Mechanism of Acetaminophen Oxidation by the Peroxidase-like

Chem. Res. Toxicol. 2009, 22, 1841–1850

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Mechanism of Acetaminophen Oxidation by the Peroxidase-like Activity of Methemoglobin Marıá I. Gonza´lez-Sańchez,† Marıá C. Manjabacas,‡ Francisco Garcıá-Carmona,§ and Edelmira Valero*,† Department of Physical Chemistry and Department of Applied Mechanics and Project Engineering, UniVersity of Castilla-La Mancha, Campus UniVersitario, E-02071-Albacete, Spain, and Department of Biochemistry and Molecular Biology A, UniVersity of Murcia, Campus de Espinardo, E-30100-Murcia, Spain ReceiVed July 24, 2009

Oxidation of acetaminophen by human methemoglobin in the presence of H2O2 has been kinetically studied in the present paper. The drug showed a protective effect against the H2O2-induced irreversible inactivation of the protein, thus indicating the competition among both ligands, H2O2 and acetaminophen for the protein. The stoichiometry of the reaction is variable and depends on relative initial concentrations of H2O2 and the drug owing to their competitive behavior. In addition and unexpectedly, the protein exhibits non Michaelian kinetics against both acetaminophen and H2O2 under steady-state conditions and shows negative co-operativity with Hill coefficients in the 0.3-0.7 range. Therefore, these data were compared to those obtained with myoglobin under similar experimental conditions, and the same results were observed. This led us to propose a mechanism for the peroxidase-like activity of hemoglobin, which accounts for the experimental results obtained herein. The steady-state rate equation for this mechanism has been obtained and is also consistent with the experimental data, thus indicating the goodness of the model proposed herein. The results presented in this work provide new insights into the oxidation mechanism of acetaminophen. Introduction 1

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is considered safe at therapeutic levels for humans with normal drug use. However, overdoses may lead to hepatic necrosis and renal damage in both humans and laboratory animals (1). Although a large percentage of the APAP dose is directly conjugated with glucuronic acid or sulfate and is excreted, a significant amount of APAP is metabolized not only by the cytochrome P450 system (2), but possibly by other oxidative peroxidase-like enzymes, such as prostaglandin H synthase (3), myeloperoxidase, chloroperoxidase, and lactoperoxidase (4). A general consensus exists as to N-acetyl-pbenzoquinone imine (NAPQI) being the main toxic metabolite formed by P450 bioactivation through a direct two-electron oxidation mechanism. However, the relevance of the radical species N-acetyl-p-benzosemiquinone imine (NAPSQI•) in the mechanism of the P450-dependent toxicity of the drug remains speculative (1). Another less-toxic metabolite is the corresponding catechol, 3′-hydroxyacetaminophen (5), the formation of which is mediated by a different form of P450 (6). In contrast, * To whom correspondence should be addressed. Phone: +34 967 59 92 00. Fax: +34 967 59 92 24. E-mail: [email protected]. † Department of Physical Chemistry, University of Castilla-La Mancha. ‡ Department of Applied Mechanics and Project Engineering, University of Castilla-La Mancha. § Department of Biochemistry and Molecular Biology A, University of Murcia. 1 Abbreviations: APAP, acetaminophen; NAPQI, N-acetyl-p-benzoquinone imine; NAPSQI•, N-acetyl-p-benzosemiquinone imine; Hb, hemoglobin; oxyHb, oxyhemoglobin; deoxyHb, deoxyhemoglobin; metHb, methemoglobin; oxoferrylHb, oxoferrylhemoglobin; ferrylHb, ferrylhemoglobin; CoI, compound I; CoII, compound II; ROS, reactive oxygen species; Mb, myoglobin; metMb, metmyoglobin; HRP, horseradish peroxidase; SOD, superoxide dismutase; DMSO, dimethyl sulfoxide; desferal, deferoxamine mesylate salt; Vss, steady-state rate; LC-ESI-MS, electrospray ionization mass spectrometry.

it is likely that peroxidase enzymes exhibit only one-electron oxidation activity through NAPSQI• under physiological conditions, as shown by ESR studies (7). Xanthine oxidase has been reported to catalyze the direct dimerization of two APAP molecules in the presence of H2O2, and neither NAPSQI• nor NAPQI acted as an intermediate in the reaction (8). More recently, tyrosinase from mushroom has been shown to be capable of oxidizing APAP to its corresponding o-quinone, 4-acetamido-o-benzoquinone (9), which thereafter evolves after a nucleophilic attack from either water or other agents present in the reaction medium, such as amino groups from amino acids (10). These studies have led to the development of a method for the quantitative determination of the drug in the presence of Besthorn’s hydrazone (11), and to a method for the enzymatic synthesis of the less toxic catecholic intermediate, 3′-hydroxyacetaminophen, using ascorbic acid in excess (12). Hemoglobin (Hb) is a tetrameric molecule composed of two R and two β subunits linked together, and each subunit contains a single heme group. The major heme protein of red blood cells is responsible for transporting molecular oxygen to tissues and shows positive allosteric co-operativity in its binding with O2. It has long since been known that Hb performs similar peroxidative reactions in the presence of H2O2 to a conventional peroxidase (13). The reaction of H2O2 with Fe(II) Hb (oxyHb and deoxyHb) and Fe(III) Hb (metHb) results in the formation of ferrylHb and oxoferrylHb, respectively, in which the iron in the heme is Fe(IV) (ferryl state) (14, 15). Both species are strong oxidizing agents and are considered the putative source of cellular and tissue damage (16, 17). Despite numerous reports on both APAP oxidation and the pseudoperoxidative activity of Hb, as far as we are aware, no studies into the oxidation of APAP catalyzed by this protein

10.1021/tx9002512 CCC: $40.75  2009 American Chemical Society Published on Web 10/12/2009

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have been reported to date. However, this may be a physiologically important matter when faced with an oxidative stress situation in which reactive oxygen species (ROS) (O2•-, H2O2, HO•) are generated; for example, in inflammation processes, aging and other disease states, including atherosclerosis, Parkinson’s Disease, and diabetes (18). It is also well-known that the redox cycling of drugs or other xenobiotics can generate ROS in red cells (19). Therefore, the aim of this work is to study the oxidation mechanism of APAP catalyzed by the pseudoperoxidase activity of metHb in the presence of H2O2. The data obtained in this work reveal, for the first time, a nonMichaelian behavior of the protein with negative co-operativity.

Gonza´lez-Sańchez et al. Scheme 1. Schematic Representation of a Possible APAP Oxidation Mechanism by metHb in the Presence of H2O2

Experimental Procedures Reagents. Human metHb, metmyoglobin (metMb) from equine skeletal muscle, catalase from bovine liver (1,700 units/mg), horseradish peroxidase (HRP) (5,000 units/mg), superoxide dismutase (SOD) from bovine erythrocytes (3,780 units/mg), APAP, EDTA disodium salt, ethanol, dimethyl sulfoxide (DMSO), thiourea, urea, deferoxamine mesylate salt (desferal), mannitol, sodium azide, sodium cyanide, trichloroacetic acid, ferrous ammonium sulfate, potasium thiocyanate, and sodium dithionite were obtained from Sigma Quı´mica (Madrid, Spain). Hydrogen peroxide 30% and L-histidine were obtained from Fluka. All the other reagents were of analytical grade and were used without further purification. Solutions were prepared with demineralized water which was purified with a Milli-Q purification system (18.2 MΩ cm) (Millipore Corp., Bedford, MA). Spectrophotometric Assays. Absorption spectra and kinetic analyses were recorded on a UV/vis Uvikon 940 (Konton Instruments, Zu¨rich, Switzerland) spectrophotometer. Temperature was controlled at 37 °C using a Hetofrig Selecta circulating bath and checked using a Checktemp 1 pocket digital thermometer obtained from Hanna Instruments with a resolution of 0.1 °C. The reaction medium contained APAP, H2O2, and metHb at the indicated concentrations in 50 mM sodium phosphate buffer (pH 7.0). The reaction was initiated by the addition of hydrogen peroxide at a final volume of 1 mL. The steady-state rate (Vss) was defined as the slope of the linear zone of the product accumulation curve. The H2O2 concentration in the stock solutions was spectrophotometrically determined at 240 nm using a molar extinction coefficient of 39.5 M-1 cm-1 (20). The H2O2 concentration in the reaction medium was titrated by the ferrous thiocyanate assay at different time intervals after the reaction had started (21). Reactions were stopped by the addition of trichloroacetic acid to obtain a final concentration of 1%. The precipitated protein was removed by centrifugation at 9,500 × g for 10 min and was filtered. The H2O2 level in the supernatant was determined by measuring the absorbance at 492 nm after reacting with 3.2 mM ferrous ammonium sulfate and 180 mM potassium thiocyanate in 0.1 M HCl. APAP concentration was spectrophotometrically determined at 240 nm using a molar extinction coefficient of 9.7 × 103 M-1 cm-1, which was calculated. Protein concentrations were also calculated spectrophotometrically with their respective molar extinction coefficients: metHb, ε ) 3.0 × 104 M-1 cm-1 at 522 nm (22), metMb, ε ) 3.3 × 103 M-1 cm-1 at 630 nm (23), and HRP, ε ) 1.02 × 105 M-1 cm-1 at 403 nm (24). The molar extinction coefficient for the reaction product at 315 nm was calculated by an end-point method. The product was generated by the chemical oxidation of APAP (2 mM) with different amounts of NaIO4 (40-400 µM). Aliquots of the reaction medium were injected into HPLC before adding the oxidant agent, as well as at the end of the reaction to check the stoichiometric conversion of APAP. The absorbance data obtained were fitted by least-squares linear regression to obtain ε315 ) 7.0 × 103 M-1 cm-1. HPLC Analysis. The biocatalytic oxidation of APAP in the presence of H2O2 was also followed in an Agilent Technologies (Waldbronn, Germany) HPLC system that was equipped with a series 1100 quaternary pump and vacuum degasser and with an

Agilent 1200 series diode array and a multiple wavelength detector SL. Separations were performed on a reversed-phase 5 µm Discovery C18 (15 cm × 4.6 mm) from Supelco (Madrid, Spain). Samples were filtered through a 0.45 µm filter prior to injection. Metabolites were eluted using a binary solvent system with a flow rate of 1.0 mL/min. Solvent A consisted in 2.8% (v/v) acetic acid in water, while solvent B was 100% methanol. After the HPLC column was equilibrated, a mixture of 95% solvent A and 5% solvent B was maintained for 7 min, followed by a linear gradient to obtain 85% solvent A and 15% solvent B in 2 min. This ratio was maintained for a further 15 min. Finally the 95:5 ratio was obtained again before the end of the chromatogram to equilibrate the column. The total sample analysis time, including equilibration and elution, was 35 min. Solvents A and B were previously filtered through a 0.22 µm filter and degassed by sonication in a Selecta Ultrasons water bath. The elution conditions were as follows: injection volume, 20 µL, and oven temperature, 30 °C. Elution was monitored at 250 nm. Calibration straight lines for APAP and its oxidation product were performed by duplicated injections of known amounts of them (0.1-3.0 mM and 40-400 µM, respectively). The reaction product was chemically synthesized by oxidating APAP with NaIO4 limitant, as described above, and was injected into HPLC at the end of the reaction (checked in the spectrophotometer until a constant absorbance was obtained). Peaks were identified by their retention times and corresponding spectra. An Agilent ChemStation B.03.01 revision was used to integrate the peak areas. Mass Spectrometry. An electrospray ionization mass spectrometry (LC-ESI/MS) analysis of samples was performed at different reaction times with an ion-trap mass spectrometer (LCQ Advantage; Thermo-Finnigan, San Jose, CA). The ion trap operated in the positive ionization mode. Mass data were acquired using the X-Calibur software, version 1.3 (Thermo-Finnigan) on the massto-charge (m/z) range 90-1000. This equipment was coupled to a Surveyor HPLC system with a diode array Surveyor detector. Separations were performed on two reversed phase 5 µm Discovery C18 (50 mm × 2.1 mm) from Supelco (Madrid, Spain) coupled in series. Samples were withdrawn from a reaction mixture at different times and stopped by the addition of trichloroacetic acid to obtain a final concentration of 5%. The precipitated protein was removed by centrifugation for 5 min at 10 000 × g, and the supernatant was filtered through a 0.45 µm filter prior to injection. The elution conditions were as follows: injection volume, 10 µL; flow rate, 200 µL/min; oven temperature, 30 °C. A binary solvent system was used for APAP oligomers separation, as described above. Polarographic Assays. Oxygen production was followed at 37 °C by a Hansatech oxygraph based on the Clark electrode, interfaced online with a PC-compatible computer. The electrode was calibrated by the use of sodium dithionite (Hansatech Instruments Ltd.). Steady-State Equations Derivation. The differential equations systems corresponding to the mechanisms shown in Schemes 1 and 2 were analytically solved under steady-state conditions with the TRAPHAER software (25), and by assuming that the only

Oxidation of Acetaminophen by Hemoglobin

Chem. Res. Toxicol., Vol. 22, No. 11, 2009 1843

Scheme 2. Proposed Mechanism for the Oxidation Reaction of APAP Catalyzed by the Peroxidase-like Activity of metHb in the Presence of H2O2

enzymatic species present at t ) 0 was metHb and that ligand species (APAP or H2O2) and the enzymatic species oxoferrylHb (Compound I, CoI) and ferrylHb (Compound II, CoII) were in rapid equilibrium with their respective enzyme-substrate complexes. Fitting Data. Steady-state rate data vs substrate concentration (Figure 7) were fitted by using the SigmaPlot Scientific Graphing Software for Windows, version 8.02 (2001, SPSS Inc.), to the Hill equation for three parameters

y)

axb c + xb b

(1)

with the following constraints: a and c > 0. These data were also fitted to a rational equation for five parameters (Figures 7A and C):

y)

a + bx + cx2 1 + dx + ex2

(2)

and to a rational equation for six parameters (Figures 7B and D)

y)

a + bx + cx2 1 + dx + ex2 + fx3

(3)

with the following constraints: a ) 0, b, c, d, e, and f > 0. The data corresponding to the formation of the ferryl state of Hb and its decay (curve 1 in Figures 6C, D and E) were fitted to the equation

y ) y0 + a

b (e-bt - e-ct) c-b

(4)

with the above-mentioned software and the following constraints: a, b, and c > 0. This equation corresponds to the time course of the ferryl state of Hb concentration by assuming first-order kinetics for its formation and decay, in agreement with the following mechanism: k+1

kd

metHb 98 (CoI f CoII) 98 MetHb + other Hb species (5)

Figure 1. Spectrophotometric recordings of the oxidation of APAP by metHb (A), metMb (B), and HRP (C) in the presence of hydrogen peroxide. The following experimental conditions were used: (A) [APAP]0 ) 3.0 mM, [H2O2]0 ) 3.0 mM, [metHb]0 ) 1.9 µM, scan speed was up to 1000 nm/min at 1 min intervals; (B) [APAP]0 ) 2.7 mM, [H2O2]0 ) 2.7 mM, [metMb]0 ) 4.1 µM, scan speed was up to 1000 nm/min at 1 min intervals; (C) [APAP]0 ) 0.4 mM, [H2O2]0 ) 0.5 mM, [HRP] ) 65.5 pM, scan speed was up to 2,000 nm/min at 24 s intervals. Insets show the same data plotted as difference spectra, constructed by subtracting spectrum 1 from the other spectra.

The parameters in eq 4 have the following meaning: y0 is the initial absorbance of the sample at 550 nm, a ) [metHb]0, b ) k+1, and c ) kd.

Results APAP oxidation by metHb in the presence of H2O2 was performed and changes in the UV/Visible spectrum were observed with time (Figure 1A). Maxima spectral changes were noted at 315 nm (increase in absorbance, see inset), as were two isosbectic points at 391 and 446 nm. These spectral characteristics at 315 nm were not observed in the absence of either APAP or H2O2 (data not shown) and can, therefore, be considered the result of drug oxidation by the pseudoperoxidase activity of Hb. These data were then compared to those obtained using metMb and HRP under the same experimental conditions (Figure 1B and 1C, respectively). Maxima spectral changes were also noted at 315 nm (increase in absorbance), thus indicating that the product formed by the three proteins could be the same. Biocatalytic APAP oxidation was also monitored by HPLC analysis. Figure 2 shows the results obtained when compared to those obtained by the chemical oxidation of APAP with NaIO4. The retention times and spectra of the products of the oxidation reaction of APAP by metHb (Figure 2A), metMb (Figure 2B), HRP (Figure 2C), and NaIO4 (Figure 2D) were the same, which indicates that these peaks should correspond to the same products. P1 was the major product of the oxidation reaction in the first minutes from the start of the reaction; taking into account its mass spectrum (Figure 2A, inset), it is an APAP

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Gonza´lez-Sańchez et al.

Figure 2. HPLC elution profile of the products formed by the oxidation of 2.0 mM APAP by metHb (A), metMb (B), HRP (C), and NaIO4 (D). The following experimental conditions were used: (A) [H2O2]0 ) 0.4 mM, [metHb]0 ) 3 µM; (B) [H2O2]0 ) 0.4 mM, [metMb]0 ) 5.3 µM; (C) [H2O2]0 ) 0.4 mM, [HRP] ) 0.26 nM; (D) [NaIO4]0 ) 4.0 mM. Samples A, B, and C were injected 4 min after the start of the reaction. Sample D was injected just after the start of the reaction. Insets in A, B, and C show the mass spectra of P1, P2, and P3, respectively. The inset in D shows the spectrum of the product P1.

Figure 3. Time course of the APAP oxidation reaction catalyzed by metHb, metMb and HRP. Experimental conditions as Figure 2.

dimer. Two other small peaks (P2 and P3) can be observed in the sample injected at this time reaction (4 min); they corresponded to an APAP dimer which differed from P1 (P2) and to an APAP trimer (P3), and their corresponding mass spectra can be seen in Figure 2B and C, insets. These data strongly suggest that the radical species NAPSQI• is the first product of the biocatalytic reaction in the three cases, which is in agreement with a peroxidase-like activity mechanism. This free radical is a highly reactive species which rapidly evolved to yield a dimer (P1) in the first minutes of the reaction. Afterward, other different oligomers also formed (data not shown). The time course of the APAP oxidation reaction was then measured at 315 nm in agreement with the spectral data above. Figure 3 shows the progress of the reaction catalyzed by metHb (curve i), metMb (curve ii) and HRP (curve iii), corresponding to the chromatograms shown in Figure 2. A lag period is observed in the product accumulation curves corresponding to

the APAP oxidation catalyzed by metHb and metMb. However, this induction period was not observed in the reaction catalyzed by HRP, despite the activity levels in all three cases being similar. This fact indicates that the lag period cannot be due to a time period that is necessary for the formation of the dimer P1 since it also formed in the reaction catalyzed by HRP, and this indicates that metHb and metMb catalyze the APAP oxidation by a different mechanism than HRP does. A steadystate phase lasting several minutes can also be seen in the three cases, which is in agreement with a major reaction product (P1) formed during this time. This fact also indicates that the inactivation of the three proteins by the action of H2O2 in the presence of the drug can be considered negligible in this time period. The steady-state was broken when the concentrations of other APAP-derived oligomers began to increase in the reaction medium, as verified by the HPLC analysis (data not shown). So all the measurements performed in the present paper were stopped at this point. The time course of the reaction between metHb and H2O2 in both the presence and absence of APAP was also followed by measuring the absorbance changes at the maximum wavelength of the So¨ret band (406 nm) (Figure 4). Decreased absorbance at the So¨ret band attained a near constant value in the presence of APAP (Figure 4, curve i), thus indicating a protective effect of the drug on the inactivation of Hb by H2O2 as previously reported for other substrates, such as the phenotiazine derivatives (26). To establish the stoichiometry of the pathway among APAP, H2O2, and metHb, the reaction was carried out until APAP or H2O2 was totally depleted in the reaction medium (Figure 5). The data obtained show a variable stoichiometry which depends on the initial reaction conditions: 1 H2O2/0.3 APAP for the data


Figure 4. Progress curves at 406 nm of the reaction of metHb with H2O2 in the presence (curve i) and absence (curve ii) of APAP (1 mM). Initial conditions were [H2O2]0 ) 1 mM and [metHb]0 ) 2.2 µM. The point on the ordinate axis represents the absorbance of metHb at 406 nm at this concentration in the absence of any reagent.

in Figure 5A and 1 H2O2/1.3 APAP for the data in Figure 5B. Once APAP had been depleted in the reaction medium, [H2O2] continued to decrease because of the inactivation of the protein by this reagent (Figure 5A). However, once H2O2 had been depleted in the reaction medium, APAP consumption was stopped (Figure 5B). The above results do not coincide with a classical peroxidase activity mechanism (Scheme 1) in which two molecules of the donor substrate (APAP in our case) were consumed by each molecule of H2O2. This fact also supports that metHb catalyzes the APAP oxidation by a different mechanism than HRP does. When Hb or Mb in the met form (i.e., in the Fe(III) heme state) reacts with either H2O2 or other peroxides, then Fe(III) oxidizes to Fe(IV). This one-electron oxidation takes place with H2O2 reduction to water, the latter being a two-electron process. The second electron participating in the H2O2 reduction comes from a tyrosine residue in the globin chain (27), which left it in the free radical state. Immediately afterward, this intermediate, the so-called CoI by analogy with peroxidases, withdraws one electron from a donor substrate to form a more stable species without the protein radical, the so-called CoII. CoI and CoII of Hb are spectrophotometrically indistinguishable (16). Figure 6 shows the results obtained from repetitive spectral differential analysis of the reaction of metHb with H2O2 in the absence (A) and presence (B) of APAP, respectively. The maxima spectral differences between Hb in the ferryl state and metHb may be seen at 494, 557, 589, and 634 nm (Figures 6A and 6B, curve 1). In the absence of the phenolic drug, the decay of the ferryl state of Hb showed three isosbectic points at 462, 525, and 611 nm, along with maxima spectral differences at 478, 551, 583, and 634 nm (curves 2-13). Figure 6B shows a faster decay when APAP was added to the reaction medium. Then the isosbectic points were observed at 459, 521, and 606 nm, and the maxima spectral differences appeared at 490, 550, 575, and 633 nm (curves 2-13). No significant increase in absorbance at 700 nm was observed during the assay time, even at the higher H2O2 initial concentrations. Figure 6C, D, and E not only show the time course of the reaction between metHb and H2O2 measured at 550 nm (curve 1 in each case) but also the effect of adding catalase and APAP to the reaction medium at different times, as indicated by the arrows. This wavelength was chosen after considering the previously obtained spectral differences. Other authors measured this reaction at 543 nm (22, 26) since Hb in the ferryl state displays an absorption maximum at this wavelength. However, the experiments performed at this wavelength in this work (data not shown) showed a lot of noise due to the excessively small


differences in absorbance observed in the corresponding spectra (see Figures 6A and B). For this reason, we decided to measure at 550 nm in agreement with the previously shown spectral differences. The rate constants for the formation and decay of the ferryl state of Hb can be obtained from the data in curves 1 (Figures 6C, D, and E), as indicated in Materials and Methods. The values obtained were k+1 ) 1.97 min-1 and kd ) 0.026 min-1. As these experiments indicate, the addition of catalase caused the ferryl state of Hb to decay faster (Figure 6C), while the addition of APAP accelerated this process (Figure 6D). The addition of catalase at more than 25 min after the reaction had started did not affect the decay of the ferryl state of Hb since H2O2 had been totally consumed, which was checked by the titration of this reagent (data not shown). The decay rate depended on the APAP concentration, which became slower as the concentration of APAP decreased (Figure 6D, curves a, b). Figure 6E shows a more marked decay of the ferryl state of Hb induced by APAP once H2O2 had been totally consumed by the addition of catalase. When an aliquot from the reaction media corresponding to curves 2a, b, and c (Figure 6E) was injected into the HPLC apparatus, a slight oxidation of APAP was detected (2-6 µM), and a small peak with the same retention time and spectrum as P1 also appeared (data not shown). This fact indicates that a small amount of Hb in the ferryl state remaining in the reaction medium was able to withdraw electrons from the drug. However, the reaction was unable to progress since H2O2 had been depleted in the medium by the addition of catalase. Therefore, the formation of new CoI molecules is not possible under these conditions. This, in turn, indicates that the presence of H2O2 in the reaction medium is essential for the oxidation reaction to progress. The experiments performed under limiting H2O2 concentrations, with an excess of APAP until the end of the reaction by depleting the limiting reagent, confirm these data when the chemical composition of the reaction media was analyzed by HPLC since no further accumulation of the reaction product was observed once H2O2 had been consumed (data not shown). To study the kinetic behavior of metHb toward APAP in depth, several experiments were performed at different initial APAP and H2O2 concentrations, and the steady-state rates obtained were plotted in a double reciprocal way (Figure 7A and B). It is easy to observe a deviation from the linear behavior predicted by the classical peroxidase mechanism (Scheme 1). The equation obtained for the accumulation of the reaction product under steady-state conditions for this reaction Scheme is a rate equation of degree 1:1 (see Appendix, eq A1). The double reciprocal plots obtained after varying the concentration of one ligand species (APAP or H2O2) while maintaining the concentration of the other constant should be straight lines. An extensive review of the scientific literature on the pseudoperoxidase activity of Hb toward different substrates only revealed a similar kinetic response of the protein toward styrene (28). In this case, complex kinetics were attributed to the presence of two potential hemoprotein catalysts (the R- and β-chains of Hb) and also to the loss of the prosthetic heme chromophore in the presence of H2O2. In the case of APAP as an electron donor for metHb-catalyzed oxidation, however, a steady state lasting several minutes (Figure 3, curve i) was reached after a very small transient phase, which indicates that protein inactivation may be considered negligible during the assay time. To explore the possibility of the non-Michaelian behavior being caused by the tetrameric structure of Hb, these experi-

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Figure 5. APAP (b) and H2O2 (O) consumption in the reaction with metHb. Initial conditions were as follows: (A) [H2O2]0 ) 0.4 mM, [APAP]0 ) 0.1 mM, [metHb]0 ) 4.7 µM; (B) [H2O2]0 ) 0.4 mM, [APAP]0 ) 1.2 mM, [metHb]0 ) 3.3 µM.

Figure 6. Spectral analysis of the reaction of metHb with H2O2 in the absence and presence of APAP. The spectrum obtained 1 min after the addition of 100 µM H2O2 has been subtracted from all the spectra to obtain maxima differences. (A) Spectrum 1, 10 µM metHb; spectra 2-13 were taken at 2 min after the addition of 100 µM H2O2 at 1 min intervals. Scan speed was up to 1000 nm/min. (B) Spectrum 1, 10 µM metHb; spectra 2-13 were taken at 0, 25, 50, 75, 100, 125, 150, 175, 225, 250, 275, and 325 s after the addition of 100 µM APAP, which was added 90 s after the addition of 100 µM H2O2. Scan speed was up to 1000 nm/min. (C) Curve 1, time course of the reaction of 10 µM metHb and 100 µM H2O2. The rest of curves in the graph represent the decay of the ferryl state of Hb by the addition of 43 U of catalase at the times indicated by the arrows. (D) Curve 1 as in C. The rest of curves in the graph represent the decay of the ferryl state of Hb by the addition of 50 µM APAP at the times indicated by the arrows (solid lines). Dashed lines correspond to the addition of APAP to provide final concentrations of 10 and 30 µM for curves a and b, respectively, 2 min after the addition of H2O2. (E) Curve 1 as in C; curve 2, decay of the ferryl state of Hb by the addition of 43 U of catalase 1.5 min after the addition of H2O2; curves a, b and c, decay of the ferryl state of Hb by the addition of 50 µM APAP at 1.0, 3.5, and 8.5 min (indicated by the arrows) after the addition of catalase.

ments were also performed under the same experimental conditions with metMb. This is a monomeric protein that also shows pseudoperoxidative activity in the presence of H2O2. As Figure 7C and D reveals, similar results were obtained, indicating that the negative co-operativity shown by metHb toward APAP and H2O2 does not correlate with either its tetrameric nature or the presence of subunits R and β, and should, therefore, be interpreted as kinetic co-operativity. All these data were then fitted to the Hill equation by nonlinear regression, and very good regression coefficients were obtained. Table 1 provides the Hill coefficients obtained, which increased with the initial H2O2 concentration and decreased with the initial APAP concentration to obtain a parallel response with metMb. These overall results indicate that both Hb and Mb show

a very strong negative kinetic co-operativity toward APAP and H2O2 (the Hill coefficients were as low as 0.31), which is an unusual kinetic behavior of the peroxidase-like activity of both proteins. The extent of co-operativity increases the higher the ratio is [APAP]/[H2O2]. However, even though the Hill equation proves useful to determine the extent of co-operativity, it cannot explain the mechanism by which this phenomenon was observed. A variety of test agents was used to further evaluate the APAP oxidation mechanism catalyzed by the peroxidase-like activity of metHb in the presence of H2O2 (Table 2). Hydroxyl radical scavengers, such as DMSO, mannitol, desferal, and thiourea, barely affected APAP oxidation, indicating that the hydroxyl radical was not involved in the Hb-mediated APAP oxidation. Urea (which is not a hydroxyl radical scavenger) was used as



Figure 7. Double reciprocal plots of steady-state rates against initial concentrations of APAP and H2O2 for the oxidation reaction catalyzed by metHb (A and B) and metMb (C and D). Experimental conditions: (A) the initial H2O2 concentrations used were (b) 0.4, (O) 1.2, (1) 2.0, (∇) 4.0, and (9) 6.0 mM, where [metHb]0 ) 1.1 µM; (B) the initial APAP concentrations used were (b) 0.5, (O) 1.0, (1) 2.0, (∇) 4.0, and (9) 6.0 mM, where [metHb]0 ) 1.1 µM; (C) the initial H2O2 concentrations used were (b) 0.5, (O) 1.0, (1) 2.0, (∇) 4.0, and (9) 6.0 mM, where [metMb]0 ) 7.2 µM; (D) the initial APAP concentrations used were (b) 0.5, (O) 1.0, (1) 2.0, (∇) 4.0, and (9) 6.0 mM, where [metMb]0 ) 8.4 µM. The points correspond to experimental data, while the lines correspond to the data obtained by the nonlinear regression analysis to eqs 2 (Figure 7A and C) and 3 (Figure 7B and D).

Table 1. Hill Coefficients for the Oxidation Reaction of APAP Catalyzed by metHb and metMb in the Presence of H2O2 [H2O2]0 (mM)

h (Figure 7A)

h (Figure 7C)

[APAP]0 (mM)

h (Figure 7B)

h (Figure 7D)

0.4 1.2 2.0 4.0 6.0

0.3341 0.3373 0.4010 0.6079 0.6082

0.4751 0.5353 0.6072 0.6080 0.6102

0.5 1.0 2.0 4.0 6.0

0.7270 0.6071 0.5329 0.5047 0.4522

0.3501 0.3469 0.3292 0.3250 0.3077

a control; no effect was observed on the steady-state rate of the oxidation reaction. However, another hydroxyl radical scavenger, ethanol, showed an inhibitory action on the steady-state product accumulation rate, which was likely the result of a nonspecific inactivation effect on the protein. L-Histidine, a free amino acid, which has been demonstrated to have an antioxidative action as an 1O2 scavenger and to be a chelator of metal ions (29), only had a slight inhibition effect on the oxidation reaction rate under study. SOD did not affect the product accumulation rate. The complete inhibition observed when catalase was included in the reaction medium indicates that H2O2 needs to be present for drug oxidation, as previously mentioned. EDTA did not affect APAP oxidation at all, indicating that free iron ions were not released from Hb during the reaction. Cyanide and azide are well-known heme-binding ligands that inhibit peroxidases which strongly inhibited the APAP oxidation reaction. Finally, a set of experiments was performed in an oxygraph in the presence and absence of APAP and SOD by measuring the variation in O2 concentration during the reaction. The

reaction of metHb with H2O2, as reported earlier (28), results in the formation of molecular oxygen (Figure 8, curve i). O2 production in the reaction medium was higher in the presence of SOD (curve ii), indicating the presence of O2•-. Despite the signal decreasing when APAP was included in the reaction medium (curve iii), the signal increased in the presence of SOD (curve iv). In these experiments, it is also possible to observe a decrease in oxygen production when APAP was added to the reaction medium (curve ii; the addition of APAP is indicated by the arrow).

Discussion This paper shows that human metHb is able to oxidize APAP in the presence of H2O2 very likely via one-electron similarly to HRP, and that the radical species NAPSQI• is probably the first product obtained from the biocatalytic process, which rapidly dimerizes to yield P1. Upon reaction with H2O2, metHb transforms into CoI. In addition, denatured Hb species finally form. However, the

1848



Table 2. Effect of Different Test Agents on APAP Oxidation by metHb in the Presence of H2O2a reagent added none (standard reaction) DMSO mannitol desferal thiourea urea ethanol L-histidine

SOD catalase EDTA sodium cyanide sodium azide

initial concentration

% standard reaction

5.0% 50.0 mM 250.0 mM 1.0 mM 1.0 mM 25.0 mM 12.0 mM 100.0 mM 2.5% 5.0% 1.0 mM 6.0 mM 18.6 U/mL 60.0 U/mL 1.0 mM 1.0 mM 2.0 mM 0.3 mM 0.75 mM 2.0 mM

100.0 92.4 100.9 99.5 110.5 98.3 102.5 100.9 99.8 73.9 66.5 96.0 93.5 100.1 0.0 98.5 4.5 2.9 32.0 21.1 13.6

a Experimental conditions were the following: [APAP]0 ) 2 mM, [H2O2]0 ) 0.4 mM, [metHb]0 ) 3.1 µM.

Figure 8. Effect of the addition of SOD and/or APAP on the oxygen production of a reaction mixture of metHb and H2O2. Initial conditions were the following: [metHb]0 ) 3.5 µM, [H2O2]0 ) 4 mM for all of the curves, [SOD] ) 45 U/ml for curves ii and iv and [APAP]0 ) 2 mM for curves iii and iv. The arrow in curve ii indicates the addition of APAP to obtain a final concentration of 2 mM.

presence of H2O2 is essential for the peroxidase-like activity of Hb to progress, even if CoI is still present in the reaction medium. The addition of APAP to Hb in the ferryl state without H2O2 being available induces a fast decay of the ferryl state of Hb (Figures 6A and B), which is dependent on the initial APAP concentration (Figures 6D and E). Nonetheless, a very small APAP transformation may be observed under this condition which progresses no further. The maximum concentration of the ferryl state of Hb in these experiments takes 1.7 min (Figures 6C-E, curve 1). This time period necessary for the formation of the ferryl state of Hb may explain the lag period observed in the progress curves of the peroxidative reaction of APAP (Figure 3). Our kinetic results reveal, for the first time, a very strong deviation from classical Michaelis-Menten kinetics of the peroxidase-like activity of metHb and metMb against both H2O2 and APAP, whose Hill coefficients are in the 0.3-0.7 range (Figure 7). The experimental data obtained in this work indicate the following: this fact cannot be (a) an artifact of the spectrophotometric method to measure the reaction progress since P1 is the major product of the oxidation reaction during the assay time; (b) because of the inactivation of the protein

since a very clear steady-state phase that lasts several minutes is seen in the progress curves; (c) because of the tetrameric nature of Hb since Mb shows a similar response. All these experimental observations lead us to believe that this phenomenon should be interpreted as kinetic co-operativity. Mechanistic kinetic co-operativity in a monomeric or oligomeric enzyme may occur in several ways (30, 31). This type of co-operativity is best considered in relation to deviations from Michaelis-Menten kinetics, and it arises as a result of the occurrence of several closed loops involving the substrate(s) in the reaction mechanism, that is, in principle, any mechanism in which the binding of a substrate can occur in two or more parallel pathways will generate a rate equation containing highorder terms at the concentration of that substrate. The data obtained in the present study show that APAP has a protective effect against the H2O2-induced inactivation of Hb (Figure 4). In addition, the stoichiometry of the APAP oxidation reaction catalyzed by Hb in the presence of H2O2 is variable and depends on the ratio of the initial APAP/H2O2 concentrations (Figure 5). These results indicate that APAP and H2O2 are competitive substrates for the same form of the protein, that is, H2O2 can also act as an electron donor for Hb molecule. The competition between H2O2 and APAP for the protein is also clearly noted when measuring O2 production since the addition of APAP leads to a decrease in the signal (Figure 8, curves ii and iii). The decay of the globin radical in CoI proceeds through mechanisms that have not yet been conclusively identified. In the absence of other reductants, the ferryl moiety, and possibly the globin radical, can oxidize another H2O2 molecule to O2•(eq 6), which consequently yields molecular oxygen by spontaneous dismutation. Moreover, CoII can be reduced to metHb by reacting with H2O2 (slow stage), which is oxidized to O2•(eq 7) (32, 33): + CoI + H2O2 f CoII + O•2 + 2H

(6)

+ CoII + H2O2 f MetHb + O•2 + 2H

(7)

Therefore, by taking into account this background and the competition observed between H2O2 and APAP, the mechanism that we propose for the metHb-catalyzed oxidation of APAP in the presence of H2O2 is shown in Scheme 2. This mechanism also accounts for the kinetic co-operativity obtained herein. In this mechanism, competition takes place between H2O2 and APAP for the oxoferryl (CoI) and ferryl (CoII) forms of the protein which, in turn, gives rise to the existence of three closed loops in the mechanism. The relative contribution of each route within the process depends on the values of the individual rate constants and initial conditions. This is the reason why the stoichiometry of the reaction is variable and depends on the initial ratio [APAP]/[H2O2], and it also explains the protective effect shown by APAP toward the H2O2-induced inactivation of Hb. The lag period which appears in the P1 progress curves (Figure 3) is not only a possible consequence of the time required by CoI and CoII to form in the reaction medium but also indicates a transient from a slow catalytic cycle to another relatively faster one (34). If we take into account the APAPinduced faster decay of the ferryl state of Hb (Figure 6), then k+5 is higher than k+9 (Scheme 2), which is in agreement with the reduction of CoII to metHb in the presence of H2O2 (eq 7) being the slow stage in the reaction of metHb with H2O2 (32, 33).



The steady-state rate equation of the mechanism indicated in Scheme 2 is shown in the Appendix (eq A9). It is a rational polynomial equation of 2:2 degree for APAP (two molecules of APAP enter the mechanism) and of 2:3 degree for H2O2 (three molecules of H2O2 enter the mechanism). This equation is in agreement with our kinetic results in which the peroxidase-like activity of Hb shows non-Michaelian behavior against both ligands, H2O2 and APAP (Figure 7). Given their role in oxygen transport and the presence of redox active Hb molecules, red blood cells generate relatively high levels of ROS (19). To counteract the potential deleterious effects of ROS, red blood cells have a well integrated network of antioxidant mechanisms to combat this oxidative stress. However, this system may be perturbed, for example, in sickle cell disease (35), acute inflammation processes, aging, arteriosclerosis, Parkinson’s Disease, diabetes, Alzheimer’s Disease, etc. There are numerous mechanisms by which oxyHb can be transformed into its non functional form, metHb: exposure of red cells to certain drugs or toxins, a deficit of antioxidant agents or genetic abnormalities, etc. All in all, a small percentage of Hb is always auto-oxidized, even under normal physiological conditions, to result in the presence of metHb in blood (36). Under oxidative stress conditions, metHb may act as a peroxidase since H2O2 levels increase and may oxidize some drugs, for example, APAP, depending on the permeability of the erythrocyte membrane toward the target molecule. In fact, the formation of reactive free radicals derived from the respective xenobiotic and a ferryl-heme oxo-complex has been shown in erythrocytes incubated with xenobiotics agents, such as hydroxylamines or phenols (37). The results here obtained show that the NAPSQI• free radical can be formed in blood and may contribute to the knowledge of APAP oxidation mechanisms. In addition, our data show clearly that the oxidation of APAP by metHb cannot be described by classical Michaelis-Menten kinetics. This is a complex response of metHb that might contribute to explain some of the results obtained for studies of oxidation of other compounds by metHb in the presence of H2O2. Acknowledgment. This work was supported by Projects PAI05-036 and PAI08-0175-8618 from the Consejerıá de Educacioń y Ciencia de la Junta de Comunidades de Castilla-La Mancha (JCCM, Spain). M.I.G.-S. has a predoctoral fellowship from JCCM associated with the first project. The authors wish to acknowledge Dr. I. Pe´rez of the Department of Molecular Biology and Biochemical Engineering at the University of Pablo de Olavide (Seville, Spain) for his expert technical assistance with the LC-ESI/MS equipment.

a2 ) k-1K2k+5

(A3)

a3 ) k+3k+5

(A4)

a4 ) k+1(K2k+5 + K4k+3)

(A5)

a5 ) k+1(k+3 + k+5)

(A6)

K2 ) k-2 /k+2

(A7)

K4 ) k-4 /k+4

(A8)

and

Steady-state rate for the accumulation of the reaction product (NAPSQI•) in Scheme 2:

Vss ) [Hb]0 b1[H][A]2 + b2[H]2[A] b3[H] + b4[A] + b5[H][A] + b6[H]2 + b7[A]2 +

(A9)

b8[H][A]2 + b9[H]2[A] + b10[H]3 where A and H correspond to APAP and H2O2, respectively. Parameters bi (i ) 1-10) have the following meaning:

b1 ) 2k+1k+3k+5K6K8

(A10)

b2 ) k+1(k+3K4K6k+9 + K2k+5k+7K8)

(A11)

b3 ) k-1K2K4K6k+9

(A12)

b4 ) k-1K2k+5K6K8

(A13)

b5 ) k+3K4K6k+9 + K2k+5k+7K8 + k+1K6K8(K2k+5 + K4k+3)

(A14)

b6 ) K2K4(k+7k+9 + k+1K6k+9 + k+1k+7K8) (A15) b7 ) k+3k+5K6K8

(A16)

b8 ) k+1K6K8(k+3 + k+5)

(A17)

b9 ) k+1(K4K6k+9 + K2k+7K8 + K2k+5K8 + k+3K4K6) (A18) b10 ) k+1K2K4(k+9 + k+7)

(A19)

K6 ) k-6 /k+6

(A20)

K8 ) k-8 /k+8

(A21)

where

Appendix Steady-state rate for the accumulation of the reaction product (NAPSQI•) in Scheme 1:

Vss ) 2[Hb]0

References

a1[H2O2][APAP] a2 + a3[APAP] + a4[H2O2] + a5[H2O2][APAP]

(A1)

where

a1 ) k+1k+3k+5

(A2)

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TX9002512

Mechanism of Acetaminophen Oxidation by the Peroxidase-like

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