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Peroxidase-catalyzed oxidation of capsaicinoids: steady-state and transient-state kinetic studies
ABB Archives of Biochemistry and Biophysics 417 (2003) 18–26 www.elsevier.com/locate/yabbi
Peroxidase-catalyzed oxidation of capsaicinoids: steady-state and transient-state kinetic studies Douglas C. Goodwin* and Kristen M. Hertwig Department of Chemistry and Program in Cell and Molecular Biosciences, Auburn University, Auburn, AL 36849-5312, USA Received 12 May 2003, and in revised form 17 June 2003
Abstract Capsaicinoids are the pungent compounds in Capsicum fruits (i.e., ‘‘hot’’ peppers). Peroxidases catalyze capsaicinoid oxidation and may play a central role in their metabolism. However, key kinetic aspects of peroxidase-catalyzed capsaicinoid oxidation remain unresolved. Using transient-state methods, we evaluated horseradish peroxidase compound I and II reduction by two prominent capsaicinoids (25 °C, pH 7.0). We determined rate constants approaching 2 107 and 5 105 M1 s1 for compound I and compound II reduction, respectively. We also determined kapp values for steady-state capsaicinoid oxidation approaching 8 105 M1 s1 (25 °C, pH 7.0). Accounting for stoichiometry, these are in excellent agreement with constants for compound II reduction, suggesting that this reaction governs capsaicinoid-dependent peroxidase turnover. Ascorbate rapidly reduced capsaicinoid radicals, assisting in the determination of the kinetic constants reported. Because ascorbate accumulates in Capsicum fruits, it may also be an important determinant for capsaicinoid content and preservation in Capsicum fruits and related products. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Capsaicin; Ferryl-oxo; Compound I; Compound II; Ascorbate; Nonivamide; Capsicum; N-Vanillylnonanamide; Free radical; Peroxidase
Capsaicinoids impart the pungent flavor of Capsicum ‘‘hot’’ pepper fruits. The most abundant capsaicinoids are capsaicin and dihydrocapsaicin (Fig. 1), typically comprising 80% of the total capsaicinoid content in Capsicum fruits [1]. Other capsaicinoids have been identified in much smaller quantities, including nordihydrocapsaicin, homocapsaicin, homodihydrocapsaicin, and nonivamide. Indeed, nonivamide, also called ‘‘synthetic capsaicin,’’ has recently been identified as a natural product [2,3]. Capsaicinoids are present in Capsicum fruits at levels up to 1% [4]. Consequently, many varieties of peppers are widely used in commercial and domestic food preparation. Indeed, over a quarter of the worldÕs population consumes hot peppers in some form on a daily basis [5]. Clearly, Capsicum fruits are important agricultural commodities with prominent ethnic and cultural significance. The desirability of these peppers for * Corresponding author. Fax: 1-334-844-6959. E-mail address: [email protected] (D.C. Goodwin).
0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0003-9861(03)00321-7
food preparation depends greatly on their capsaicin content. Pungency and the general nociceptive effects of capsaicinoids result from their interaction with the vanilloid receptor type 1, a non-selective ion channel that opens in response to noxious heat (>48 °C) [6,7]. Capsaicinoids lower the temperature threshold of the receptor, leading to gating of the channel at room temperature [6]. This is followed by a refractory period in which there is neuronal resistance not only to additional capsaicinoid exposure, but also to other painful stimuli [5]. This forms the basis for the extensive use of capsaicinoid-based topical agents for the treatment of acute (e.g., muscle fatigue and soreness) or chronic pain (e.g., diabetic neuropathy, fibromyalgia, or rheumatoid arthritis) [5,8,9]. At high doses, capsaicinoids can cause respiratory depression in mammalian systems [10–13]. They are also neurotoxic, preventing the intra-axonal transport of nerve growth factor in neonatal dorsal root ganglionic neurons [14]. The capsaicinoids have also been evaluated
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compound I, leaving the ferryl-oxo intermediate compound II and one equivalent of substrate radical (A ) (reaction (2)). A second equivalent of AH reduces compound II to ferric peroxidase, yielding a second equivalent of A and a second equivalent of H2 O (reaction (3)) [26]. ð1Þ ð2Þ ð3Þ
Fig. 1. Diagram representing the structures of some common capsaicinoids as well as the generation and subsequent reactions of capsaicinoid radicals. Capsaicin and dihydrocapsaicin are the most abundant naturally occurring capsaicinoids. Nonivamide is observed at low levels in natural sources but is widely available as a synthetic preparation.
for their potential as anticarcinogens, co-carcinogens, or carcinogens. However, data in this regard remain inconclusive [4]. Clearly, the metabolism of capsaicinoids has important ramifications. Peroxidases and other hemoproteins (e.g., cytochromes P450) occupy central roles in capsaicinoid catabolism [15–18]. In the maturing pepper fruits of Capsicum annuum cultivars, the degradation of capsaicinoids has been correlated with a rise in peroxidase activity [19]. Plant peroxidases catalyze capsaicinoid oxidation [20–24] leading to polymerization to form lignin-like products [25]. Two dimeric species, 5,50 dicapsaicin and 40 -O-5-dicapsaicin ether, have been identified, and further oxidation results in the formation of high molecular weight polymers [25] (Fig. 1). In the typical catalytic cycle, H2 O2 oxidizes ferric peroxidase (also called resting or native enzyme) by two electrons to form a ferryl-oxo porphyrin radical intermediate (compound I) (reaction (1)). An exogenous reducing substrate (AH) reduces the porphyrin radical of
Although capsaicinoids are known to be reducing substrates for plant peroxidases (horseradish and C. annuum) [21–24], several kinetic aspects of the process remain poorly defined. The reactions of peroxidase compounds I and II with capsaicinoids have not been directly measured. Furthermore, a broad range of polymeric products (dimers, multimers, and protein-capsaicinoid copolymers) are formed (each product with its own absorption characteristics), making a meaningful correspondence between product formation and direct interaction between the peroxidase and the capsaicinoid substrate difficult to establish. This has hampered the assignment of steady-state parameters for capsaicinoid oxidation by peroxidases. To help address these deficiencies, we evaluated the reactions of the archetypal plant peroxidase (horseradish peroxidase) with two typical capsaicinoids (capsaicin and nonivamide) by transient-state and steady-state kinetic methods. To avoid problems that have hindered these types of studies, we used the properties of ascorbate as an efficient reductant of phenoxyl radicals. For our transient-state kinetic studies, ascorbate maintained pseudo-first order conditions even with very low concentrations of capsaicinoid, allowing for the effective determination of rate constants for the very rapid reduction of peroxidase compound I. For our steady-state kinetic studies, ascorbate allowed us to forgo problematic spectrophotometric procedures in favor of a chronometric method. As a result, we have determined rate constants for the direct reaction of capsaicinoids with peroxidase compounds I and II and apparent second-order rate constants for oxidation of capsaicinoids by peroxidase. Our studies have also revealed that peroxidase-generated capsaicinoid radicals are very rapidly reduced by ascorbate, preventing polymerization. These data provide valuable information for understanding the metabolism of capsaicinoids by peroxidases and the potential influence that antioxidants like ascorbate may have in that process. Thus, these results may have significant implications because of the great cultural,
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agricultural, and neuropharamcological importance of the capsaicinoids.
Materials and methods Materials Hydrogen peroxide (30%), capsaicin, nonivamide, peroxidase (Type VI), and ascorbate (ACS grade) were purchased from Sigma (St. Louis, MO). All buffers and media were prepared using Millipore (Q-PakII)-purified water (18.2 MX/cm resistivity). Concentrations of H2 O2 and peroxidase were determined spectrophotometrically at 240 nm (e ¼ 39:4 M1 cm1 ) [27] and 403 nm (e ¼ 102,000 M1 cm1 ) [26], respectively. Transient-state kinetics The reactions of peroxidase compounds I and II with capsaicinoids were monitored using an SX.18MV stopped-flow spectrophotometer (Applied Photophysics, Surrey, UK) in sequential-mixing mode. All reactions were carried out at pH 7.0 and 25 °C. To obtain spectra, peroxidase compound I was formed by pre-mixing equal volumes of 8 lM peroxidase with 7.6 lM H2 O2 in a delay line. After a 100 ms delay, the contents of the delay line were mixed with 4 lM capsaicinoid and reaction progress was monitored by diode array detector. Final reactions contained 2 lM peroxidase compound I, 2 lM capsaicinoid, and 50 mM phosphate buffer, pH 7.0. For some measurements, ascorbate was used to maintain pseudo-first-order conditions with respect to capsaicinoid. For these reactions, the 4 lM capsaicinoid solution also contained 100 lM ascorbate, giving a final ascorbate concentration of 50 lM. Spectra for compound II reduction to ferric enzyme were collected by first forming compound I as above. Compound I was then mixed with an equal volume of 20 lM capsaicinoid and spectra were collected by diode array. Following the very rapid conversion of compound I to compound II (<40 ms), compound II reduction to ferric enzyme was recorded. To obtain rate constants for compound I reduction, ferric peroxidase (4 lM) was pre-mixed with an equal volume of 3.8 lM H2 O2 . Following a 200 ms delay, this solution was mixed with an equal volume of 100 lM ascorbate and 0.4–40 lM capsaicin or nonivamide. Ascorbate was added to ensure pseudo-first-order conditions with respect to capsaicinoid [28]. The final concentrations of peroxidase, ascorbate, and phosphate buffer, pH 7.0 were 1 lM (predominantly compound I), 50 lM, and 50 mM, respectively. Final concentrations of capsaicinoid ranged from 0.2 to 20 lM. Reduction of compound I was measured at 411 nm, an isosbestic wavelength for compound II and ferric peroxidase [26].
Rate constants for compound II reduction were obtained by the same procedure except that higher concentrations of capsaicinoid were present (20– 400 lM) and the reactions were monitored at 428 nm, an isosbestic wavelength for ferric peroxidase and compound I [26]. Due to the much higher concentrations of capsaicinoid in these experiments, addition of ascorbate was not required to maintain psuedofirst-order conditions. Steady-state kinetics Steady-state capsaicinoid oxidation was evaluated using our stopped-flow instrument in single-mixing mode. One syringe contained peroxidase (100 nM) in 200 mM phosphate buffer, pH 7.0. The second syringe contained 500 lM H2 O2 , varying concentrations of capsaicinoid, and, when appropriate, varying concentrations of ascorbate. Spectra were recorded by diode array and rate constants were determined from singlewavelength measurements. All reactions were carried out at pH 7.0 and 25 °C. Capsaicinoid oxidation by peroxidase leads to a series of dimer and polymer products [25], and corresponding increase in absorbance across the UV–visible spectrum [21], complicating the estimation of steady-state kinetic parameters for these reactions. To circumvent this problem, we used a chronometric method [29], including ascorbate in reactions to rapidly reduce peroxidase-derived radicals. Ascorbate efficiently prevented free-radical polymerization, giving rise to a pronounced lag in the appearance of capsaicinoid oxidation products. Practically, complete consumption of ascorbate had to occur before any polymerized products were observed. Thus, the lag time (s) was used to calculate the initial rate (v0 ) for capsaicin oxidation by peroxidase: v0 ¼ 2½ascorbate =s:
ð1Þ
Rodrıguez-L opez and coworkers point out that ascorbate can be consumed in a reaction with H2 O2 (k ¼ 1:03 M1 s1 ). The initial rate can be corrected for these effects, where k is the rate constant for reaction between ascorbate and H2 O2 [29], v0 ¼ f2½ascorbate ð1 sk½H2 O2 Þg=s:
ð2Þ
For each concentration of capsaicinoid, a series of s values was obtained corresponding to a series of ascorbate concentrations. Dividing each ascorbate concentration by the corresponding s produced a set of v0 values that was independent of the concentration of ascorbate. From these, an average v0 was determined for each capsaicinoid concentration. Each v0 was divided by the concentration of enzyme present and these v0 =½E T values were plotted against the concentration of capsaicinoid to obtain apparent second-order rate constants.
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Results and discussion Transient-state kinetics Reduction of peroxidase compounds I and II by capsaicinoids has been assumed, but the reactions have not been directly observed nor the rate constants determined. We used sequential mixing stopped-flow methods to evaluate these reactions. Reaction of peroxidase compound I with equimolar capsaicin resulted in a rapid shift in absorption maximum from 403 to 418 nm with an isosbestic point near 398 nm. New absorption peaks were also detected at 529 and 557 nm (data not shown). Nearly identical results were obtained when compound I was reacted with equimolar capsaicin and 50 lM ascorbate (Fig. 2). Whether collected in the presence or absence of ascorbate, spectra were consistent with the conversion of peroxidase compound I to compound II. Both reactions occurred roughly within the same time frame, but single exponential behavior was observed only in the presence of ascorbate (Fig. 3). The same results (spectral and kinetic) were observed when nonivamide was used in place of capsaicin. Ascorbate has been used as a tool for evaluating peroxidase kinetics [28,29]. In many cases, the rate constant for reaction of ascorbate with a particular free radical far exceeds the rate constant for generation of
Fig. 2. Visible absorption spectra for conversion of peroxidase compound I to compound II in the presence of capsaicin and ascorbate. The first spectrum shown was recorded 3.8 ms after mixing peroxidase compound I with capsaicin/ascorbate. Subsequent spectra were recorded 17, 29, 42, 55, 68, 81, 114, 165, 237, and 339 ms after mixing. The final reaction (25 °C) contained 1.8 lM peroxidase compound I, 2 lM capsaicin, 50 lM ascorbate, and 50 mM phosphate buffer, pH 7.0. Arrows indicate the direction of absorbance changes with time.
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that radical by peroxidase compound I or II. So, when ascorbate is present, the substrate radical is reduced to the parent substrate as rapidly as it is formed by the peroxidase. Thus, the concentration of the reducing substrate remains constant, even though it is present at equimolar or submolar concentrations compared to the peroxidase. In this way, pseudo-first-order conditions can be maintained and rate constants measured even for very rapid peroxidase reactions [28]. Using a constant concentration of ascorbate (50 lM), we evaluated the effect of capsaicinoid concentration on the conversion of peroxidase compound I to compound II. Single exponential behavior was observed over the entire concentration range tested (0.2–20 lM) for both capsaicin and nonivamide. A linear increase in kobs was observed for each capsaicinoid, and second-order rate constants approaching 2 107 M1 s1 were calculated from each line (Table 1). Within error, the y-axis intercepts were zero, suggesting that compound I reduction by capsaicinoids is irreversible and confirming that compound I reduction by ascorbate is relatively slow. In the presence of 10 lM capsaicin, peroxidase compound II was formed within 40 ms. Spectral shifts then occurred over 2 s with new isosbestic points (410, 453, and 521 nm) and absorption maxima (403 and 495 nm) (Fig. 4), consistent with the reduction of compound II to ferric peroxidase. In this case, the reaction was slow enough to measure using pseudo-first-order capsaicinoid concentrations (i.e., P 10 [peroxidase]). Addition of ascorbate was not required. Single exponential behavior was observed for compound II reduction by capsaicin and nonivamide (e.g., Fig. 4, inset). There was linear dependence of kobs on capsaicinoid concentration, giving second-order rate constants of 4.6 105 and 3.9 105 M1 s1 for nonivamide and capsaicin, respectively (Table 1). Within error, y-intercepts were zero, suggesting that compound II reduction by capsaicinoids is also irreversible. Due to their limited solubility in neutral, aqueous solution, we did not obtain kinetic data for capsaicin or nonivamide concentrations exceeding 200 lM. These data demonstrate that capsaicinoids are effective reductants for peroxidase compounds I and II. The rate constants reported here are within the range of those reported for other phenolic compounds [30,31]. Rate constants for the reduction of peroxidase compound II by capsaicinoids were about 40-fold less than those measured for compound I reduction. With rate constants of roughly 4 105 M1 s1 , compound II reduction represents the lowest of the three principal reactions of the peroxidase catalytic cycle. As such, under most conditions (i.e., [H2 O2 ] P 0.1 [capsaicinoid]), one would expect this to be the ratelimiting step for peroxidase-catalyzed capsaicinoid oxidation.
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Fig. 3. Effect of ascorbate on reduction of peroxidase compound I to compound II by capsaicin. The rate of compound II formation (monitored at 412 nm) was followed after reaction of 1.8 lM peroxidase compound I with 2 lM capsaicin (A) or 2 lM capsaicin and 50 lM ascorbate (B). Both reactions were carried out at 25 °C in 50 mM phosphate buffer, pH 7.0. Both curves were fit to single exponential equations.
Table 1 Transient-state rate constants and steady-state parameters for oxidation of capsaicinoidsa Method
Constant (M1 s1 )
Capsaicinoid Nonivamide
Capsaicin
Vanillylamine
Transient-state
k2 k3
(1.8 0.2) 10 (4.6 0.2) 105
(1.5 0.1) 10 (3.9 0.1) 105
1.3 105 3.9 103
Steady-state
kapp
(7.6 0.7) 105
(6.6 0.7) 105
NDb
a b
7
7
All rate constants were determined at 25 °C and pH 7.0. Not determined.
Steady-state kinetic studies Consistent with previous reports [21–25], incubation of capsaicinoids with peroxidase and H2 O2 under multiple turnover (i.e., steady-state) conditions led to the accumulation of dimers and other polymeric products as indicated by the increase in absorbance across the UV– visible spectrum (Fig. 5A). Up to 200 lM, there was a linear increase in rate with increasing concentrations of either capsaicin (Fig. 5B) or nonivamide. Though the relative rate of capsaicinoid oxidation was easily measured spectrophotometrically, assignment of apparent second-order rate constants to these data was complicated by the fact that capsaicinoid oxidation leads to the formation of a mixture of polymerized products [25]. This is a difficulty because the increases in absorbance cannot be kinetically correlated with the direct electron transfer between the capsaicinoid substrate and the
peroxidase [32]. Previous studies have shown that this difficulty can be averted in evaluating the oxidation of some phenolics (e.g., coniferyl alcohol) because the disappearance of the starting material can be directly observed at UV wavelengths [32]. Unfortunately, this is much more difficult with most capsaicinoids because the formation of the dimer/polymer oxidation products leads to increases in absorption across visible and UV wavelengths (Fig. 5A) [21,22]. To avoid these difficulties and obtain accurate apparent second-order rate constants for peroxidase-catalyzed capsaicinoid oxidation, we used a chronometric method that relies on ascorbate as a free radical scavenger. Our own studies on compound I reaction with capsaicinoids provide strong evidence that ascorbate very rapidly reduces capsaicinoid radicals. Indeed, even under conditions where the concentration of capsaicinoid was fivefold less than that of compound I, the full
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Fig. 4. Visible absorption spectra for reduction of peroxidase compound II to ferric enzyme by capsaicin. The first spectrum shown was recorded 39.7 ms after mixing peroxidase compound I with capsaicin. Subsequent spectra were recorded 53, 65, 78, 109, 157, 227, 326, 467, 664, 943, 1340, and 1890 ms after mixing. The final reaction (25 °C) contained 1.8 lM peroxidase compound I, 10 lM capsaicin, and 50 mM phosphate buffer, pH 7.0. Arrows indicate the direction of absorbance changes with time.
expected amplitude for conversion of compound I to compound II was observed and this conversion occurred with a single exponential. Furthermore, RodrıguezL opez et al. [29] successfully developed and used this approach to evaluate the kinetic parameters of oxidation for a series of peroxidase reducing substrates.
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This chronometric method relies on two essentials. First, the apparent second-order rate constant for peroxidase-catalyzed oxidation of reducing substrates (in this case 105 –106 M1 s1 ) is much less than that for reduction of the corresponding substrate radicals by ascorbate (107 –109 M1 s1 ) [33]. Second, direct oxidation of ascorbate by the peroxidase is very slow. Therefore, in reactions containing peroxidase, H2 O2 , capsaicinoid, and ascorbate, ascorbate should be consumed first, and the time required for ascorbate consumption (s) should depend directly on the rate of capsaicinoid radical production by the peroxidase. This rate, of course, depends upon the concentration of capsaicinoid in the reaction. If ascorbate reduces capsaicinoid radicals as rapidly as they are formed, the concentration of capsaicinoid will remain constant for as long as ascorbate remains in the reaction. After ascorbate is consumed, capsaicinoid radicals generated by the peroxidase will not be reduced and will polymerize instead. Our data indicate that these essentials were in place for our studies (Fig. 6). The effect of ascorbate on capsaicin polymerization was monitored at two wavelengths. At 326 nm (Fig. 6A), absorbance changes were due exclusively to capsaicin polymerization. At 285 nm (Fig. 6B), capsaicin polymerization was also detected as an increase in absorbance, but at this wavelength it was also possible to monitor ascorbate consumption as a decrease in absorbance. Without ascorbate, capsaicin polymerization over time was observed at both wavelengths. The inclusion of 160 lM ascorbate completely prevented capsaicin polymerization for 80 s (Fig. 6A). During this 80-s lag, ascorbate was consumed at a constant rate (Fig. 6B)
Fig. 5. Steady-state oxidation of capsaicin by peroxidase. Panel A shows the spectra collected during the oxidation of capsaicin by peroxidase. The reaction contained 0.1 mM capsaicin, 0.25 mM H2 O2 , 50 nM peroxidase, and 100 mM phosphate buffer, pH 7.0. Panel B shows the effect of capsaicin concentration on the rate of its oxidation by peroxidase. Reactions contained 0.25 mM H2 O2 , 50 nM peroxidase, 100 mM phosphate buffer, pH 7.0, and 10 lM capsaicin (d), 40 lM capsaicin (j), 100 lM capsaicin (s), or 200 lM capsaicin (). All reactions were carried out at 25 °C.
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Fig. 6. Effect of ascorbate on the peroxidase-catalyzed accumulation of capsaicin oxidation products. The accumulation of capsaicin oxidation products was measured at 326 nm (panel A) and 285 nm (panel B) in the presence (a) and absence (b) of 160 lM ascorbate. All reactions contained 50 nM peroxidase, 250 lM H2 O2 , 100 lM capsaicin, and 100 mM phosphate buffer, pH 7.0. All reactions were carried out at 25 °C.
after which the accumulation of capsaicin oxidation products was observed at both wavelengths. Importantly, the maximum rate of capsaicin polymer formation was the same in the presence and absence of ascorbate. This confirmed that the concentration of capsaicin remained constant and the concentration of H2 O2 did not become a limiting factor during the lag period. Finally, the minimum absorbance values observed at 285 nm in the presence (t ¼ 82 s) and absence (t ¼ 0) of ascorbate were identical (Fig. 6B), confirming that very near complete consumption of ascorbate was required before capsaicinoid oxidation products could accumulate. Increasing the capsaicinoid concentration against a constant concentration of ascorbate decreased the lag time (s) and increased the rate of capsaicinoid polymerization following the lag period (data not shown). Conversely, increasing the ascorbate concentration against a constant capsaicinoid concentration linearly increased s, but had no effect on the rate of capsaicinoid polymerization after the lag phase (e.g., Fig. 7, inset). These data confirmed that ascorbate would be effective for the chronometric determination of apparent secondorder rate constants for steady-state capsaicinoid oxidation by peroxidases. Individual v0 =½E T values were determined directly from the magnitude of s values induced by increasing concentrations of ascorbate at constant capsaicinoid concentration (e.g., Fig. 7, inset). Within the limits of capsaicinoid solubility, plots of v0 =½E T versus capsaicinoid concentration were linear for nonivamide (Fig. 7) and capsaicin. The apparent second-order rate constants obtained were 7.6 105 and 6.6 105 M1 s1 , respectively (Table 1). The rate constant for oxidation of ferric peroxidase by H2 O2 to form compound I is 1.7 107 M1 s1 [34,35]. Our rate constants for compounds I and II reduction by capsaicinoids are 2 107 and 4 105 M1 s1 , respectively, suggesting that compound II reduction will be rate-determining for peroxidase-catalyzed capsaicinoid
Fig. 7. Ascorbate-based chronometric determination of steady-state rate constants for peroxidase-catalyzed oxidation of capsaicinoids. Individual v0 =½E T values were obtained based on the duration (s) of the ascorbate-dependent lag in capsaicinoid polymer accumulation. The inset shows a typical series of traces used to determine a single v0 =½E T value. The reactions pictured contained 50 nM peroxidase, 150 lM nonivamide, 250 lM H2 O2 , 100 mM phosphate buffer, pH 7.0, and 0 lM (a), 40 lM (b), 60 lM (c), 80 lM (d), or 100 lM ascorbate (e). The v0 =½E T values determined as above were then plotted against the concentration of capsaicinoid (e.g., nonivamide) and an apparent second-order rate constant was obtained from the slope. All reactions were carried out at 25 °C.
oxidation under most conditions (i.e., [H2 O2 ] P 0.1 [capsaicinoid]). This is strongly supported by the fact that our kapp values for steady-state capsaicinoid oxidation (accounting for stoichiometry) are in excellent agreement with the rate constants for compound II reduction by the same compounds. Accounting for methoxy substitution at the 2-position, our steady-state parameters are in good agreement with recent studies on oxidation of catechols by peroxidase. These authors suggest that a hydrophobic substituent at the 4-position is generally preferred by plant peroxidases [29]. Thus, t-butylcatechol is oxidized considerably more rapidly by compound II than dopamine.
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Likewise, capsaicin and nonivamide are effective peroxidase substrates while vanillylamine is much less so ([23] and Table 1). It has been suggested that conversion of compound II to ferric peroxidase is a two-step process: Binding of reducing substrate followed by electron transfer to the ferryl-oxo heme [29,36]. Neither our transient-state studies of compound II reduction nor our steady-state studies indicated hyperbolic dependence of rate on capsaicinoid concentration as would be expected for such a mechanism, especially if compound II reduction is rate-determining. However, these previous studies also indicate that Km values derived from such hyperbolic data are rather high (>0.6 mM). Our studies were limited by the poor water solubility of capsaicinoids. Thus, we were unable to access capsaicinoid concentrations likely to reveal hyperbolic rate dependence. These studies provide further evidence that ascorbate is a highly effective tool for kinetic studies on peroxidase-catalyzed oxidations. For our purposes, ascorbate was essential for the evaluation of compound I reduction and peroxidase-catalyzed capsaicinoid oxidation in the steady-state. That ascorbate was used effectively in these endeavors demonstrates that it is a highly efficient scavenger of capsaicinoid radicals. This may have important implications for capsaicinoid metabolism. Clearly, ascorbate prevents the accumulation of capsaicin oxidation products (dimers, polymers, etc.). Upon the resulting consumption of ascorbate, capsaicinoid radical polymerization ensues. Consequently, ascorbate concentration may be an important determinant for the metabolism of capsaicinoids in plant tissues and other environments. In a similar vein, ascorbate has been suggested as a regulator of coniferyl alcohol oxidation by peroxidases in Vigna angularis epicotyls [37]. Consistent with this hypothesis, there is a notable correlation between the factors that influence the levels of capsaicinoids and ascorbate in Capsicum fruits. Capsaicinoids are located primarily within the placental tissue [1]. Ascorbate is also present in this tissue [38,39]. Increased light exposure increases levels of ascorbate and capsaicinoids in pepper fruits [1,38,40–42]. Indeed, this effect is so dramatic that fruit from the upper parts of the plant (where there is greater light exposure) contains higher levels of both compounds [38,40]. The effects of mineral fertilizers also suggest an integral role for ascorbate in capsaicinoid metabolism. Mineral fertilizer supplementation increases the yield of ascorbate and capsaicinoids in the pepper fruits [38,43,44] and simultaneously decreases lignin-like products [43]. Because lignins are indicative of the oxidative metabolism of phenolics (e.g., capsaicinoids), it is reasonable to suggest that the increase in capsaicinoids may be due to the ascorbate-dependent inhibition of oxidative capsaicinoid metabolism by the peroxidases.
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Peroxidases are effective catalysts for capsaicinoid oxidation and appear to play a pivotal role in the metabolism of these compounds in Capsicum. However, to our knowledge, the reactions between the capsaicinoids and the mammalian peroxidases have never been evaluated. We suggest that such an investigation may provide valuable insight into some of the physiological impacts of capsaicinoid consumption (e.g., the role of capsaicinoids in cancer biology). In conclusion, we have evaluated the oxidation of two typical capsaicinoids by the archetypal plant peroxidase (horseradish peroxidase) using transient- and steadystate kinetic methods. Capsaicinoids are effective reductants for peroxidase compound I and compound II, with the latter representing the rate-determining factor for capsaicinoid-dependent peroxidase turnover. The close agreement between rate constants for compound II reduction (obtained by transient-state methods) and our steady-state kinetic data support compound II reduction as the rate-limiting step. Ascorbate rapidly reduces capsaicinoid radicals. Thus, ascorbate was a helpful tool for the determination of the rate constants reported here. Given the close association of ascorbate and capsaicinoids in Capsicum fruits, we suggest that capsaicinoid levels across varieties and cultivars of Capsicum are dependent upon, among other things, the counterbalancing effects of peroxidase activities and ascorbate levels.
Acknowledgments This work was supported by funds from the Department of Chemistry, the College of Science and Mathematics, and the Office of the Vice President for Research at Auburn University. K.M.H. was supported by a Cell and Molecular Biosciences Undergraduate Summer Research Fellowship and an Auburn University Undergraduate Research Fellowship.
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[9] R. Dubner, Pain 47 (1991) 247–248. [10] T. Glinsukon, V. Stitmunnaithum, C. Toskulkao, T. Buranawuti, V. Tangkrisanavinont, Toxicon. 18 (1980) 215–220. [11] T. Snyman, M.J. Stewart, V. Steenkamp, Forensic Sci. Int. 124 (2001) 43–46. [12] D.F. Billmire, C. Vinocur, M. Ginda, N.B. Robinson, H. Panitch, H. Friss, D. Rubenstein, J.F. Wiley, Pediatrics 98 (1996) 961–963. [13] C.A. Reilly, J.L. Taylor, D.L. Lanza, B.A. Carr, D.J. Crouch, G.S. Yost, Toxicol. Sci., 2003, in press. [14] D.C. Taylor, F.K. Pierau, J. Szolcsanyi, Cell Tissue Res. 240 (1985) 569–573. [15] C.A. Reilly, W.J. Ehlhardt, D.A. Jackson, P. Kulanthaivel, A.E. Mutlib, R.J. Espina, D.E. Moody, D.J. Crouch, G.S. Yost, Chem. Res. Toxicol. 16, 2003, in press. [16] P.M. Gannett, P. Iversen, T. Lawson, Bioorg. Chem. 18 (1990) 185–198. [17] M.S. Miller, K. Brendel, T.F. Burks, I.G. Sipes, Biochem. Pharmacol. 32 (1983) 547–551. [18] Y.J. Surh, S.H. Ahn, K.C. Kim, J.B. Park, Y.W. Sohn, S.S. Lee, Life Sci. 56 (1995) PL305–PL311. [19] M. Contreras-Padilla, E.M. Yahia, J. Agric. Food Chem. 46 (1998) 2075–2079. [20] A. Boersch, B.A. Callingham, F. Lembeck, D.F. Sharman, Biochem. Pharmacol. 41 (1991) 1863–1869. [21] M.A. Bernal, A.A. Calderon, M.A. Pedreno, R. Munoz, A. Ros Barcelo, F. Merino de Caceres, J. Agric. Food Chem. 41 (1993) 1041–1044. [22] M.A. Bernal, A.A. Calderon, M.A. Pedreno, R. Munoz, A. Ros Barcelo, F. Merino de Caceres, J. Food Sci. 58 (1993) 611–613. [23] M.A. Bernal, A.A. Calderon, M.A. Ferrer, F. Merino de Caceres, A. Ros Barcelo, J. Agric. Food Chem. 43 (1995) 352–355. [24] F. Pomar, M.A. Bernal, J. Diaz, F. Merino, Phytochemistry 46 (1997) 1313–1317. [25] M.A. Bernal, A. Ros Barcelo, J. Agric. Food Chem. 44 (1996) 3085–3089.
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Peroxidase-catalyzed oxidation of capsaicinoids: steady-state and transient-state kinetic studies Douglas C. Goodwin* and Kristen M. Hertwig Department of Chemistry and Program in Cell and Molecular Biosciences, Auburn University, Auburn, AL 36849-5312, USA Received 12 May 2003, and in revised form 17 June 2003
Abstract Capsaicinoids are the pungent compounds in Capsicum fruits (i.e., ‘‘hot’’ peppers). Peroxidases catalyze capsaicinoid oxidation and may play a central role in their metabolism. However, key kinetic aspects of peroxidase-catalyzed capsaicinoid oxidation remain unresolved. Using transient-state methods, we evaluated horseradish peroxidase compound I and II reduction by two prominent capsaicinoids (25 °C, pH 7.0). We determined rate constants approaching 2 107 and 5 105 M1 s1 for compound I and compound II reduction, respectively. We also determined kapp values for steady-state capsaicinoid oxidation approaching 8 105 M1 s1 (25 °C, pH 7.0). Accounting for stoichiometry, these are in excellent agreement with constants for compound II reduction, suggesting that this reaction governs capsaicinoid-dependent peroxidase turnover. Ascorbate rapidly reduced capsaicinoid radicals, assisting in the determination of the kinetic constants reported. Because ascorbate accumulates in Capsicum fruits, it may also be an important determinant for capsaicinoid content and preservation in Capsicum fruits and related products. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Capsaicin; Ferryl-oxo; Compound I; Compound II; Ascorbate; Nonivamide; Capsicum; N-Vanillylnonanamide; Free radical; Peroxidase
Capsaicinoids impart the pungent flavor of Capsicum ‘‘hot’’ pepper fruits. The most abundant capsaicinoids are capsaicin and dihydrocapsaicin (Fig. 1), typically comprising 80% of the total capsaicinoid content in Capsicum fruits [1]. Other capsaicinoids have been identified in much smaller quantities, including nordihydrocapsaicin, homocapsaicin, homodihydrocapsaicin, and nonivamide. Indeed, nonivamide, also called ‘‘synthetic capsaicin,’’ has recently been identified as a natural product [2,3]. Capsaicinoids are present in Capsicum fruits at levels up to 1% [4]. Consequently, many varieties of peppers are widely used in commercial and domestic food preparation. Indeed, over a quarter of the worldÕs population consumes hot peppers in some form on a daily basis [5]. Clearly, Capsicum fruits are important agricultural commodities with prominent ethnic and cultural significance. The desirability of these peppers for * Corresponding author. Fax: 1-334-844-6959. E-mail address: [email protected] (D.C. Goodwin).
0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0003-9861(03)00321-7
food preparation depends greatly on their capsaicin content. Pungency and the general nociceptive effects of capsaicinoids result from their interaction with the vanilloid receptor type 1, a non-selective ion channel that opens in response to noxious heat (>48 °C) [6,7]. Capsaicinoids lower the temperature threshold of the receptor, leading to gating of the channel at room temperature [6]. This is followed by a refractory period in which there is neuronal resistance not only to additional capsaicinoid exposure, but also to other painful stimuli [5]. This forms the basis for the extensive use of capsaicinoid-based topical agents for the treatment of acute (e.g., muscle fatigue and soreness) or chronic pain (e.g., diabetic neuropathy, fibromyalgia, or rheumatoid arthritis) [5,8,9]. At high doses, capsaicinoids can cause respiratory depression in mammalian systems [10–13]. They are also neurotoxic, preventing the intra-axonal transport of nerve growth factor in neonatal dorsal root ganglionic neurons [14]. The capsaicinoids have also been evaluated
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compound I, leaving the ferryl-oxo intermediate compound II and one equivalent of substrate radical (A ) (reaction (2)). A second equivalent of AH reduces compound II to ferric peroxidase, yielding a second equivalent of A and a second equivalent of H2 O (reaction (3)) [26]. ð1Þ ð2Þ ð3Þ
Fig. 1. Diagram representing the structures of some common capsaicinoids as well as the generation and subsequent reactions of capsaicinoid radicals. Capsaicin and dihydrocapsaicin are the most abundant naturally occurring capsaicinoids. Nonivamide is observed at low levels in natural sources but is widely available as a synthetic preparation.
for their potential as anticarcinogens, co-carcinogens, or carcinogens. However, data in this regard remain inconclusive [4]. Clearly, the metabolism of capsaicinoids has important ramifications. Peroxidases and other hemoproteins (e.g., cytochromes P450) occupy central roles in capsaicinoid catabolism [15–18]. In the maturing pepper fruits of Capsicum annuum cultivars, the degradation of capsaicinoids has been correlated with a rise in peroxidase activity [19]. Plant peroxidases catalyze capsaicinoid oxidation [20–24] leading to polymerization to form lignin-like products [25]. Two dimeric species, 5,50 dicapsaicin and 40 -O-5-dicapsaicin ether, have been identified, and further oxidation results in the formation of high molecular weight polymers [25] (Fig. 1). In the typical catalytic cycle, H2 O2 oxidizes ferric peroxidase (also called resting or native enzyme) by two electrons to form a ferryl-oxo porphyrin radical intermediate (compound I) (reaction (1)). An exogenous reducing substrate (AH) reduces the porphyrin radical of
Although capsaicinoids are known to be reducing substrates for plant peroxidases (horseradish and C. annuum) [21–24], several kinetic aspects of the process remain poorly defined. The reactions of peroxidase compounds I and II with capsaicinoids have not been directly measured. Furthermore, a broad range of polymeric products (dimers, multimers, and protein-capsaicinoid copolymers) are formed (each product with its own absorption characteristics), making a meaningful correspondence between product formation and direct interaction between the peroxidase and the capsaicinoid substrate difficult to establish. This has hampered the assignment of steady-state parameters for capsaicinoid oxidation by peroxidases. To help address these deficiencies, we evaluated the reactions of the archetypal plant peroxidase (horseradish peroxidase) with two typical capsaicinoids (capsaicin and nonivamide) by transient-state and steady-state kinetic methods. To avoid problems that have hindered these types of studies, we used the properties of ascorbate as an efficient reductant of phenoxyl radicals. For our transient-state kinetic studies, ascorbate maintained pseudo-first order conditions even with very low concentrations of capsaicinoid, allowing for the effective determination of rate constants for the very rapid reduction of peroxidase compound I. For our steady-state kinetic studies, ascorbate allowed us to forgo problematic spectrophotometric procedures in favor of a chronometric method. As a result, we have determined rate constants for the direct reaction of capsaicinoids with peroxidase compounds I and II and apparent second-order rate constants for oxidation of capsaicinoids by peroxidase. Our studies have also revealed that peroxidase-generated capsaicinoid radicals are very rapidly reduced by ascorbate, preventing polymerization. These data provide valuable information for understanding the metabolism of capsaicinoids by peroxidases and the potential influence that antioxidants like ascorbate may have in that process. Thus, these results may have significant implications because of the great cultural,
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agricultural, and neuropharamcological importance of the capsaicinoids.
Materials and methods Materials Hydrogen peroxide (30%), capsaicin, nonivamide, peroxidase (Type VI), and ascorbate (ACS grade) were purchased from Sigma (St. Louis, MO). All buffers and media were prepared using Millipore (Q-PakII)-purified water (18.2 MX/cm resistivity). Concentrations of H2 O2 and peroxidase were determined spectrophotometrically at 240 nm (e ¼ 39:4 M1 cm1 ) [27] and 403 nm (e ¼ 102,000 M1 cm1 ) [26], respectively. Transient-state kinetics The reactions of peroxidase compounds I and II with capsaicinoids were monitored using an SX.18MV stopped-flow spectrophotometer (Applied Photophysics, Surrey, UK) in sequential-mixing mode. All reactions were carried out at pH 7.0 and 25 °C. To obtain spectra, peroxidase compound I was formed by pre-mixing equal volumes of 8 lM peroxidase with 7.6 lM H2 O2 in a delay line. After a 100 ms delay, the contents of the delay line were mixed with 4 lM capsaicinoid and reaction progress was monitored by diode array detector. Final reactions contained 2 lM peroxidase compound I, 2 lM capsaicinoid, and 50 mM phosphate buffer, pH 7.0. For some measurements, ascorbate was used to maintain pseudo-first-order conditions with respect to capsaicinoid. For these reactions, the 4 lM capsaicinoid solution also contained 100 lM ascorbate, giving a final ascorbate concentration of 50 lM. Spectra for compound II reduction to ferric enzyme were collected by first forming compound I as above. Compound I was then mixed with an equal volume of 20 lM capsaicinoid and spectra were collected by diode array. Following the very rapid conversion of compound I to compound II (<40 ms), compound II reduction to ferric enzyme was recorded. To obtain rate constants for compound I reduction, ferric peroxidase (4 lM) was pre-mixed with an equal volume of 3.8 lM H2 O2 . Following a 200 ms delay, this solution was mixed with an equal volume of 100 lM ascorbate and 0.4–40 lM capsaicin or nonivamide. Ascorbate was added to ensure pseudo-first-order conditions with respect to capsaicinoid [28]. The final concentrations of peroxidase, ascorbate, and phosphate buffer, pH 7.0 were 1 lM (predominantly compound I), 50 lM, and 50 mM, respectively. Final concentrations of capsaicinoid ranged from 0.2 to 20 lM. Reduction of compound I was measured at 411 nm, an isosbestic wavelength for compound II and ferric peroxidase [26].
Rate constants for compound II reduction were obtained by the same procedure except that higher concentrations of capsaicinoid were present (20– 400 lM) and the reactions were monitored at 428 nm, an isosbestic wavelength for ferric peroxidase and compound I [26]. Due to the much higher concentrations of capsaicinoid in these experiments, addition of ascorbate was not required to maintain psuedofirst-order conditions. Steady-state kinetics Steady-state capsaicinoid oxidation was evaluated using our stopped-flow instrument in single-mixing mode. One syringe contained peroxidase (100 nM) in 200 mM phosphate buffer, pH 7.0. The second syringe contained 500 lM H2 O2 , varying concentrations of capsaicinoid, and, when appropriate, varying concentrations of ascorbate. Spectra were recorded by diode array and rate constants were determined from singlewavelength measurements. All reactions were carried out at pH 7.0 and 25 °C. Capsaicinoid oxidation by peroxidase leads to a series of dimer and polymer products [25], and corresponding increase in absorbance across the UV–visible spectrum [21], complicating the estimation of steady-state kinetic parameters for these reactions. To circumvent this problem, we used a chronometric method [29], including ascorbate in reactions to rapidly reduce peroxidase-derived radicals. Ascorbate efficiently prevented free-radical polymerization, giving rise to a pronounced lag in the appearance of capsaicinoid oxidation products. Practically, complete consumption of ascorbate had to occur before any polymerized products were observed. Thus, the lag time (s) was used to calculate the initial rate (v0 ) for capsaicin oxidation by peroxidase: v0 ¼ 2½ascorbate =s:
ð1Þ
Rodrıguez-L opez and coworkers point out that ascorbate can be consumed in a reaction with H2 O2 (k ¼ 1:03 M1 s1 ). The initial rate can be corrected for these effects, where k is the rate constant for reaction between ascorbate and H2 O2 [29], v0 ¼ f2½ascorbate ð1 sk½H2 O2 Þg=s:
ð2Þ
For each concentration of capsaicinoid, a series of s values was obtained corresponding to a series of ascorbate concentrations. Dividing each ascorbate concentration by the corresponding s produced a set of v0 values that was independent of the concentration of ascorbate. From these, an average v0 was determined for each capsaicinoid concentration. Each v0 was divided by the concentration of enzyme present and these v0 =½E T values were plotted against the concentration of capsaicinoid to obtain apparent second-order rate constants.
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Results and discussion Transient-state kinetics Reduction of peroxidase compounds I and II by capsaicinoids has been assumed, but the reactions have not been directly observed nor the rate constants determined. We used sequential mixing stopped-flow methods to evaluate these reactions. Reaction of peroxidase compound I with equimolar capsaicin resulted in a rapid shift in absorption maximum from 403 to 418 nm with an isosbestic point near 398 nm. New absorption peaks were also detected at 529 and 557 nm (data not shown). Nearly identical results were obtained when compound I was reacted with equimolar capsaicin and 50 lM ascorbate (Fig. 2). Whether collected in the presence or absence of ascorbate, spectra were consistent with the conversion of peroxidase compound I to compound II. Both reactions occurred roughly within the same time frame, but single exponential behavior was observed only in the presence of ascorbate (Fig. 3). The same results (spectral and kinetic) were observed when nonivamide was used in place of capsaicin. Ascorbate has been used as a tool for evaluating peroxidase kinetics [28,29]. In many cases, the rate constant for reaction of ascorbate with a particular free radical far exceeds the rate constant for generation of
Fig. 2. Visible absorption spectra for conversion of peroxidase compound I to compound II in the presence of capsaicin and ascorbate. The first spectrum shown was recorded 3.8 ms after mixing peroxidase compound I with capsaicin/ascorbate. Subsequent spectra were recorded 17, 29, 42, 55, 68, 81, 114, 165, 237, and 339 ms after mixing. The final reaction (25 °C) contained 1.8 lM peroxidase compound I, 2 lM capsaicin, 50 lM ascorbate, and 50 mM phosphate buffer, pH 7.0. Arrows indicate the direction of absorbance changes with time.
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that radical by peroxidase compound I or II. So, when ascorbate is present, the substrate radical is reduced to the parent substrate as rapidly as it is formed by the peroxidase. Thus, the concentration of the reducing substrate remains constant, even though it is present at equimolar or submolar concentrations compared to the peroxidase. In this way, pseudo-first-order conditions can be maintained and rate constants measured even for very rapid peroxidase reactions [28]. Using a constant concentration of ascorbate (50 lM), we evaluated the effect of capsaicinoid concentration on the conversion of peroxidase compound I to compound II. Single exponential behavior was observed over the entire concentration range tested (0.2–20 lM) for both capsaicin and nonivamide. A linear increase in kobs was observed for each capsaicinoid, and second-order rate constants approaching 2 107 M1 s1 were calculated from each line (Table 1). Within error, the y-axis intercepts were zero, suggesting that compound I reduction by capsaicinoids is irreversible and confirming that compound I reduction by ascorbate is relatively slow. In the presence of 10 lM capsaicin, peroxidase compound II was formed within 40 ms. Spectral shifts then occurred over 2 s with new isosbestic points (410, 453, and 521 nm) and absorption maxima (403 and 495 nm) (Fig. 4), consistent with the reduction of compound II to ferric peroxidase. In this case, the reaction was slow enough to measure using pseudo-first-order capsaicinoid concentrations (i.e., P 10 [peroxidase]). Addition of ascorbate was not required. Single exponential behavior was observed for compound II reduction by capsaicin and nonivamide (e.g., Fig. 4, inset). There was linear dependence of kobs on capsaicinoid concentration, giving second-order rate constants of 4.6 105 and 3.9 105 M1 s1 for nonivamide and capsaicin, respectively (Table 1). Within error, y-intercepts were zero, suggesting that compound II reduction by capsaicinoids is also irreversible. Due to their limited solubility in neutral, aqueous solution, we did not obtain kinetic data for capsaicin or nonivamide concentrations exceeding 200 lM. These data demonstrate that capsaicinoids are effective reductants for peroxidase compounds I and II. The rate constants reported here are within the range of those reported for other phenolic compounds [30,31]. Rate constants for the reduction of peroxidase compound II by capsaicinoids were about 40-fold less than those measured for compound I reduction. With rate constants of roughly 4 105 M1 s1 , compound II reduction represents the lowest of the three principal reactions of the peroxidase catalytic cycle. As such, under most conditions (i.e., [H2 O2 ] P 0.1 [capsaicinoid]), one would expect this to be the ratelimiting step for peroxidase-catalyzed capsaicinoid oxidation.
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Fig. 3. Effect of ascorbate on reduction of peroxidase compound I to compound II by capsaicin. The rate of compound II formation (monitored at 412 nm) was followed after reaction of 1.8 lM peroxidase compound I with 2 lM capsaicin (A) or 2 lM capsaicin and 50 lM ascorbate (B). Both reactions were carried out at 25 °C in 50 mM phosphate buffer, pH 7.0. Both curves were fit to single exponential equations.
Table 1 Transient-state rate constants and steady-state parameters for oxidation of capsaicinoidsa Method
Constant (M1 s1 )
Capsaicinoid Nonivamide
Capsaicin
Vanillylamine
Transient-state
k2 k3
(1.8 0.2) 10 (4.6 0.2) 105
(1.5 0.1) 10 (3.9 0.1) 105
1.3 105 3.9 103
Steady-state
kapp
(7.6 0.7) 105
(6.6 0.7) 105
NDb
a b
7
7
All rate constants were determined at 25 °C and pH 7.0. Not determined.
Steady-state kinetic studies Consistent with previous reports [21–25], incubation of capsaicinoids with peroxidase and H2 O2 under multiple turnover (i.e., steady-state) conditions led to the accumulation of dimers and other polymeric products as indicated by the increase in absorbance across the UV– visible spectrum (Fig. 5A). Up to 200 lM, there was a linear increase in rate with increasing concentrations of either capsaicin (Fig. 5B) or nonivamide. Though the relative rate of capsaicinoid oxidation was easily measured spectrophotometrically, assignment of apparent second-order rate constants to these data was complicated by the fact that capsaicinoid oxidation leads to the formation of a mixture of polymerized products [25]. This is a difficulty because the increases in absorbance cannot be kinetically correlated with the direct electron transfer between the capsaicinoid substrate and the
peroxidase [32]. Previous studies have shown that this difficulty can be averted in evaluating the oxidation of some phenolics (e.g., coniferyl alcohol) because the disappearance of the starting material can be directly observed at UV wavelengths [32]. Unfortunately, this is much more difficult with most capsaicinoids because the formation of the dimer/polymer oxidation products leads to increases in absorption across visible and UV wavelengths (Fig. 5A) [21,22]. To avoid these difficulties and obtain accurate apparent second-order rate constants for peroxidase-catalyzed capsaicinoid oxidation, we used a chronometric method that relies on ascorbate as a free radical scavenger. Our own studies on compound I reaction with capsaicinoids provide strong evidence that ascorbate very rapidly reduces capsaicinoid radicals. Indeed, even under conditions where the concentration of capsaicinoid was fivefold less than that of compound I, the full
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Fig. 4. Visible absorption spectra for reduction of peroxidase compound II to ferric enzyme by capsaicin. The first spectrum shown was recorded 39.7 ms after mixing peroxidase compound I with capsaicin. Subsequent spectra were recorded 53, 65, 78, 109, 157, 227, 326, 467, 664, 943, 1340, and 1890 ms after mixing. The final reaction (25 °C) contained 1.8 lM peroxidase compound I, 10 lM capsaicin, and 50 mM phosphate buffer, pH 7.0. Arrows indicate the direction of absorbance changes with time.
expected amplitude for conversion of compound I to compound II was observed and this conversion occurred with a single exponential. Furthermore, RodrıguezL opez et al. [29] successfully developed and used this approach to evaluate the kinetic parameters of oxidation for a series of peroxidase reducing substrates.
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This chronometric method relies on two essentials. First, the apparent second-order rate constant for peroxidase-catalyzed oxidation of reducing substrates (in this case 105 –106 M1 s1 ) is much less than that for reduction of the corresponding substrate radicals by ascorbate (107 –109 M1 s1 ) [33]. Second, direct oxidation of ascorbate by the peroxidase is very slow. Therefore, in reactions containing peroxidase, H2 O2 , capsaicinoid, and ascorbate, ascorbate should be consumed first, and the time required for ascorbate consumption (s) should depend directly on the rate of capsaicinoid radical production by the peroxidase. This rate, of course, depends upon the concentration of capsaicinoid in the reaction. If ascorbate reduces capsaicinoid radicals as rapidly as they are formed, the concentration of capsaicinoid will remain constant for as long as ascorbate remains in the reaction. After ascorbate is consumed, capsaicinoid radicals generated by the peroxidase will not be reduced and will polymerize instead. Our data indicate that these essentials were in place for our studies (Fig. 6). The effect of ascorbate on capsaicin polymerization was monitored at two wavelengths. At 326 nm (Fig. 6A), absorbance changes were due exclusively to capsaicin polymerization. At 285 nm (Fig. 6B), capsaicin polymerization was also detected as an increase in absorbance, but at this wavelength it was also possible to monitor ascorbate consumption as a decrease in absorbance. Without ascorbate, capsaicin polymerization over time was observed at both wavelengths. The inclusion of 160 lM ascorbate completely prevented capsaicin polymerization for 80 s (Fig. 6A). During this 80-s lag, ascorbate was consumed at a constant rate (Fig. 6B)
Fig. 5. Steady-state oxidation of capsaicin by peroxidase. Panel A shows the spectra collected during the oxidation of capsaicin by peroxidase. The reaction contained 0.1 mM capsaicin, 0.25 mM H2 O2 , 50 nM peroxidase, and 100 mM phosphate buffer, pH 7.0. Panel B shows the effect of capsaicin concentration on the rate of its oxidation by peroxidase. Reactions contained 0.25 mM H2 O2 , 50 nM peroxidase, 100 mM phosphate buffer, pH 7.0, and 10 lM capsaicin (d), 40 lM capsaicin (j), 100 lM capsaicin (s), or 200 lM capsaicin (). All reactions were carried out at 25 °C.
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Fig. 6. Effect of ascorbate on the peroxidase-catalyzed accumulation of capsaicin oxidation products. The accumulation of capsaicin oxidation products was measured at 326 nm (panel A) and 285 nm (panel B) in the presence (a) and absence (b) of 160 lM ascorbate. All reactions contained 50 nM peroxidase, 250 lM H2 O2 , 100 lM capsaicin, and 100 mM phosphate buffer, pH 7.0. All reactions were carried out at 25 °C.
after which the accumulation of capsaicin oxidation products was observed at both wavelengths. Importantly, the maximum rate of capsaicin polymer formation was the same in the presence and absence of ascorbate. This confirmed that the concentration of capsaicin remained constant and the concentration of H2 O2 did not become a limiting factor during the lag period. Finally, the minimum absorbance values observed at 285 nm in the presence (t ¼ 82 s) and absence (t ¼ 0) of ascorbate were identical (Fig. 6B), confirming that very near complete consumption of ascorbate was required before capsaicinoid oxidation products could accumulate. Increasing the capsaicinoid concentration against a constant concentration of ascorbate decreased the lag time (s) and increased the rate of capsaicinoid polymerization following the lag period (data not shown). Conversely, increasing the ascorbate concentration against a constant capsaicinoid concentration linearly increased s, but had no effect on the rate of capsaicinoid polymerization after the lag phase (e.g., Fig. 7, inset). These data confirmed that ascorbate would be effective for the chronometric determination of apparent secondorder rate constants for steady-state capsaicinoid oxidation by peroxidases. Individual v0 =½E T values were determined directly from the magnitude of s values induced by increasing concentrations of ascorbate at constant capsaicinoid concentration (e.g., Fig. 7, inset). Within the limits of capsaicinoid solubility, plots of v0 =½E T versus capsaicinoid concentration were linear for nonivamide (Fig. 7) and capsaicin. The apparent second-order rate constants obtained were 7.6 105 and 6.6 105 M1 s1 , respectively (Table 1). The rate constant for oxidation of ferric peroxidase by H2 O2 to form compound I is 1.7 107 M1 s1 [34,35]. Our rate constants for compounds I and II reduction by capsaicinoids are 2 107 and 4 105 M1 s1 , respectively, suggesting that compound II reduction will be rate-determining for peroxidase-catalyzed capsaicinoid
Fig. 7. Ascorbate-based chronometric determination of steady-state rate constants for peroxidase-catalyzed oxidation of capsaicinoids. Individual v0 =½E T values were obtained based on the duration (s) of the ascorbate-dependent lag in capsaicinoid polymer accumulation. The inset shows a typical series of traces used to determine a single v0 =½E T value. The reactions pictured contained 50 nM peroxidase, 150 lM nonivamide, 250 lM H2 O2 , 100 mM phosphate buffer, pH 7.0, and 0 lM (a), 40 lM (b), 60 lM (c), 80 lM (d), or 100 lM ascorbate (e). The v0 =½E T values determined as above were then plotted against the concentration of capsaicinoid (e.g., nonivamide) and an apparent second-order rate constant was obtained from the slope. All reactions were carried out at 25 °C.
oxidation under most conditions (i.e., [H2 O2 ] P 0.1 [capsaicinoid]). This is strongly supported by the fact that our kapp values for steady-state capsaicinoid oxidation (accounting for stoichiometry) are in excellent agreement with the rate constants for compound II reduction by the same compounds. Accounting for methoxy substitution at the 2-position, our steady-state parameters are in good agreement with recent studies on oxidation of catechols by peroxidase. These authors suggest that a hydrophobic substituent at the 4-position is generally preferred by plant peroxidases [29]. Thus, t-butylcatechol is oxidized considerably more rapidly by compound II than dopamine.
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Likewise, capsaicin and nonivamide are effective peroxidase substrates while vanillylamine is much less so ([23] and Table 1). It has been suggested that conversion of compound II to ferric peroxidase is a two-step process: Binding of reducing substrate followed by electron transfer to the ferryl-oxo heme [29,36]. Neither our transient-state studies of compound II reduction nor our steady-state studies indicated hyperbolic dependence of rate on capsaicinoid concentration as would be expected for such a mechanism, especially if compound II reduction is rate-determining. However, these previous studies also indicate that Km values derived from such hyperbolic data are rather high (>0.6 mM). Our studies were limited by the poor water solubility of capsaicinoids. Thus, we were unable to access capsaicinoid concentrations likely to reveal hyperbolic rate dependence. These studies provide further evidence that ascorbate is a highly effective tool for kinetic studies on peroxidase-catalyzed oxidations. For our purposes, ascorbate was essential for the evaluation of compound I reduction and peroxidase-catalyzed capsaicinoid oxidation in the steady-state. That ascorbate was used effectively in these endeavors demonstrates that it is a highly efficient scavenger of capsaicinoid radicals. This may have important implications for capsaicinoid metabolism. Clearly, ascorbate prevents the accumulation of capsaicin oxidation products (dimers, polymers, etc.). Upon the resulting consumption of ascorbate, capsaicinoid radical polymerization ensues. Consequently, ascorbate concentration may be an important determinant for the metabolism of capsaicinoids in plant tissues and other environments. In a similar vein, ascorbate has been suggested as a regulator of coniferyl alcohol oxidation by peroxidases in Vigna angularis epicotyls [37]. Consistent with this hypothesis, there is a notable correlation between the factors that influence the levels of capsaicinoids and ascorbate in Capsicum fruits. Capsaicinoids are located primarily within the placental tissue [1]. Ascorbate is also present in this tissue [38,39]. Increased light exposure increases levels of ascorbate and capsaicinoids in pepper fruits [1,38,40–42]. Indeed, this effect is so dramatic that fruit from the upper parts of the plant (where there is greater light exposure) contains higher levels of both compounds [38,40]. The effects of mineral fertilizers also suggest an integral role for ascorbate in capsaicinoid metabolism. Mineral fertilizer supplementation increases the yield of ascorbate and capsaicinoids in the pepper fruits [38,43,44] and simultaneously decreases lignin-like products [43]. Because lignins are indicative of the oxidative metabolism of phenolics (e.g., capsaicinoids), it is reasonable to suggest that the increase in capsaicinoids may be due to the ascorbate-dependent inhibition of oxidative capsaicinoid metabolism by the peroxidases.
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Peroxidases are effective catalysts for capsaicinoid oxidation and appear to play a pivotal role in the metabolism of these compounds in Capsicum. However, to our knowledge, the reactions between the capsaicinoids and the mammalian peroxidases have never been evaluated. We suggest that such an investigation may provide valuable insight into some of the physiological impacts of capsaicinoid consumption (e.g., the role of capsaicinoids in cancer biology). In conclusion, we have evaluated the oxidation of two typical capsaicinoids by the archetypal plant peroxidase (horseradish peroxidase) using transient- and steadystate kinetic methods. Capsaicinoids are effective reductants for peroxidase compound I and compound II, with the latter representing the rate-determining factor for capsaicinoid-dependent peroxidase turnover. The close agreement between rate constants for compound II reduction (obtained by transient-state methods) and our steady-state kinetic data support compound II reduction as the rate-limiting step. Ascorbate rapidly reduces capsaicinoid radicals. Thus, ascorbate was a helpful tool for the determination of the rate constants reported here. Given the close association of ascorbate and capsaicinoids in Capsicum fruits, we suggest that capsaicinoid levels across varieties and cultivars of Capsicum are dependent upon, among other things, the counterbalancing effects of peroxidase activities and ascorbate levels.
Acknowledgments This work was supported by funds from the Department of Chemistry, the College of Science and Mathematics, and the Office of the Vice President for Research at Auburn University. K.M.H. was supported by a Cell and Molecular Biosciences Undergraduate Summer Research Fellowship and an Auburn University Undergraduate Research Fellowship.
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