Anal. Chem. 1994, 66, 2710-2718
This Research Contribution is in Commemoration of the Life and Science of I . M. Kolthoff (1894-1993).
Spectrophotometric and Electrochemical Kinetic Studies of Poly(ethylene glycol)-Modified Horseradish Peroxidase Reactions in Organic Solvents and Aqueous Buffers Liu Yangt and Royce W. Murray' Kenan Laboratories of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290 The dynamics of the horseradish peroxidase-catalyzed oxidation of catechol and ferrocene derivative reductants by the oxidants tert-butyl hydroperoxide and hydrogen peroxide at saturating concentrations have been studied in aqueousbuffers and in the organic solvents ethyl acetate and 1,t-dichlorobenzene. The enzyme, modified with poly(ethy1ene glycol) (PEG) so as to be soluble in both aqueous and organic solvents, catalyzes the same reactions in organic and aqueous solutions but more slowly in organic solutions. Comparisons between spectrophotometric and electrochemical kinetic procedures confiim the methodology. The enzyme-reductant reactionrates are first order in reductant and in enzyme at low turnover rates in the organic media, but level off at high turnover rates. Influenceson reaction rates of reductant redox potential, steric bulk, hydrophobicity, and charge, and of buffer, electrolyte, and pH are also discussed. It is now appreciated that enzymes can function as reaction catalysts in organic as well as in aqueous media.lI2 Organicphase biocatalysis offers increased organic substrate solubility, enhanced enzyme thermostability, substrate specificity differing from that of aqueous media, shifts of thermodynamic equilibria, and suppression of water-dependent side reactions. Organic-phase enzyme sensors enable analyte-specific detection of poorly water soluble substances and extend the variety of possible designs of biosensors.3" An understanding of solvent-induced alteration of enzyme reactions, and an anticipation of optimal solvent and substrate choices for organic-phase enzyme catalysis and biosensor design, depend upon a better understanding of organic-phase reaction dynamics than now exists. In previous kinetic studies of organic-phase enzyme reactions, enzymes were, owing to their insolubility, either suspended in the solution7or adsorbed onto glass beadse8q9 Interfacial kinetics can be difficult to t Present address: Bioanalytical Systems, 2701 Kent Avenue, West Lafayette, IN 47906. (1) Klibanov, A. M. Trends Biochem. Sci. 1989, 14, 141-144. (2) Dordick, J. S. Curr. Opin. Biotechnol. 1991, 2, 401407. (3) (a) Wang, J.; Wu, L. H.; Angnes, L. Anal. Chem. 1991.63, 2993-2994. (b) Wang, J.; Lin, Y.; Chen, W. Electroanalysis 1993,5, 23-28. (c) Wang, J.; Lin, Y . Anal. Chim. Acta 1993,271.53-58, (d) Wang, J.; Lin, Y . ;Chen, L. Analyst 1993, 118, 277-280. (4) Schubert, F.;Saini,S.;Turner,A. P. F. A m / . Chim. Acta 1991,245,133-138. ( 5 ) Saini, S.; Hall, G. F.; Downs,M. E. A,; Turner, A. P. F. Anal. Chim. Acta
1991, 249, 1-15. (6) (a) Hall, G. F.; Turner, A. P. F. Anal. Lett. 1991.24, 1375-1388. (b) Hall, G. F.; Best, D. J.; Turner, A. P. F. Anal. Chim. Acta 1988, 213, 113-119. (7) Parida, S.; Dordick, J. S . J. Am. Chem. SOC.1991, 113, 2253-2259.
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study quantitatively due to uncertainties in reproducing reaction conditions, in knowing the quantity of reactive enzyme, and in controlling variables useful in assessing reaction order and mechanism. We sought, accordingly, a kinetic design based on an organic-solubilized enzyme whose concentration is known and manipulable and chose a poly(ethy1ene glycol)modified enzyme that is soluble in both aqueous and organic media. The hydrophilic nature of poly(ethy1ene glycol) enables aqueous-phase modification of enzyme while its hydrophobic nature solubilizes modified enzyme in organic solvents. PEG modification has been applied to various enzymes and proteins, and it has been shown that thusly modified enzymes remain catalytically active.10-12 The enzyme chosen is horseradish peroxidase, modified with poly(ethy1ene glycol). We have examined its catalytic reaction kinetics in the organic solvents ethyl acetate and 1,2dichlorobenzene, and for comparison, in aqueous buffers. Horseradish peroxidase has been studied extensively in aqueous buffers and has been used as a model enzyme in aqueousphase enzymology, and in analytical biochemistry as a coenzyme of oxidase sensors. Horseradish peroxidase is biocatalytically stable in various organic solventsand catalyzes identical reactions in aqueous and organic solvents.8~9 The present study uses large peroxide concentrations, seeking oxidant saturation of enzyme reaction kinetics and delivery of reaction rate control to the reaction of the oxidized form of the PEG-modified horseradish peroxidase with a series of model reductants. The reductants include catechols and ferrocene derivatives; the peroxides are rert-butyl hydroperoxide and hydrogen peroxide. For the reductants 4-methylcatechol and tetrabromocatechol, the enzyme-reductant reaction rate was, as a methodology check, measured in ethyl acetate and 1,2-dichlorobenzene solvents using both spectrophotometric and electrochemical procedures. The rates of reaction of PEG-modified horseradish peroxidase with reductants in ethyl acetate and in 1,Zdichlorobenzene solutions are much slower than are the corresponding reactions in (8) (a) Ryu, K.; Dordick, J . S . J. Chem. Soc. 1989, 111, 8026-8027. (b) Ryu, K.; Dordick, J. S . Biochemistry 1992, 31, 2588-2598. (9) Kazandjian, R.Z.; Dordick, J. S.; Klibanov, A. M.Blorechnol. Bioeng. 1986, 28, 417421.
(IO) Takahashi, K.; Nishimura, H.; Yoshimoto, T.; Saito, Y.; Inada, Y.Blochem. Biophys. Res. Commun. 1984, 121,261-265. (1 1) Takahashi, K.; Ajima, A.; Yoshimoto, T.; Inada, Y. Biochem. Biophys. Res. Commun. 1984,125,761-766. (12) Sakurai, K.;Kashimoto, K.; Kcdera, Y . ;Inada, Y. Biotechnol. Lett. 1990,12, 685-688.
0003-2700/94/0366-27 10$04.50/0 0 1994 American Chemical Society
aqueous buffers. Variations in the rate constants suggest the importance of enzyme-reductant binding, and of the reductant's redox potential, steric bulk, hydrophobicity, and charge in the organic medium. Effects of pH, buffer composition, and supporting electrolyte are also described.
EXPERIMENTAL SECTION Peroxidase (Type VI, from horseradish) and methoxypoly(ethylene glycol) (average MW 5000, activated with cyanuric chloride) obtained from Sigma were used as received. This horseradish peroxidase was modified with methoxypoly(ethylene glycol) as described in the literaturelo and the lyophilized dry product stored at 0 OC prior to use. The concentration of PEG-modified enzyme solutions was estimated spectrophotometrically at 403 nm (E403 = 1 X lo5 M-' cm-I).lO The aqueous-phase activity of the modified enzyme was measured by spectrophotometric assay of its 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) oxidase activityl3 as 100 f 15% of the activity of unmodified horseradish peroxidase, based on the molar concentration of the enzyme and at the same substrate concentrations. (The high activity of the modified enzyme may in part be due to a further purification of the enzyme during the ultrafiltration step in the enzyme modification procedure.) PreviouslyIo PEGmodified horseradish peroxidase was observed to be 70% as active as the unmofidied enzyme, using a weight-based concentration. tert-Butyl hydroperoxide (t-BuOOH, 90% contains 5% H2O and 5% t-BuOH) was purchased from Aldrich, anhydrous t-BuOOH (3 M in toluene, over molecular sieve) from Fluka, and hydrogen peroxide (30%) from Fisher. t-BuOOH and H202 solutions were stored at 0 OC prior to use. Ethyl acetate solutions of H202 were prepared fresh for each assay by mixing small volumes of the aqueous H202 solution with anhydrous ethyl acetate (v/v
c
.-m -
4.8E-05
c
0
.-wc
1.8E-03
ar
v)
-0 .-c
3.6E-07
0
1
.-c
0
1.8E-07
h
0
Figure9. 4-Methylcatechol reductant and modlfied peroxidase enzyme concentrationdependenciesof spectrophotometricallydetermined Initial reaction velocities. In 1,2dlchlorobenzene saturated with aqueous trls buffer, [H202] = 0.02 M and [Bu4NPFe] = 20 mM. (A) [HRP] = 0.043 pM; (B) [4-methylcatechol] = 1.4 mM.
h
0 W v)
4.OE-05
1 . c
.-0
-0W > c
,2 c
2.OE-05
0
m
0,
a:
-.-m .-c
-c
O.OE+OO O.OE+OO
2.OE-03
4.OE-03
4-Methylcatechol Conc. (M)
Figure 8. 4Methylcatechol reductant concentration dependency of spectrophotometrically determlned Initial reaction velocity. In 1,2dichlorobenzene saturated with aqueous trls buffer, [HRP] = 0.039 pM, [t-BuOOH] = 0.26 M, and [Bu4NPFe] = 20 mM.
reductant (A) and peroxidase enzyme (B) concentration dependencies of spectrophotometrically obtained reaction velocities; the rate constants are kl2 = 1.8 (fO.l) X lo5and 1.2 (fO.l) X lo5 M-I s-l, respectively. These rate constants are reasonably close to that from Figure 8, where t-BuOOH is the oxidant, as they should be if the reaction is saturated with and independent of oxidant. Notice however that Figure 9A achieves a higher reaction velocity than the plateau in Figure 8, which suggests some dependency on the oxidant identity. An earlier of horseradish peroxidase reaction kinetics in 50% (v/v) methanol-10 mM phosphate suggested a preequilibrium mechanism for the oxidation of the enzyme by peroxide
HRP + ROOH F? compound 0
-
compound I
(6)
where 0 is an association complex of enzyme and peroxide which forms less strongly with bulkier peroxide oxidants. In the present case, hydrogen peroxide would be expected to (45) Chang, C. S.;Yamautki, I.; Sinclair, R.; Khalid, S.;Powers, L. Biochemisrry 1993, 32,923-928.
associate more strongly with the enzyme on the basis of both less steric bulk and greater hydrophilicity. That is, the difference between Figures 8 and 9A may reflect differences in oxidant binding by enzyme, in competition with that of the reductant. The above interpretation of reductant-enzyme adduct dissociation constants and turnover numbers must therefore be tempered with some uncertainty concerning the oxidant. The comparisons between different catechols and ferrocenes, on a relative basis, are unaffected by this uncertainty since the peroxide oxidant concentrations were constant in each set of measurements. Reaction Kinetics for k12as a Function of Buffer Composition and pH. Buffer composition and pH commonly exert influence on enzyme-catalyzed reactions in aqueous media but little is known about their effects in nonwater environments. Table 3 presents a comparison of buffer composition in aqueous and ethyl acetate media, based on spectrophotometric experiments and 4-methylcatechol reductant. Table 3 shows that rate constants for different aqueous buffer systems at pH 7 are very close to each other and to the value obtained in unbuffered water. Reactions 1-3 should involve no net consumption or production of protons, so the latter observation is not entirely surprising. In ethyl acetate solutions saturated with aqueous solutions of the different buffers, or just with water, there is a -2-fold variation in the rate constants. Thus, the nonaqueous reaction has at best a mild dependence on buffer composition and on buffer presence, at pH 7. Detailed s t ~ d i e s ~ ~in~aqueous ~ 3 ~ ~ 6phase have shown a significant pH dependence of the horseradish peroxidase reaction kinetics, which slow at high pH. It has been suggested that this effect is connected with the ionizable amino acid residues around the active site of the enzyme, whose state of protonation may affect both coordination structureof the active site4’ and electrostatic interactions with charged substrates. Table 4 shows no change in enzyme activity toward 4-methylcatechol between pH 4.7 and 7, but a substantial loss in (46) Matsumura-Inouc, T.; Kuroda, K.; Umczawa, Y.; Achiba, Y. J. Chem. Sm., Faraday Trans. 1989.85. 857-866. (47) Fukuzumi, S.; Mochizuki, S.; Tanaka, T. Inorg. Chem. 1989,242459-2463.
Analytical Chemistry, Vol. 66, No. 17, September 1, 1994
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Table 3. Buffer Dependency of Rate Constants for the Oxidation of 4-Methylcatechoi by t-BuOOH-Saturated HRP buffer
rate constant k1zu (M-Ls-')
tris (50 mM, pH 7)
phosphate (50 mM, pH 7)-Bu4NPF6 (20 mM)d phosphate (50 mM, pH 7)
in ethyl acetate bufferb in aqueous buffef
1.2 (f0.2) X lo4 7.1 (f0.2) X los
8.4 (*0.3)
X
Hz0
6.3 (f0.1) x 103 6.8 (f0.3) X los
lo3
5.8 (f0.1) x 103
7.3 (f0.4)
X
10s
Spectrophotometric results of this work. Tolerances are 90% confidence intervals; N - 1 degrees of freedom. In ethyl acetate saturated with the aqueous solutions shown at the top row. [HRP] = 73.4 nM; [r-BuOOH] = 0.9 M. In a ueous solutions shown at the top row. [HRP] = 40.7 nM; [t-BuOOH] = 0.23 M. For ethyl acetate with Bu4NPF6 and saturated with phosphatetuffer. (1
Table 4. Buffer pH Dependency of Reaction Rate for the Oxldatlon of Medttator-Reductants by tBuOOH-Saturated HRP
buffer (50 mM tris) pH apparent pH of ethyl acetate-tris' rate constant klz (M-1 s-I) for 4-methyl~atechol~ ethyl acetate-trisc tris bufferd rate constant klz (M-I s-1) for ferrocenecarboxylic acidb ethyl acetate-trisc tris bufferf apparent pH of 1,2-dichl~robenzenetris~ rate constant klz (M-1. s-I) for tetrabromocatechol in dichlorobenzene-trisb
4.7 6.5
7.0 8.0
10.8 8.8
dry solvent 7.0
9.4 (ko.8) x 103 6.6 (*1.0) X los
9.4 (ho.7) x 103 5.6 (f0.8) X 10'
9.2 (fo.6) x 103
1.2 (f0.1) x 103
g
4.7 p 0 . 2 ) x io4 1.6 (f0.2) X lo6 3 7.9 (i1.o) x 103
4.5 (f0.2) x 104 7.7 (f0.5) X los 3 7.2 (*o.a) 103
4.7 (f0.1) x io4 g 3 8.1 ( ~ 1 . 0 x ) 103
*
7.1 (10.6) x 103
a pH measured with glass pH electrode for tris-saturated organic solvents. Spectrophotometric experiment results of this work. Tolerances are 90% confidence intervals; N - 1 degrees of freedom. [4-Methylcatechol = 7.8 mM; [HRP] = 22 nM; [t-BuOOH] = 0.22 M. [4-Methylcatechol] =0.23M. e [Ferrocenecarboxylicacid]=0.4mM;[HRP] = 15nM; [t-BuOOH]=0.44M.f[Ferrocenecarboxylic [HzOz] = 0.35 mM. 8 Reaction rates less than 1 X 10-8 M s under the experiment condition; rate constant not trophotometric results of this work. [Tetrabromocatechol] = 0.45-4 mM; [H PI = 0.95 pM; [t-BuOOH] = 0.26 M. Tolerances are 90% confi ence intervals; N - 1 degrees of freedom.
R
enzyme activity at pH 10.8. There is a larger difference in enzyme activity toward ferrocenecarboxylic acid between pH 4.7 and 7.0; the HRP active site (p& = 5.343)should be more positive at pH 4.7 than at pH 7.0 whereas ferrocenecarboxylic acid is substantially anionic at both pH values. The fast, pH-dependent reaction of ferrocenecarboxylic acid may thus arise from efficient (electrostatic) association with the enzyme at pH 4.7, compensating for the relatively modest reducing potential of this reductant. In organic solvents saturated with tris buffers at different pH, however, Table 4 shows that the reaction rate is independent of the buffer pH for both 4-methylcatechol and ferrocenecarboxylic acid reacting in ethyl acetate and for tetrabromocatechol in 1,2-dichlorobenzene. Glass electrodebased pH measurements indicate changes in the apparent organic solvent pH values that are much smaller than those of the originating aqueous buffer, which may explain the relative independence of reaction rate on buffer pH in ethyl acetate and dichlorobenzene. The apparent pH of 1,2dichlorobenzene-tris solutions could be driven up when the organic-solublebase trioctylamine was added, and in this case, significant decreases in enzyme-tetrabromocatechol reaction rates were observed. It is not clear, however, whether this is a pH effect or one of specific enzyme inhibition by the amine. Finally, Table 4 also shows that when no water is added to the ethyl acetate solvent, the enzyme activity is still observable but is substantially depressed compared with that in aqueous buffer-saturated ethyl acetate. This is consistent
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Analytical Chemistry, Vol. 66,No. 17, September 1, 1994
with earlier observations1J7regarding maintaining hydration of the enzyme. CONCLUSIONS Rate constants for reactions of solubilized horseradish peroxidase with reductants in organic solvents have been measured for the first time. Comparisons between spectrophotometry and microelectrode voltammetry show that either methodology is capable of producing reliable kinetic results in the organic phases. These studies are meant to illustrate an approach to understanding the function of enzymes in organic phases, including their potential function in biosensor applications. The reactions studied do not show clean dependencies of kinetics on reaction free energy as manifested in reductant redox potential, unlike previous aqueous-phase investigation^^^ that followed Marcus relations. The reaction scheme is obviously more complex and includes a reductantenzyme binding step and, possibly, competition with binding of oxidant. ACKNOWLEDGMENT Scientific Parentage of the Author. R. W .Murray, Ph.D. under R. C. Bowers, Ph. D. under I. M. Kolthoff. Received for revlew February 4, 1994. Accepted M a y 17, 1994.' Abstract published in Aduance ACS Abstracts, July 1, 1994.
