et Biophysica A~ta ELSEVIER
Biochimica et Biophysica Acta 1206 (1994) 272-278
Inactivation of horseradish peroxidase by phenol and hydrogen peroxide: a kinetic investigation K a t h y J. B a y n t o n a, J a t i n d e r K. B e w t r a b, N i h a r B i s w a s b, K e i t h E. T a y l o r a,. a Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ont. N9B 3P4, Canada b Department of Civil and Environmental Engineering, University of Windsor, Windsor, Ont. NgB 3P4, Canada Received 23 November 1993
Inactivation of horseradish peroxidase (HRP) was examined in the presence of hydrogen peroxide alone and in the presence of hydrogen peroxide plus phenol. HRP is inactivated upon exposure to hydrogen peroxide (H202) by the combination of two possible pathways, dependent upon hydrogen peroxide concentration. At low H202 concentrations (below 1.0 mM in the absence of phenol), inactivation is predominantly reversible, resulting from the formation and accumulation of catalytically inert intermediate compound III. As H202 concentrations increase, an irreversible mechanism-based inactivation process becomes predominant. The overall inactivation comprised of both processes exhibits a second-order inactivation rate constant (kapp) of 0.023 + 0.005 M-is - 1 at pH 7.4 and 25°C. In the presence of both hydrogen peroxide fixed at 0.5 mM and phenol, HRP was inactivated in an irreversible, time- and phenol concentration-dependent process, also mechanism-based, with a kapp of 0.019 ___0.004 M-is-1.
Key words: Horseradish peroxidase; Suicide inactivation; Phenol; Hydrogen peroxide
Horseradish peroxidase undergoes mechanism-based or suicide inactivation:  under conditions of 'excess' H 2 0 2 both in the presence and absence of donor substrates (AH 2) [1,2];  from attack by radical intermediates [3-7] produced by oxidation of the donor substrate during the peroxidase cycle . Reaction schemes, based on works presented by Arnao et al. [1,2] and Nakajima and Yamazaki , incorporating inactivation pathways operating both in the presence and absence of donor substrates are presented in Fig. la and b, respectively. When H 2 0 2 is present in excess over donor substrates or alone, inactivation can occur by one of two possible pathways, one reversible, one irreversible, which are reported to be partitioned at HRP intermediate compound I [1,2,9]. The 'reversible' inactivation pathway, so-called because fully active native enzyme recovers slowly from
Abbreviations: HRP, horseradish peroxidase; 4-AAP, 4-aminoantipyrine; HDCBS, 3,5-dichloro-2-hydroxybenzenesulfonic acid; AH 2, donor substrate; TNM, tetranitromethane * Corresponding author. Fax: + 1 (519) 9737050. 0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 1 6 7 - 4 8 3 8 ( 9 4 ) 0 0 0 3 5 - F
the relatively inert intermediate compound III that accumulates via compound II during this process (Fig. lb) [1,9,10], is predominant . Compound III formation follows from compound II along one of two simultaneous pathways, distinguished by the inhibitory effect of superoxide radical scavenger tetranitromethane (TNM) over one path but not the other [9,10]. In the predominant, TNM-sensitive path, compound II is reduced to a transient intermediate, which either reacts with a superoxide radical generated in this reaction to yield compound III, or with an equivalent of H202 producing compound I [9,10]. From compound I, more compound III may form as previously described, or the second, slower pathway along which HRP is inactivated in an irreversible, mechanism-based reaction, may be followed [1,2,9]. The appearence of a green intermediate referred to as compound P670 or compound IV [1,2,11,12] accompanies inactivation. Amao et al. suggest that for every two reversible inactivations, one irreversible inactivation event occurs . However, concentrations of H202 greater than those typically present under assay conditions were studied and details of the events occurring at these lower concentrations are not yet available. At low H202 to donor substrate concentration ratios, oxidation of donor molecules produces free radical inter-
K.J. Baynton et al. / Biochimica et Biophysica Acta 1206 (1994) 272-278
mediates [5,8] which diffuse away from the enzyme's active site [8,13] where they may react with:  another radical ;  a neutral donor molecule or product of radical coupling [6,18]; or,  the enzyme itself [7,15]. Mechanism-based inactivation of HRP by radicals produced during incubation with phenylhydrazine , alkylhydrazines , and sodium azide  have been previously observed. Inactivation was also examined in the presence of H202 plus a phenol  at a high H202 (5.0 mM) to phenol (0.1 mM) concentration ratio. The present steady-state kinetic investigation of HRP inactivation mediated by both oxidant (H202) and donor substrates (i.e., phenol) under conditions typical for a number of activity determinations (i.e., low H202:donor) is of practical interest, particularly because of the enzyme's amenability to a number of applications ranging from enzyme markers in histo- and immunochemical techniques [16,17,26] to removal of toxic organics from industrial waste effluents [6,18,19] and soils [20,21].
2. Materials and methods
2.1. Materials Horseradish peroxidase (HRP) ((donor: hydrogen peroxide oxidoreductase: EC 188.8.131.52) Grad II, R.Z. 2) was obtained as a lyophilizate from Boehringer-Mannheim, Canada and contained predominantly isoenzyme C. Stock solutions were prepared in 0.1 M sodium phosphate buffer (NaPP) (pH 7.4) and dialysed against distilled water. Enzyme concentration was determined spectrophotometrically based on heme absorbance using an oo404 of 102000 M -1 cm -1 . Catalase (EC 184.108.40.206. from bovine liver) was purchased from Sigma. 3,5-dichloro-2-hydroxybenzenesulfonic acid (HDCBS), 4-aminoantipyrene (4-AAP) and phenol crystals (99% purity or greater) were purchased from Aldrich. All solutions were prepared as needed in 0.1 M sodium phosphate buffer (pH 7.4). Hydrogen peroxide (60%, w / v ) was supplied by BDH. Stock solutions were prepared fresh prior to use. Disodium hydrogen phosphate (Na 2HPO4) and sodium dihydrogen phosphate (Nail 2PO4) for phosphate buffer were supplied by BDH. All stock solutions and buffers were prepared in distilled water. 2.2. Instrumentation and equipment
Spectrophotometric measurements were performed on a Hewlett-Packard diode array spectrophotometer Model 8451. Dialyses were carried out using 42 mm dialysis capsules and 12000-14000 molecular weight cut-off (MWCO) membranes supplied by InstruMed. 2.3. Inactivation experiments
1-1202 Compound I ~ ~P670
. Compound II
(b) Fig. 1. Possiblecatalyticand inactivationpathwaysof horseradishperoxidase in the presence (a) and absence (b) of donor substrates compiled from work done by Nakajima and Yamazaki and Arnao et al. [1,2]. AH2 = donor molecule;A'= free radical intermediate;O~= superoxide radical.
H202-mediated inactivation was initiated by the addition of an aliquot of H202 to a 1 ml solution of enzyme at a given concentration (100-1000 nM). A 20-50 ml aliquot was withdrawn from the incubation solution at specific times and plunged into a cuvette containing final concentrations of 2.4 mM 4-AAP, 9.0 mM HDCBS and 100 mM H202 in a final volume of 1 ml. Enzyme concentrations in the activity assay were designed to be 5-10 nM, yielding sensitive and reproducible results. Activity was measured as an increase in the absorbance at 510 nm (e510 = 25 000 M -~ cm-1 based on peroxide)  at room temperature (pH 7.4). Results permitted the determination of the percentage remaining activity vs. time compared to the activity of a control solution containing enzyme incubated alone. Selected samples were dialysed for three-hour periods against a buffer containing catalase to determine if any lost activity could be recovered. Inactivation of HRP in the presence of phenol was monitored as follows. To test tubes containing solutions of HRP yielding the desired concentration in a volume of 5 ml (25-100 nM), an aliquot of phenol was added from a prepared stock. Phenol concentration was determined from
K.J. Baynton et al. / Biochiraica et Biophysica Acta 1206 (1994) 272-278
the absorbance value at 272 nm using an extinction coefficient of 1300 M -1 cm -1 (unpublished data). Hydrogen peroxide was added to initiate the inactivation process. At specific times, aliquots (0.1-0.4 ml) were removed from the incubation solutions into 1 ml cuvettes containing 0.6-0.9 ml of an activity reagent to yield final concentrations of 0.84 mM 4-AAP, 10.0 m M phenol and 0.4 mM H 2 0 2. Final enzyme concentrations were 1 - 5 0 nM. Activity was measured at room temperature (pH 7.4), as an increase in the absorbance at 510 nm ( e 5 1 0 = 6 0 0 0 M-1 c m - l b a s e d on peroxide) (unpublished data). The activity reagent used in these experiments did not contain HDCBS but, rather, phenol in large excess as coupling partner for AAP in order to avoid competition in the coupling which would have arisen between H D C B S in a reagent and variable amounts of phenol arriving with the enzyme sample. Percentage remaining activity vs. time was determined compared to controls containing enzyme alone and enzyme plus phenol.
2.4. Inactivation rate constant determination Pseudo-first order inactivation rate constants (kob s) were calculated using an enzyme kinetics program EnzfitterTr'k Best fit of percent remaining activity vs. time data obtained from inactivation experiments to double- and single-exponential decay curves described by equations y = A l e x p - k l t + A 2 e x p - k 2 t and y = A e x p - k t , respectively, were determined using non-linear regression analysis. Double exponential fits were used for enzyme inactiviation in the presence of peroxide alone in order to obtain rate constants for the fast phase of the clearly biphasic activity vs. time plots where the fast phase amplitudes varied with peroxide concentration. In contrast, inactivation in the presence of peroxide plus phenol did not show biphasic behaviour and, therefore, the simpler function was fit to these data.
3. R e s u l t s
3.1. H20 2-mediated inactivation Incubation of HRP with various concentrations of H 202 resulted in a time-dependent loss in activity as depicted in Fig. 2. Above 0.01 m M H202, the curves obtained exhibited two increasingly distinct regions of activity: (1) a rapid burst of inactivation which was complete within 5 min with magnitude dependent upon H 2 0 2 concentration; (2) a second region which follows' abruptly from the first where activity changes little from the last activity test taken at the end of the 'rapid phase' over the ensuing 5 to 60 min and on to 24 h at peroxide concentrations < 0.10 mM. At H202 concentrations above 0.10 mM, enzyme solutions lost all activity by 24 h. Only at 0.001 m M H202 (stoichiometric to 10-fold higher concentration than enzyme) did enzyme activity completely recover by 24 h
Fig. 2. Inactivation progress curves for exposure of 100aM HRP to various concentrations of H202, 25°C (pH 7.4). H202 concentration: (0) 0.001 raM, (11) 0,025 raM, (ll) 0.075 mM, (,~) 0.1 raM, ( v ) 0.5 mM, (×) 1 raM, (+) 5 mM, and (*) 25 raM.
Data in Table 1 indicates the participation of reversible and irreversible inactivation processes. Duplicate samples were incubated for 1 h with specific concentrations of H 2 0 2 and then dialysis was started with one of each pair and incubation continued. Dialysis in the presence of catalase served to remove any excess H 2 0 2 that had not yet been consumed by the enzyme. Following 3 h of dialysis all samples recovered at least 2- to 3-times the level of activity observed at the onset of dialysis but never 100% of the original activity. The amount of activity regained from dialysis was greater for samples exposed to lower H 2 0 2 concentrations. It was noted that upon the completion of dialysis at three hours, neither inactivation nor recovery occurred during the subsequent 3 - 2 4 hour period. The results of these experiments support the existence of two inactivation pathways: (a) one reversible, which is notably operative at concentrations of H202beTable 1 Percentage residual enzyme activity vs. H202 concentration H202 (mM) Residualactivity (%) No dialysis With dialysis lh a 24h lh a 3h b 24h c 0.5 1.0 5.0 10.0 25.0 50.0
34.0 24.3 3.80 3.30 2.00 1.30
0.50 0.60 0.00 0.00 0.00 0.00
35.2 24.7 4.00 3.30 2.00 1.30
70.6 36.2 15.2 9.40 6.20 6.30
70.8 35.9 13.9 10.3 5.70 6.40
Comparison of the mean percentage residual activities of undialysed and dialysed triplicate samples of 100nM HRP solutions exposed for one hour to specific concentrations of H20 2. At this time, all samples were tested for residual activity. a Undialysed samples were left to incubate at room temperature for 24 hours, at which time residual activity was tested. Dialysed samples were checked for residual activity immediately following 3 h of dialysis. b As above but after 24 h further incubation. c At room temperature after dialysis was complete (25°C, pH 7.4).
K.J. Baynton et al. / Biochimica et Biophysica Acta 1206 (1994) 272-278 4.75
( 4.50 -
30 40 Time (sec.)
Fig. 3. Least-squares linearized plots of percentage remaining activity vs. time data for selected H202 concentrations. (HRP concentration = 100
nM at 25°C (pH 7.4)). H202 concentration:(0) 0.1 mM, (1:3)0.5 mM, (11) 0.75 mM, and (O) 1 mM. low 1.0 mM; (b) one irreversible which becomes increasingly prominent at H202 concentrations of 1.0 mM and greater. Rapid inactivation over time periods of 30 to 60 s demonstrated pseudo first-order, inactivation kinetics dependent on H202 concentration as shown in Fig. 3. This figure is useful as a graphical presentation but kobs (the pseudo-first order inactivation rate constant) [3,22] values were obtained from the fast component in a fit of the data by non-linear regression to a double-exponential decay model. This approach is similar to that undertaken by Arnao et al. . Fig. 4 shows the calculated kobs values plotted vs. H202 concentration. For H202 concentrations above those presented in Fig. 4, kobs values could not be reliably measured with the instrumentation at hand since in many instances, inactivation was virtually complete in 5 s. Indeed, the kobs depicted in Fig. 4 are likely to be underestimates of the true values; despite this they still serve to illustrate the dependence of inactivation rate on H202
Fig. 5. Least-squares linearized plots of percentage remaining activity vs. time data obtained from experiments in which triplicate samples of 50 nM HRP were incubated with phenol at specific concentrations in the presence of 0.5 mM H202 (pH 7.4 at 25°C). Phenol concentration: (11) 0.2 raM, ( 0 ) 0.5 mM, ( O ) 0.75 mM, (zx) 1 mM, and ( n ) 2 mM.
concentration. A second-order inactivation rate constant, kapp, was calculated from the slope in Fig. 4 to be 0.023 + 0.005 M - i s - t . Double-reciprocal plots of rate vs. inhibitor concentration curves are frequently used as tools to examine complex formation between inhibitor and enzyme [4,22]; such a plot (not shown) yielded: from the x-intercept, an inhibitor binding constant (Kiapp) of (4.4 + 1.04) mM; and, from the y-intercept, the limiting rate constant or maximum rate of inactivation when all enzyme molecules are occupied with inhibitor (kinact) , determined to be (0.20 + 0.08)s -1. The large value of Klapp suggests that H202 associates very poorly with the enzyme prior to the step in which a subsequent covalent or irreversible change at the enzyme's active site results in inactivation . Still, inactivation is 51 times faster than that observed by Arnao et al.  for similar concentrations of H202 and enzyme (kinact = 0.0039 s -t compared to 0.2 s-1 obtained in this study).
3.2. Inactivation in the presence of phenol
Fig. 4. Peroxide-dependence of kobs values for the rapid phase of inactivation obtained from double-exponentiai decay analysis performed on percentage remaining activity vs. time data obtained at specific H202 concentrations. (HRP concentration = 100 nM, 25°C (pH 7.4)). The linear-regression line is shown ( r = 0.8323)
Data representing the loss of HRP activity over time, in the presence of both phenol and H202, and fit to singleexponential decay curves, are presented in Fig. 5. The data for these inactivation curves were obtained from triplicate experiments and are typical of the inactivation observed at all concentrations of phenol, H202 and enzyme examined (HRP = 25, 50, and 100 nM, respectively; phenol = 0.2 to 2.0 mM; and, H202 = 0.5 and 1.0 mM). The inactivation process was time-dependent, irreversible and essentially complete by 5 min. Residual activities at all H202 and HRP concentrations, in the presence of phenol concentrations > 0.5 mM, were less than 10% by 5 min. At the lowest phenol concentration (0.2 mM), inactivation of HRP at 25 and 50 nM (but not 100 nM) in the presence of H202 at both concentrations was less severe and followed an initial activation of enzyme activity (i.e., typically 10-20% greater than the control activity during the first 30
K.J. Baynton et al. / Biochimica et Biophysica Acta 1206 (1994) 272-278
4.1. H20 2-mediated inactivation
In the absence of reductant substrates and at high H 2 0 2 concentrations, HRP is inactivated in a time- and H 2 0 2 c o n c e n t r a t i o n dependent process, exhibiting suicide or mechanism-based inactivation kinetics. Similar behaviour was observed in a previous study of H2OE-mediated inactivation under identical experimental conditions of temperature, pH and concentrations of enzyme and H202 . However, differences exist in the shapes of the inactivation curves, in the magnitudes and rates of inactivation at s p e c i f i c H 2 0 2 concentrations, and between the maximal rate of inactivation (kinac t) in comparison with the present study. Inactivation observed in our study, at all concentrations of H202, was more rapid and extensive over identical reaction time periods. A plausible explanation may lie with the different enzyme preparations employed: the Boehringer-Mannheim Grad II enzyme used in this study contains predominantly isoenzyme C, while the Sigma Type IX HRP used by Arnao et al.  is composed exclusively of basic isoenzymes. Differences in kinetic behaviour among the HRP isozymes towards H202 has been well documented . The inactivation process (Fig. 2 and Table 1) involves the participation of two pathways: one reversible and the other irreversible--which may or may not function independently of each other and whose individual contribution to the overall inactivation process appears to depend on H202 concentration. Amao et al.  have proposed a partition to exist between the two pathways at compound I. Their model, based on studies at high H202 concentrations (1 to 50 mM), invokes a further partition between these two inactivation pathways and a catalatic reaction in which H202 is consumed with relatively little harm to the enzyme (Fig. lb). Kinetic parameters determined under their reaction conditions, accounting for a 3-pathway partition, predicted that reversible inactivation leading to production and accumulation of compound III would be favoured over irreversible inactivation and, further, that the catalatic reaction would be the predominant pathway of the three. Evidence suggesting that a catalatic pathway was in operation in the present study was not immediately obvious but such a path could conceivably account for the slow phase of inactivation. A recent review , however, questions the existence of HRP's catalatic activity under conditions other than in the presence of iodide ion. Our study points to reversible inactivation operating predominantly at H202 concentrations ranging up to 1.0 mM. However, even as H202 concentration increased within this range, an irreversible inactivation process became more prevalent until it began to dominate at 1.0 mM and above. One observation is consistent between this and Arnao et al.'s study  : a partition does appear to exist between the twO inactivation pathways. Further evidence
Fig. 6. A plot of kobs values (pseudo first-order inactivation rate constart0 obtained from single-exponential decay analysis of % remaining activity vs. time data collected during exposure of enzyme (25 nM) to HzO 2 (1.0 mM) and varying concentrations of phenol (25°C, pH 7.4). The linear regression line is shown (r = 0.9903).
s of the inactivation reaction). In such cases, exponential decay was computed from the highest point on the relative activity curve. Control experiments demonstrated that catalytic turnover of phenol in the presence of peroxide is a necessary prerequisite in order for inactivation to occur. Enzyme solutions exposed to both phenol and H202 turned progressively darker brown. Visible absorption spectra of these solutions exhibited an absorbance maximum at 400 nm which has been attributed to the oxidation of p,p'-biphenol to form p-diphenoquinone . The pseudo-first order inactivation rate constants were determined from the fits such as in Fig. 5. Plots of kob~ vs. phenol concentration, exemplified by Fig. 6, were used to estimate second-order rate constants (k~pp) (see Table 2) according to the equation: kobs = k~pp(I), where I represents the phenol concentration. From data in Table 2, a mean second-order inactivation constant value of (0.019 + 0.004) M - l s-1 was determined.
Table 2 Second-order inactivation rate constant obtained at various concentrations of H202 and enzyme [HRP] (nM)
[H 202 ] (mM)
25 25 50 50 100 100
0.5 1.0 0.5 1.0 0.5 1.0
1.87 + 0.24 1.88 + 0.35 1.85 + 0.30 1.87 + 0.13 1.39 5:0.60 2.71 + 0.27
( × 102M - l s- 1)
Values of kapp, the second-order inactivation rate constant, obtained from the slopes of plots of k~s vs. phenol concentration at given HRP and H202 concentrations indicated (T = 25"C, pH 7.4; phenol concentration: 0.2-2.0 raM). The uncertainties were calculated from the standard error of triplicate determinations of values of kobs.
K.J. Baynton et al. / Biochimica et Biophysica Acta 1206 (1994) 272-278
of a partition at compound I comes from kinetic studies of the formation of compound III from compound II in the presence of excess H202 performed by Nakajima and Yamazaki . The HRP intermediate at which partitioning may occur could not be identified in this study. However, qualitative spectroscopic examinations designed to follow changes in the native enzyme's visible spectrum upon addition of H202 at specific concentrations (data not shown) suggested that reversible inactivation leading to a steady-state formation of compound III was prevalent below 1.0 mM H202; above this range, the compound III spectrum was readily distinguishable within the first few seconds but declined concommitant with an increase in absorbance at 670 nm, indicating the presence of inactive HRP compound P670 . Due to differences in the magnitude of the extinction coefficients of various HRP intermediates, the rapidity of the reaction being observed and the instability of at least two of HRP's reaction intermediates (compound's I and II) , these observations are only qualitative. Nevertheless, a dependence of enzyme intermediate formed, and thus the pathway operating, under given concentrations of H202 relative to enzyme are indicated. Together, these observations are consistent with the existence of a partition between or possibly among reaction/inactivation pathways.
4.2. Inactivation in the presence of phenol and 1-1202 Enzyme inactivation observed in the presence of n 2 0 2 and phenol closely resembles mechanism-based inactivation of HRP by phenylhydrazine in the presence of H 2 0 2 . In both studies, inactivation was time-based and dependent on inhibitor concentration (phenol and phenylhydrazine). Peroxide-dependent turnover of substrate was required for inactivation to occur. Ma and Rokita  observed a slower HRP (24 nM) inactivation in the presence of > 50-fold H202 relative to phenol concentration at 25°C (pH 7.0). A kinact of 0.211 min -1 was obtained under anaerobic conditions; slight inactivation potentiation was observed in the presence of oxygen, with a kinact of 0.331 min -1. It was speculated that potentiation was due to the formation, from a reaction between a molecule of phenol and oxygen, of an extremely reactive peroxy radical species. However, a complete absence of oxygen from their anaerobic solutions could not be guaranteed. Unlike Ma and Rokita's study, HRP inactivation observed in this study and in the presence of phenylhydrazine  was too fast to enable determination of the kinetic parameters kinact and K~ using steady-state spectrophotometric techniques.A possible explanation may lie in the ratios of H202 to phenol/phenylhydrazine used as well as the reactive intermediate generated. Activation of enzyme activity observed at the lower concentrations of phenol and enzyme has been previously documented  but poorly understood and remains so
today. Despite its ambiguity, this phenomenon of phenolenhanced enzyme activity has been employed to increase the sensitivity of chemiluminescence techniques . Studies aimed at determining the chemical nature of radical intermediates formed during the peroxidase reaction should shed light on this process and could be exploited in a number of applications in clinical and industrial processes where enhanced enzyme activity is frequently desirable. Despite the difficulty encountered in determining kinetic parameters, it is clear that inactivation observed in the present study satisfies the criteria of mechanism-based (suicide) inactivation:  inactivation was irreversible over the observation period;  substrate turnover was mandatory for inactivation to occur; and,  rate of inactivation was dependent on the inhibitor concentration, in this case a phenol. Thus substrate turnover by horseradish peroxidase is a double-edged sword: under certain reaction conditions, catalysis does not result in significant enzyme mortality; under other conditions, enzyme inactivation is rapid and frequently non-recoverable. Despite the wealth of information available concerning the kinetics of this ubiquitously employed enzyme, much still needs to be discerned concerning mechanistic details. For researchers interested in employing HRP in applications including removal of toxic substances from industrial effluents and contaminated soils [6,18-21], these studies are of primary importance to the conversion of hazardous materials into biologically inaccessible and manageable compounds with minimal productivity loss to catalyst. Studies on modelling of the overall process of phenol removal by peroxidases, including the inactivation pathways reported here, are continuing in this laboratory.
Acknowledgments Thanks are gratefully extended to the Natural Sciences and Engineering Research Council of Canada (NSERC), the Institute for Chemical Science and Technology (ICST) and the University Research Incentive Fund (URIF) of Ontario for financial support.
References  Arnao, M.B., Acosta, M., del Rio, J.A. and Garcia-Canovas, F. (1990) Biochim. Biophys. Acta 1038, 85-89.  Arnao, M.B., Acosta, M., del Rio, J.A., Varon, R. and GarciaCanovas, F. (1990) Biochim. Biophys. Acta 1041, 43-47.  Ortiz de Montellano, P.R., David, S.K., Ator, M.A. and Tew, D. (1988) Biochemistry 27, 5470-5476.  Ator, M.A., David, S.K. and Ortiz de Montellano, P.R. (1987) J. Biol. Chem. 262, 14 954-14 960.  Ator, M.A. and Ortiz de Monteilano, P.R. (1987) J. Biol. Chem. 262, 1542-1551.
K.J. Baynton et al. / Biochimica et Biophysica Acta 1206 (1994) 272-278
 Klibanov, A.M. and Morris, E.D. (1981) Enzyme Microbiol. Technol. 3, 119-122.  Ma, X. and Rokita, S.E. (1988) Biochem. Biophys. Res. Commun. 157, 160-165.  Yamazaki, I., Mason, H.S. and Piette, L. (1960) J. Biol. Chem. 235, 2444-2449.  Nakajima, R. and Yamazaki, I. (1987) J. Biol. Chem. 262, 25762581.  Adediran, S.A. and Lambeir, A.M. (1989) Eur. J. Biochem. 186, 571-576.  Yamazaki, I. and Yokota, K.N. (1973) Mol. Cell. Biochem. 2, 39-52.  Chance, B. (1949) Arch. Biochem. Biophys. 21, 416-430.  Danner, D.J., Brignac, P.J., Arceneux, D. and Patel, V. (1973) Arch. Biochem. Biophys. 156, 759-763.  Tripathi, G.N.R. and Schuler, R.H. (1984) J. Chem. Phys. 81, 113-121.  Lindsay, R.H., Gaitan, E., Jolley, R.L., Cooksey, R.C. and Hill, J. (1986) Abstr. Pap. - Am. Chem. Soc. 191, envr. 21.  Artiss, J.A., Draisey, T.F., Thibert, R.J. and Taylor, K.E. (1979) Microchem. J. 26, 487-505.
 Conyers, S.M. and Kidwell, D.A. (1991) Anal. Biochem. 192, 207-211.  Dec, J. and Bollag, J.M. (1990) Arch. Environ. Contam. Toxicol. 19, 543-558.  Matoney, S.W., Marem, J., Mallevaille, J. and Fiesinger, F. (1986) Environ. Sci. Technol. 20, 249-253.  Simmons, K.E., Minard, R.D. and Bollag, J.M. (1988) Soil Sci. J. 52, 1356-1360.  Bollag, J.M. (1990) in Chemical and Biochemical Detoxification of Hazardous Waste (Glaser, J.A., ed.), Lewis, Philadelphia, PA.  Walsh, C. (1979) Enzymatic Reaction Mechanisms, pp. 88-89, W.H. Freeman, San Francisco, CA.  Sawahata, T. and Neal, R.A. (1982) Biochem. Biophys. Res. Commun. 109, 988-994.  Kay, E., Shannon, L.M. and Lew, J.Y. (1967) J. Biol. Chem. 242, 2470-2473.  Dunford, H.B. (1990) in Peroxidases in Chemistry and Biochemistry (Everse, J., Everse, K.E. and Graham, M., eds.), Vol. II, CRC Press, Boca Raton, FL.  Thorpe, G.H.G, Kricka, L.J., Mose|ey, S.B. and Whitehead, T.P. (1985) Clin. Chem. (Winston-Salem, NC) 31, 1335-1341.