-assisted column electrolysis, although for rigorous aspects of the kinetics of the redox reaction between HRP and the mediator, the comproportionation reaction between the ferric form and compound I and vice versa should be taken into consideration.
CONCLUDING REMARKS the further oxidation (λmax ) 402, 405, and 398 nm at E ) 0.550, 0.715, and 0.850 V, respectively). These results strongly support the generation of an intermediate redox state, most probably compound II. Thus, we tried to fit the theoretical Nernstian curve (eq 12) for a two-step one-electron-transfer mechanism to the A402-E data by means of a nonlinear least-squares method. The regression curve given by the solid line in Figure 3 reproduces the data well. E°′ values are E°′1 ) 0.720 and E°′2 ) 0.750 vs Ag/AgCl at pH 7.0 (sd = 0.007 V for each as the goodness of the fitting). Almost identical results were obtained by similar analyses at other wavelengths. The molar fractions of the ferric form, compound II, and compound I at a given E can be given by [ferric]e/[HRP]o ) 1/(η1η2 + η1 + 1), [compound II]e/[HRP]o ) η1/(η1η2 + η1 + 1), and [compound I]e/[HRP]o ) η1η2/(η1η2 + η1 + 1), respectively; for example, [ferric]e/[HRP]o ) 49%, [compound II]e/[HRP]o ) 41%, and [compound I]e/[HRP]o ) 10% at E ) 0.715 V using the E°′1 and E°′2 values evaluated here. Thus, a pure spectrum of compound II can be extracted on the basis of eq 12 by subtracting the contribution of the ferric form and compound I from the mixed spectrum B in Figure 4. The extracted spectrum given in the inset of Figure 4 shows a strong Soret band with λmax of 418 nm and two broad bands in the visible region around 550 nm and is very close to those reported in the literature.24,28 However, the evaluated E°′ values were slightly positive than reported ones obtained by the OTTLSET with the same mediator (E°′1 ) 0.672 V (0.869 V vs NHE) and E°′2 ) 0.700 V (0.897 V vs NHE)).24 The reason for the deviation was not clarified, but an increase in the column temperature due to electrolysis might cause an increase in E°′1 and E°′2, since positive values of the entropy S () F dE°′/ dT) were reported in the literature for the redox reaction of HRP.24 From the analytical viewpoint, however, it should be pointed out that the data analysis in the literature24 was based on a one-step electron-transfer model (see eq 10), which is applicable only to the case of E°′1 , E°′2 but not to HRP. Such inappropriate treatments of the data as well as the instability of the mediator during equilibration (1-1.5 h 24) under the OTTLSET conditions seem to cause some errors in the estimation of E°′ values. 4694 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998
It can be concluded that the mediator-assisted CFCESET is a novel technique for the indirect redox regulation of proteins and then the determination of the redox potentials. The CFCESET produces very stable background spectra and the employment of an FIA mode in the introduction of protein samples provides an benefit in the subtraction of large absorption of mediators. This allows the use of an increased concentration of mediators, which will shorten the time required to reach the equilibrium. Strictly speaking, however, it would be important to keep the concentration of mediators as low as possible because they tend to interact with proteins in such a way to influence the potential. In addition, the upper limit of the mediator concentration would be restricted by Michaelis-Menten-type steady-state kinetics. Elongation of the column length and/or decrease in the flow rate would be beneficial to attain the redox equilibrium within the electrolysis time. Another and probably much more important factor in the CFCESET is the selection of suitable mediators with a large kf and with an E°′M close to E°′P as in the case of OTTLSET. This may mean that the present technique has limited utility for the general cases. The CFCESET may be also utilized for kinetic study of the reactions between proteins and mediators, since the reaction becomes pseudo first order (see Principles section). In the kinetic study, the concentrations of mediators should be kept sufficiently low compared with the Michaelis constant to simplify the reaction kinetics between proteins and mediators. For rigorous kinetic study, it would be better to incorporate a pre-electrolysis column electrode in order to adjust the solution potential of a redox buffer just before mixing with a protein sample, although even in a single column electrolysis instrument the time required to adjust the solution potential to the electrode potential would be negligible compared with the time for the indirect column electrolysis of proteins. In this study using HRP as a model sample, no nonspecific adsorption on the column electrode surface was observed. However, adsorption of proteins may be encountered occasionally, which would cause the tailing of peaks. In such cases, simple data treatment using peak-top total spectra would not be sufficient.
Another appropriate mathematical correction, such as normalization of absorbance based on the area of an FIA peak, may be required. One of the disadvantages of the present technique is that it requires a relatively large amount of protein samples (5 × 10-8 mole for 15 injections in this work) compared with the OTTLSET, although the samples used can be recovered by gel filtration for example. Decrease of the inner diameter of the electrolysis column will be essential in order to minimize the protein sample dilution in the column (∼200-fold dilution in the present column) and then to reduce the amount of samples.
ACKNOWLEDGMENT This work was supported in part by Grant-in-Aids for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
Received for review June 8, 1998. Accepted September 2, 1998. AC980621Z
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