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A comparison of the peroxidase-catalyzed and electrochemical oxidation of uric acid
Bioelectrochemistry and Bioenergetics, 9 (1982) 39-60 A section of J. Electroanal. Chem., and constituting Vol. 141 (1982) Elsevier Sequoia S.A., Lausanne-Printed in The Netherlands
COMPARISON OF THE PEROXIDASE-CATALYZED ELECTROCHEMICAL OXIDATION OF URIC ACID
479-A
R.N. GOYAL Department (Manuscript
*, ANNA
of Chemistry, received
BRAJTER-TOTH
l*,
University of Oklahoma,
August
GLENN
DRYHURST
Norman,
l
AND
** and N.T. NGUYEN
OK 73019 (U.S.A.)
7th 1981)
SUMMARY The oxidation of uric acid by hydrogen peroxide in the presence of type VIII peroxidase has been studied between pH 5.2 and 8. Intermediates generated in the reaction have been characterized in terms of their U.V. spectra and kinetics of decay. In addition at least one U.V.-absorbing intermediate has been trapped, converted to its trimethylsilyl derivative and identified by gas chromatography-mass spectrometry. This intermediate is l-carbohydroxy-2,4,6,8-tetraza-3,7-dioxo-4-ene-bicyclo-(3,3, O)-octane. At pHa7 the product is allantoin while at lower pH S-hydroxyhydantoin-5-carboxamide is also formed as a major product. The intermediates and products formed and spectral and kinetic measurements observed during and after peroxidase-catalyzed oxidation of uric acid are virtually identical to those noted upon electrochemical oxidation. It has thus been concluded that the mechanisms of electrochemical and enzymic oxidation of uric acid are, in a chemical sense, indentical.
INTRODUCTION
In recent reports from this laboratory it has been demonstrated that the electrochemical and peroxidase-cat,alyzed oxidation of uric acid [ 1,2] and various N-methylated uric acids [2-41 appear to proceed, in a chemical sense, by very similar if not identical mechanisms at around physiological pH. We have recently investigated the electrochemical oxidation of uric acid over a wide pH range (pH 1.5-9.5) in phosphate buffers using thin-layer spectroelectrochemistry to generate and study intermediates [5,6]. Some intermediates and all major products were separated and analyzed by gas chromatography-mass spectrometry (GC-MS). On the basis of these studies it has been concluded that the electrooxidation of uric acid is significantly more complex than was originally reported. At pH values > pK, for uric acid the electrochemical mechanism seems to be the most straightforward. Thus, the monoanion of uric acid [I, equation (l)] is electro-
l l l
Permanent address: Department of Chemistry, Roorkee University, Roorkee, India. * Current address: Department of Chemistry, University of Maine, Orono, Maine, U.S.A. ** To whom correspondence and reprint requests should be directed.
0302-4598/82/0000-OOOOo/$
02.75 0 1982 Elsevier Sequoia
S.A.
Peak
I,
-2 e-- 2 Hz +2P+2H+ Peak
Ic
I:
1
4 H,O,H+
(HOC)
1-co* 0 Allantoin
oxidized in a quasi-reversible 2e--2H+ reaction to a very unstable quinonoid-diimine (II, equation l), having a maximal half-life at around pH 8 (t,,, = 22 ms), which rapidly disappears in a pseudo first order hydration reaction [l] to give an anionic imine-alcohol [III, equation (l)]. This intermediate undergoes a ring contraction reaction to give l-carbohydroxy-2,4,6,8-tetraaza-3,7-dioxo-4-ene-bicyclo-(3,3,0)octane [BCA, equation (l)]. The intermediate III and BCA [equation (l)] may be inferred by analysis of absorbance versus time (A verms f) curves for decay of intermediate species generated upon electrooxidation of uric acid in a thin-layer cell containing an optically-transparent reticulated vitreous carbon (RVC) electrode. BCA decomposes to a single organic product, allantoin [equation (l)]. At pH a 6 phosphate appears to have no significant effect on the basic mechanism shown in equation (1). Between pH 3 and 5.6 a rather more complex reaction scheme appears to occur. In the presence of very low concentrations of phosphate (H,POi) neutral uric acid [l, equation (2)] is electrooxidized in a quasi-reversible 2e--2H+ reaction to the neutral quinonoid-diimine [II, equation (2)] which is very rapidly hydrated to the two isomeric imine-alcohols III, and III, [equation (2)]. Hydration of imine-alcohol III, [equation (2)] leads to the diol [VIII, equation (2)] and hence to 5-hydroxy-hydantoin-5-carboxamide [equation (2)] and, at pH 3 and below, alloxan [equation (2)]. Imine-alcohol III, [equation (2)] undergoes a ring contraction to give BCA [equation (2)] and hence allantoin. The values of k, and k, shown in equation (2) were determined by analysis of A versus t curves obtained following electrooxidation of uric acid in a thin-layer cell. In the presence of high concentrations of phosphate between pH 3 and 5.6 the putative quinonoid diimine [II, equation (3)] seems to be
-2
Ht-2
e-
I
H2O
(Illb)
1
1
-H+
“20
0
(VIII)
(2) H2O
k2
+ COOH
0 OH OH 0 Alloxan
(VI)
1
H,O 402 -NH3
“2$ f=O WJ H2N
1
-cop
0
5 Hydroxyh&antoin -5-carboxamide
(VIII
Allanloin
42
;2H+-2e-.
xkNho
+2Ht+2e-
“z-
o
,
;~‘f’-&>~
t OH-
u
(II) H20
In
HzP04
(Ilk)
H2Po4k,
H20 1
OH-
+
0 0 (Illb)
H2P04 (IV)
H2P04-
“20 i
(3) 0 0 (BCA)
Alloxan H20
H$
1 k,
t=o H&
H2N
0
5-Hydroxyh&ntoin -5-carboxamide 1 -co2
0 Allantoin
43
preferentially attacked by H,PO; rather than by water to give the phosphorylated intermediates III, and III, [equation (3)]. Intermediate III, [equation (3)] is further attacked by H,PO; (or water) to give VIII [equation (3)] and hence 5-hydroxyhydantoin-5-carboxamide or, at pH 3, small amounts of alloxan [equation (3)]. Analysis of A versus t curves indicates that three distinct intermediates are involved. This has been interpreted as shown in equation (3). Thus, intermediate III, [equation (3)] is rapidly hydrated to give IV [equation (3)] which undergoes a ring contraction to V [equation (3)J characterized by the pseudo first order rate constant k,. Intermediate V [equation (3)] then loses phosphate to give BCA [k,, equation (3)] which hydrates [k3, equation (3)] leading ultimately to allantoin. The work reported here was undertaken to investigate the enzymic oxidation of uric acid catalyzed by peroxidase. In particular, we were interested to find out if the detailed mechanisms elucidated from electrochemical studies were applicable to the enzyme catalyzed reactions. This study was undertaken not only to obtain fundamental information about the enzymic reaction but also to further justify our long-held contention that electrochemical studies of the redox mechanisms of biomolecules can provide uniquely valuable guidance and insights into the overall chemical pathways followed by substrate molecules in enzymic redox reactions. EXPERIMENTAL
Chemicals
Uric acid (Calbiochem) was used without additional purification. Type VIII peroxidase (R, = 2.8), isolated from horseradish peroxidase, and catalase (activity of 2000 units mg-‘) were obtained from Sigma. High phosphate buffers were prepared from reagent grade chemicals (NaH,PO, and Na,HPO,) and had an ionic strength of 0.5 M [7]. Low phosphate buffers used for trapping intermediates and for some kinetic studies were 0.5 M in NaCl plus 5 mM Na,HPO,, the pH being adjusted to the desired value by addition of HCl or NaOH. N,O-bis-trimethylsilylacetamide (BSA), N,N-trimethylsilyltrifluoroacetamide (BSTFA) and silylation grade pyridine and acetonitrile were obtained from Pierce Chemical Co. Deuterated BSA (d,-BSA) was obtained from Merck (St. Louis). Apparatus
Conventional electrochemical equipment was used [5,6]. U.V. studies of the enzymic oxidation of uric acid utilized either a Hitachi 100-80 microprocessor-controlled spectrophotometer or a Harrick Rapid Scanning Spectrophotometer. GC-MS studies employed a Hewlett-Packard Model 5985B instrument using packed columns (1.8 m X 2 mm i.d., SE-30). Details of the GC-MS procedure employed have been described elsewhere [5].
44
Procedure for enzymic oxidation
of uric acid
Stock solutions of type VIII peroxidase (0.4 pLM*), H,O, (600 pLM) and uric acid (600 pLM) were prepared fresh each day in an appropriate buffer. Normally, 0.7 cm3 each of the uric acid and peroxidase solutions were mixed in a 1.0 cm quartz spectrophotometer cell. The enzymic reaction was initiated by addition of 0.7 cm” of the H,O, stock solution. The course of the oxidation was monitored by repetitively recording spectra of the solution. When approximately 95% of the uric acid had been oxidized the reaction was terminated by addition of 0.7 cm3 of buffer solution containing 0.7 mg catalase. The reference cell for spectrophotometric studies contained all buffers, reagents and enzymes contained in the sample cell but did not contain uric acid. Isolation and identification
of intermediates
and products
The procedures used and information obtained to isolate and identify intermediates and products formed on thin-layer electrochemical oxidation of uric acid have been described in detail [5,6] and will not be repeated here. In order to identify some intermediates and the major organic products formed on enzymic oxidation of uric acid low phosphate buffers at pH 5.2 and 7.5 were used. The U.V.-absorbing intermediate was trapped in the following way: 0.04 cm3 of 3 mM uric acid in one of the latter buffers was placed in a small beaker and to it was added 0.04 cm3 of 4 PM peroxidase in the same buffer. The oxidation was initiated by addition of 0.05 cm3 of 6mM H,O,, again in the same buffer. After time periods ranging from 30 s to 2 min the solution was transferred from the beaker into a small vial (Reacti-vial, 2cm3 volume, Pierce Chemical Co.) maintained at -78°C (dry ice-acetone). The sudden and rapid decrease in the temperature of the reaction mixture served to quench the oxidation reaction and to prevent extensive decomposition of the U.V.-absorbing intermediate. The frozen solution was then dried by lyophilization with the vial maintained at temperatures below 0°C at all times. The dried mixture was then derivatized with BSA (10 mm3) in pyridine (100 mm3) in a sealed vial at 12O’C for 15 min. The derivatized mixture was then analyzed by GC-MS using, typically, a 5 mm3 sample injection. GC-MS studies revealed that maximal intermediate could be trapped when the enzyme reaction was quenched after 60-90 s. This observation was in accord with spectral (U.V.) studies of the oxidation reaction under the reaction conditions used to trap the U.V.-absorbing intermediate. A similar procedure was employed to identify the products of the enzymic reaction except that the oxidation was allowed to proceed for 15-20 minutes. The solution was then frozen and dried by lyophilization. Silylation of the product mixture was carried out with BSTFA (100 mm3) in acetonitrile (100 mm3) for 15 min
l
Based on a molecular
weight of 40,000
at 12O’C. Typically, GC-MS.
5 mm3 of the resulting
derivatized
mixture
was analyzed
by
RESULTS
The peroxidase-catalyzed oxidation of uric acid by H,O, was studied both in the low and high phosphate buffers. In low phosphate buffers the enzymic oxidation may be studied at pH between about 5 and 9 while in the high phosphate buffers it may be studied at pH between 5 and 8. At higher and lower pH values the enzymic oxidation proceeds very slowly. The electrochemical oxidation of uric acid has been studied in detail at pH between about 1.5 and 10 and has been reported elsewhere [6]. These results will not be described in detail here but for reference purposes typical cyclic voltammograms obtained at pH 7.5 in low (Fig. 1A) and high (Fig. 1B) phosphate buffers are shown in Fig. 1. Peak I, is the 2e--2H + electrooxidation of uric acid to a diimine and peak I, corresponds to the reverse reaction. Peak II, is thought to be due to reduction of one or more of the U.V.-absorbing intermediates formed by hydration of the latter species, i.e., intermediates III and/or BCA [(equation (l)]. Apart from the slight
-1.0
I
-0.5 I
0 I
I/ (V vs. s.c.e )
0.5 I 1
Fig. I. Cyclic voltammograms at the PGE of I m M uric acid at pH 7.5. Voltammogram (A) was obtained in 0.5 M NaCl plus 5 mM Na,HPO, and voltammogram (B) in phosphate buffer (NaH2P04-Na2HP0,) having an ionic strength of 0.5 M. Sweep rate: 200 mV s ‘_
46
broadening of peaks I, and I, in the low phosphate buffers and the appearance of a small adsorption post-peak after peak I, it appears that phosphate has only a minor effect on the electrochemical behavior [6]. The primary oxidation peak I, of uric acid is pH dependent with the peak potential, U, in high phosphate buffers being given by the expression: U, (pH 4-9) = [ 1.030 - 0.086 pH]V [4]. Considerable information concerning both the electrochemical and enzymic oxidations of uric acid was obtained from spectral studies of the reactions. Electrochemical oxidations were carried out in a thin-layer cell containing an opticallytransparent reticulated vitreous carbon (RVC) electrode. Enzymic oxidations were carried out in a conventional 1 cm quartz spectrophotometric cell. Some typical spectra obtained upon electrochemical and enzymic oxidations of uric acid at pH 7.5 in high phosphate buffer having an ionic strength of 0.5 M are presented in Fig. 2. Uric acid exhibits two U.V. absorption bands at A,,, = 290 nm and 230 nm (curves 1, Fig. 2A and 2C). Initiation of the enzymic and electrochemical oxidations causes the band at 290 nm to decrease and, correspondingly, the absorbance at 315-330 nm increases. The band at 230 nm, however, shifts to shorter wavelengths and grows. Curves 2 in Fig. 2 show the spectra when there is about maximal concentration of the U.V.-absorbing intermediate in the solution. After scanning curve2 in Figs. 2A and 2C the oxidation is terminated. In the electrochemical experiment this is done simply by open-circuiting the RVC electrode. In the enzymic experiment catalase is added (both to sample and reference cells). In both cases the intermediate bands decrease as noted between curves 2 and 3 in Figs. 2B and 2D. In the electrochemical experiment A versus t curves monitored at either the long or short wavelength band of the intermediate increase for a few seconds after termination of the electrolysis before starting to decrease (Fig. 3). This effect could not be observed in the enzyme system because of the time required to add and disperse the catalase necessary to terminate the reaction. Above pH 7.5, in high phosphate buffers, the enzymic oxidation of uric acid proceeded extremely slowly. However, in low phosphate buffers (0.5 M NaCl + 5 mM Na,HPO, adjusted to required pH with NaOH or HCl) the enzymic oxidation proceeded quite rapidly at pH values up to ca. 9.0. In both the high and low phosphate buffers at pH 2 7 within the latter restrictions, the thin-layer spectroelectrochemical and enzymic oxidations exhibited spectral behavior similar to that shown in Figs. 2 and 3. At pH values below the pK, of uric acid (5.75 [S]) somewhat different spectral behavior was noted. Upon electrochemical oxidation in high phosphate buffer at pH 5.2, for example, the U.V. bands of uric acid (X,, = 286 nm, 231 nm and 198 nm) decrease. The spectral changes shown between curves 1 and 2 in Fig. 4A occur over the course of 76 s electrolysis. If the electrolysis is terminated after scanning curve 2 in Fig. 4A then the changes shown in Fig. 4B occur. Thus, between about 330 nm and about 205 nm the absorbance first increases. Curve3 in Fig. 4B represents the spectrum of the solution at the point when this absorbance has reached its maximal value. The entire spectrum then decreases and curve 4 in Fig. 4B shows the spectrum 20 min after curve 3 was recorded. A clearer picture of the A versus t changes which occur following termination of the electrolysis is seen in Fig. 5. Thus, at 280 nm,
I
I
325
340
300
260
220
340
275
300
260
225
220
Fig. 2. Oxidation of uric acid in phosphate buffer (NaH,PO,-Na,HPO,) pH 7.5 having an ionic strength of 0.5 M. A, B: 200 PM uric acid, 200 uLM H,O, and 0.133 gM peroxidase. C, D: 1 mM uric acid undergoing electrooxidation at 0.8 V in a thin-layer cell containing an optically-transparent reticulated vitreous carbon electrode. In A and B each spectral sweep had a duration of 60 s with a 15 s gap between each spectral scan. In C and D each spectral sweep had a duration of 19 s and 38 s, respectively, with no significant time interval between each scan. Curves (1) are the initial spectrum of uric acid. Curves (2) represent the spectrum of the solution containing approximately maximal concentration of the U.V-absorbing intermediate. Curves (3) show the decay of the intermediate after termination of the electrochemical or enzymic oxidation.
where the changes are quite small (Fig. 5A) then upon terminating the electrolysis an initial decrease of absorbance occurs followed by an increase and then a further decrease. At 220 nm a sigmoidal increase of absorbance first occurs followed by a decrease (Fig. 5B). The spectral behavior observed upon enzymic oxidation of uric acid (Figs. 6A and 6B) was essentially identical to that observed on electrochemical oxidation (Fig. 4A, 4B). The spectra shown in Fig. 6B, particularly at short wavelengths, are somewhat distorted compared to those shown in Fig. 4B because of
400
600
Fig. 3. Absorbance at 308 nm nersu.s time curve obtained during and after electrochemical oxidation of I m M uric acid at 0.8 V in phosphate buffer pH 7.0 having an ionic strength of 0.5 M. The potential was applied at time zero and turned off after 50 s (arrow).
320
260
200
3zo
260
200
Fig. 4. Spectra observed on electrochemical oxidation of 1 mM uric acid in phosphate buffer pH 5.2 having an ionic strength of 0.5 M at 0.8 V in a thin-layer cell containing an optically transparent reticulated vitreous carbon electrode. Curves 1 are the initial spectrum of uric acid. (A) Spectra observed during the electrooxidation; after scanning curve 2 the electrolysis was terminated. Spectral sweeps of 19 s are shown. (B) Spectra observed after terminating the electrolysis. The changes observed between curves 2 and 3 first occur (sweeps of 38 s are shown). After scanning curve 3 approximately IS min elapsed before curve 4 was recorded.
49
Fig. 5. Absorbance at (A) 280 nm and (B) 220 nm oerswl time curves observed during and after electrochemical oxidation of I m M uric acid in phosphate buffer pH 5.2 (ionic strength=0.5 M) at 1.O V in a thin-layer cell. The potential was applied at time zero and then turned off after 50 s (arrow).
300
260
200
I
I
300
260
h(nm)
I 200
Fig. 6. Spectra observed upon enzymic oxidation of uric acid in phosphate buffer pH 5.2 (ionic strength=05 M). Initial solution contained 200 pM uric acid, 200 pM H20z and 0.13 PM type VIII peroxidase in a total volume of 2.1 cm3. (A) Spectra observed during the oxidation. Curve I is the initial spectrum of uric acid and spectral sweeps of 19 s duration are shown. After scanning curve 2 the oxidation was terminated by addition of catalase (0.7 cm3 having a concentration of I mg/cm3). (B) Spectra observed after termination of the electrolysis. Curve 2 is the first spectrum with the absorbance increasing to curve 3 (I9 s repetitive sweeps). Curve 4 was recorded approximately 10 min after curve 3.
50
the absorbance of catalase. A ~er~u.s t curves observed following termination of the enzymic oxidation of uric acid at pH 5.2 were similar to those shown in Figs. 5A and 5B. However, electrolyses may be stopped simply by open-circuiting the RVC working electrode so that A versus t curves (Figs. 5A, 5B) may be observed immediately following termination of the oxidation. In the enzyme-catalyzed reaction the oxidation is terminated by adding catalase and then stirring the solution to disperse the latter enzyme and to dislodge bubbles that form. This typically takes about 30 s. Thus, it was not possible to observe the initial decrease of absorbance noted, for example at 280 nm, following termination of the electrochemical oxidation. Thus, at pH 5.2 in high phosphate buffers at both 280 nm and 220 nm only an increase followed by a decrease in absorbance could be observed. In low phosphate buffers at pH 5.2 somewhat different spectral behavior was noted (Fig. 7A, B). Thus, upon initiation of both the electrochemical (Fig. 7A) and enzymic (Fig. 7B) oxidations the uric acid band at 283 nm begins to decrease. The band at 226 nm, however, immediately begins to grow and to shift to shorter wavelengths. Upon terminating the oxidations the absorbance between about 300 nm and 200 nm decreases. However, A versus t curves monitored at 220 nm revealed that following termination of the electrochemical oxidation the absorbance continued to increase for about 5-10 s and then decreases. Because of the time required to stop the enzymic reaction the latter phenomena could not be observed in the enzymic process. Following complete decay of the intermediate species generated
320
260
200
350
300
250
200
Fig. 7. Oxidation of uric acid in low phosphate buffer (0.5 M NaCl plus 5 m M Na,HPO,) pH 5.2. (A): I mM uric acid undergoing electrochemical oxidation at 0.8 V in a thin-layer cell containing an optically-transparent reticulated vitreous carbon electrode. Each spectral scan took 19 s with no significant time lapse between sweeps. Curve I is the initial spectrum of uric acid, the electrolysis being initiated following completion of this scan. (B): 200 p M uric acid, 200 p M HzO, and 0. I33 P M peroxidase. Each spectral scan took 60 s with a 15 s interval between scans. Curve I is the spectrum taken immediately after initiation of the enzymic oxidation.
electrochemically or enzymically the end product exhibited an U.V. band at X,,, = 204 nm. The behavior noted at pH 5.2 in low and high phosphate buffers upon both electrochemical and enzymic oxidation of uric acid was also observed at pH 5.6. The A versus t curves observed following electrochemical oxidation of uric acid in high phosphate buffers in a thin-layer cell containing an optically-transparent RVC electrode at pH 5.2 and 5.6 (Fig. 5) are indicative of a three-step process of the type shown in equation (4). At 220 nm (Fig. 5A), for example,
A must react to give a less strongly absorbing intermediate, B, which subsequently reacts to give a more strongly absorbing intermediate C. The latter then decomposes to weakly absorbing D. The scheme shown in equation (4) should result in A versus t behavior predicted by equation (5) [9] assuming that all steps are first order reactions. The (Y’Sin equation (5) are constants dependent upon the initial A = a, exp( -k,t)
+ (Y*exp( -k2t)
+ a3 exp( -k3t)
(5)
concentration and molar absorptivities of the various intermediates [9]. In low phosphate buffers at pH 5.2 and 5.6 and in both high and low phosphate buffers at pH 2 6 A versus t curves are indicative of only a two-step reaction. Analysis of the A versus t curves obtained following electrochemical oxidation of uric acid to obtain the first order rate constants shown in equations (l-5) was carried out using a non-linear least squares computational method which has been described elsewhere [6]. A portion of these results relevant to this report is presented in Table 1. The results obtained by a similar analysis of A versus t curves obtained following enzymic oxidation of uric acid are also shown in Table 1. In many instances the values of k, (Table 1) could not be evaluated in the enzymically oxidized solutions because of the time required to add and diperse the catalase required to terminate the reaction. Furthermore, analysis of A versus t curves recorded following electrochemical oxidation of uric acid generally gave considerable uncertainties in the rate constant values for the faster processes (Table 1). Such uncertainties were also noted in analyses of A versus t curves following enzymic oxidation. However, the rate constants measured for the last or slowest kinetic step appear to be less uncertain. A comparison of the latter rate constants measured following both electrochemical and enzymic oxidation reveals that they are very similar. The minor differences noted between these rate constants measured following electrochemical and enzymic oxidation of uric acid are probably related to the fact that the composition of the solutions are somewhat different. For example, the enzymically oxidized solution contains significant amounts of both peroxidase and catalase and any inorganic/organic contaminants present in these enzymes. However, it is possible to conclude from the spectral and kinetic studies that following both electrochemical and enzymic oxidation of uric acid, intermediates are formed which exhibit virtually identical spectral behavior and qualitatively similar kinetic behavior. A direct
-t-lPJln 8888 dddd tlulj 8888 6666
s&m~~
dddddddd $1 tl tl $1 $1 +I +I $1
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53
comparison of all first order rate constants kinetic step which may be observed exhibits Characterization
of intermediates
is not possible but the last or slowest the same rate constant.
and products
Some intermediates and all major organic products formed on electrochemical and enzymic oxidation of uric acid were characterized by carrying out the reactions in the low phosphate buffers. Electrochemical oxidations were performed in thin-layer cells, enzymic oxidations were carried out with relatively high concentrations of uric acid, H,O, and peroxidase (see EXPERIMENTAL). Intermediates were trapped by allowing the oxidations to proceed until maximal concentrations of U.V.-absorbing intermediates were present. Then, the reactions were quenched by rapidly cooling the reaction solutions to -78°C (dry ice-acetone). The samples were then lyophilized and, when dry, derivatized (silylated) with BSA in pyridine at 120°C. Since large excesses of phosphate interfere with the silylation reaction only the low phosphate buffers could be employed. A gas chromatogram (GC) of the intermediate trapped upon peroxidase oxidation of uric acid at pH 7.5 is shown in Fig. 8A. The GC shown in Fig. 8B is that obtained when a blank mixture (i.e., containing no uric acid) is carried through the same procedure. The multiple peaks noted in Fig. 8B are undoubtedly due to the silylated derivatives of the degradation products of peroxidase. No attempts were made to identify these peaks. The most noticeable difference between the GC in Fig. 8A and that in Fig. 8B is the peak which occurs at a retention time (t,<) of 28.7 min. Electron impact (EI) and chemical ionization (CI)* mass spectra of the species responsible for this peak are shown in Figs. 9A and 9B, respectively. The EI mass spectrum (Fig. 9A) shows a very small ion at m/e = 472 and a larger ion at m/e = 457 indicating loss of CH,. The latter suggests that the ion at m/e I= 472 is the molecular ion [9]. This conclusion was confirmed by the CI mass spectrum shown in Fig. 9B which exhibits a large ion at m/e= 473 corresponding to the expected (M + H)+ peak. In addition an ion at m/e = 501 corresponding to the (M + C,Hs)+ species characteristically formed in CI mass spectrometry using methane as the reactant gas. The base peak (m/e = 457) corresponds to the (M-CHs)+ species. The number of trimethylsilyl groups substituted onto the compound of molecular weight 472 was determined by derivatizing the enzymically generated and trapped intermediate with deuterated BSA (d,-BSA). The EI mass spectrum of the species responsible for the GC peak at t, - 28.7 min (Fig. 8A) exhibited a small peak at m/e= 508 (M+) and a larger peak at m/e= 490 (M-CD3)+. That the peak at m/e = 508 was the molecular ion was confirmed by CI mass spectrometry where a large peak at m/e = 509 (M + H)+ was formed (Fig. 9C) along with characteristic peaks at m/e = 490 (M-CD,)+, 537 (M + C,HS)+ and 549 (M t- C,H,)+. The difference in mass between the deuterated silyl derivative (MW = 508) and the normal silyl derivative (MW = 472) corresponds to 36 amu and proves that the *Using
methane
as the reactant
gas.
B
,,,,
~__ 17
19
21
23
25
27
29
31
33
35
17
19
21
23
25
27
29
31
33
35
Fig. 8. Total ion current chromatograms obtained intermediate formed upon peroxidase oxidation of sample prepared in an identical fashion to (A) except SE-30 on chromosorb W was used with He as the spectrometer was in the electron impact mode using
upon GC-MS of (A) the trapped U.V.-absorbing uric acid in low phosphate buffer pH 7.5 and (B) without uric acid. A 1.8 mX2 mm i.d. column of 3X carrier gas at a flow rate of 10 cm3/min. The mass an electron beam energy of 70 eV.
trapped intermediate species is substituted with four trimethylsilyl groups [lo] and hence has a molecular weight of 184. The various mass spectra reported above for the derivatized intermediate generated upon peroxidase-catalyzed oxidation of uric acid agree with those obtained for the intermediate generated by electrochemical oxidation of uric acid [5,6]. If the U.V.-absorbing intermediate(s) generated upon peroxidase oxidation of uric acid at pH 7.5 was allowed to decompose and then the solution was lyophilized, derivatized and analyzed by GC-MS, the chromatogram shown in Fig. 10A was obtained. The EI mass spectrum of the component eluted under peak 1 (Fig. 10A) is shown in Fig. 10B. This spectrum agrees with that observed for pentasilylated allantoin. GC peak 2 in Fig. 10A is due to the tetrasilylated derivative of uric acid. The U.V.-absorbing intermediate formed on peroxidase-catalyzed oxidation of uric acid in low phosphate buffer pH 5.2 was trapped and derivatized as described above. GC-MS of the silylated mixture gave the results shown in Fig. 11. Peak 1 in the gas chromatogram shown in Fig. 1lA gave a mass spectrum which was identical
55
A
280
300
1: t
320
340
360
380
400
420
440
480
460
500
1
057 I+-CH3 .J
80
60
0
IM+HI+ 473
4
J
5 %
40
t
2
280
300
320
340
360
360
400
420
440
460
480
500
540
560
100--
490
L'lc"'+
80 -
C
IMtHl+
20 -
I 340
360
380
400
1I
I 420
440
460
480
500
520
Fig. 9. (A) EI mass spectrum, (B) CI mass spectrum (methane reagent gas) of silylated intermediate formed upon peroxidase catalyzed oxidation of uric acid in low phosphate buffer pH 7.5 (peak under arrow in Fig. 8A). (C) CI mass spectrum of same intermediate derivatized by deuterated d,-BSA.
to that of authentic Shydroxyhydantoin-Scarboxamide (Fig. 11 B). Peak 2 gave the mass spectrum expected for allantoin (Fig. 11C) and peak 3, the major peak, gave a mass spectrum (Fig. 11 D) essentially identical to that observed for the tetrasilylated intermediate species observed at pH 7.5 (Fig. 9B). If the intermediate is allowed to decompose before lyophilization and derivatization peak 3 shown in Fig. 11A
56
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I IL 500
1 520
Fig. IO. (A) Total ion current chromatogram of the silylated product obtained after peroxidase-catalyzed oxidation of uric acid in low phosphate buffer pH 7.5. (B) EI (70 eV) mass spectrum of component eluted under peak 1 in (A).
disappears and the peaks due to 5-hydroxyhydantoin-5-carboxamide and allantoin grow. These GC-MS results clearly indicate the formation of an U.V.-absorbing intermediate at both pH 5.2 and 7.5 having a molecular weight of 184 and possessing four silylatable sites. Both electrochemical and peroxidase-catalyzed oxidation of uric acid gives rise to intermediates which have identical U.V. spectra (Figs. 2,4,6,7) and a molecular weight of 184. The mass spectra of the silylated intermediate trapped following both the electrochemical and enzymic oxidation of uric acid are also identical. On the basis of the kinetic analysis of A versus t curves and the time required to generate and trap the intermediate we have concluded that the intermediate identified is BCA [equations (1) and (2)]. However, BCA and its precursor imine-alcohol [III, equation 1 and III,, equation (2)J have exactly the same molecular weight and molecular formula and possess four silylatable positions. Thus, it is possible that a mixture of imine-alcohol and BCA are in fact trapped. These would be expected to exhibit very similar chromatographic behavior and mass spectra. The ultimate end products of both enzymic and electrochemical oxidation of uric acid oxidation in low phosphate buffers at pH > and < pK, of uric acid are also identical. In addition the spectral and kinetic behaviors observed in high phosphate buffers upon electrochemical and enzymic oxidation of uric acid are virtually identical.
A
16 loo-
aO-s
20
24
28
32
36
40
44
432
M-CH3 /
B
60-2 z 40-z
C
Fig. I I. (A) Total ion current chromatogram of the silylated mixture obtained after trapping the U.V.-absorbing intermediate(s) generated upon peroxidase-catalyzed oxidation of uric acid in low phosphate buffer pH 5.2; (B) CI mass spectrum of GC peak I (5-hydroxyhydantoin5carboxamide; (C) CI mass spectrum of GC peak 2 (allantoin) and (D) CI mass spectrum of GC peak 3 (intermediate of molecular weight 184).
58
A further piece of evidence that the enzymic reaction parallels the electrochemical oxidation is obtained from cyclic voltammetric experiments. Voltammogram A in Fig. 12 was obtained with uric acid in low phosphate buffer pH 7.5. Peak 1, corresponds to the 2e--2H + electrooxidation of uric acid to the quinonoid-diimine, peak I, to the reverse reaction. Peak II, is due to reduction of the putative imine-alcohol [III, equation (I)] and/or BCA. That this is so has been previously demonstrated by Wrona et al. [4] who found in high phosphate buffers that if the U.V.-absorbing intermediate is generated in a thin-layer cell then application of a potential corresponding to reduction peak II, causes a significant increase in the rate intermediate or of disappearance of the latter intermediate, i.e., the U.V.-absorbing intermediates
-1.5
I
-1.0
are reducible
-0.5
0.0
I I I u (V/S s CC)
in the peak II, reaction.
0.5
I
The voltammogram
shown
in
10
1
la
a.
A- A
04 1
::5 %-0
“C
‘c
-S B
Fig. 12. Cyclic voltammograms at the PGE in low phosphate buffer (0.5 M NaCl+ 5 m M Na, HPO,) pH 7.5. (A) 0.6 m M uric acid, initial sweep towards positive potentials. (B) 0.56 PM type VIII peroxidase. 0.6 m M H,O, initial sweep towards positive potentials. (C) 1 m M uric acid, 0.56 PM peroxidase. 0.6 m M H,O,, 30 s after initiation of the enzymic oxidation: initial sweep towards negative potentials. Sweep rate: 200 mV s-‘.
59
Fig. 12B is for a mixture of peroxidase and H,O, and clearly no significant oxidation or reduction peaks may be observed. Figure 12C is a voltammogram taken about 30 s after initiation of the enzymic oxidation of uric acid. Reduction peak II, is clearly present without having electrochemically oxidized uric acid. Thus, the intermediate species responsible for peak II, is clearly generated in the enzymic reaction. When the enzymic reaction is complete and sufficient time has elapsed for BCA to decompose to allantoin then peak II, disappears. CONCLUSIONS
It appears to be quite evident from this investigation between about pH 5.2 and 8 in buffers containing both low and high concentrations of phosphate that the enzymic oxidation of uric acid by H,O, catalyzed by type VIII peroxidase and the electrochemical oxidation yields intermediates that are spectrally, kinetically and analytically identical. These intermediates then decompose to identical end-products. Peroxidase catalysis is generally characterized by a one-electron oxidation of substrate molecules [11] and indeed Howell and Wyngaarden [ 121 have proposed that the oxidation of uric acid with methemoglobin and hydrogen peroxide leads to formation of a radical intermediate. This conclusion was based largely on the fact that uric acid methylated at the N(7) or N(9) position may be oxidized in the presence of methemoglobin/H,O, whereas 1,3,7,9_tetramethyluric acid is not. Thus, it was concluded that an unsubstituted N(7) or N(9) position is necessary to permit oxidation to occur and that the oxidation proceeded via a dehydrogenation reaction to give a radical. However, recent results from this laboratory [2] reveal that 7,9-dimethyluric acid may be oxidized by peroxidase/H,O, and hence the premise upon which the latter mechanism was proposed cannot be correct. The electrochemical studies carried out on uric acid reveal that it is oxidized in a quasi-reversible 2e‘--2H+ reaction. Unfortunately, it is not possible to decide whether this process in fact occurs via two very fast sequential 1 e‘ processes. The electrochemical and enzyrnic studies described in this and earlier reports from this laboratory certainly support the conclusion that both types of oxidations lead to the same intermediates and endlproducts. Thus, although the electrochemical investigations do not provide direct information concerning the mechanism of the initial enzyme oxidation they do provide considerable insights into the initial, unstable products of the biological process, i.e. diimine [II, equations (1), (2) and (3)], imine-alcohol [III, equations (1) and (2)] and BCA (equations (1), (2) and (3)] species. The mechanisms presented in equations (I)-(3), which were deduced on the basis of electrochemical experiments [I-6], are clearly quite complex. There are undoubtedly modified forms of these mechanisms which could be advanced to account for the observed spectral, kinetic and analytical information. However, at this time these mechanisms appear to be quite plausible and account well for the experimental observations. It is our view that such detailed mechanisms would be very difficult to elucidate based solely upon studies of the enzymic reactions and support our contention that electrochemical and related methodologies can provide
60
uniquely valuable insights and guidance nisms of enzyme processes.
into chemical
aspects
of the redox mecha-
ACKNOWLEDGEMENTS
This work was supported by the National Institutes of Health through Grant No. GM-21034-07. Additional support was provided by the Research Council of the University of Oklahoma. REFERECES 1 2 3 4 5 6 7 8 9 10
H.A. Marsh and G. Dryhurst, J. Electroanal. Chem., 95 (1979) 81. M.Z. Wrona and G. Dryhurst, Biochim. Biophys. Acta, 570 (1979) 371. R.N. Goyal, M.Z. Wrona and G. Dryhurst, Bioelectrcchem. Bioenerg., 7 (1980) 433. M.Z. Wrona, J.L. Owens and G. Dryhurst, J. Electroanal. Chem., 105 (1979) 295. A. Brajter-Toth and G. Dryhurst, J. Electroanal. Chem., 122 (1981) 205. R.N. Goyal, A. Brajter-Toth and G. Dryhurst, J. Electroanal. Chem., 131 (1981) 181. G.D. Christian and W.C. Purdy, J. Electroanal. Chem., 3 (1962) 363. F. Bergman and S. Dikstein, J. Amer. Chem. Sot., 77 (1955) 691. S.W. Benson, The Foundations of Chemical Kinetics, McGraw-Hill, New York, 1960, Chap. 3. J.A. McCloskey in Basic Principles in Nucleic Acid Chemistry, P.O.P. T’so (Editor), Academic Press, New York, 1974, Vol. I, p. 209. 11 I. Yamazaki in Molecular Mechanisms of Oxygen Activation, 0. Hayaishi (Editor), Academic Press, New York, 1974, Chap. 13. 12 R.R. Howell and J.B. Wyngaarden, J. Biol. Chem., 235 (1960) 3544.
COMPARISON OF THE PEROXIDASE-CATALYZED ELECTROCHEMICAL OXIDATION OF URIC ACID
479-A
R.N. GOYAL Department (Manuscript
*, ANNA
of Chemistry, received
BRAJTER-TOTH
l*,
University of Oklahoma,
August
GLENN
DRYHURST
Norman,
l
AND
** and N.T. NGUYEN
OK 73019 (U.S.A.)
7th 1981)
SUMMARY The oxidation of uric acid by hydrogen peroxide in the presence of type VIII peroxidase has been studied between pH 5.2 and 8. Intermediates generated in the reaction have been characterized in terms of their U.V. spectra and kinetics of decay. In addition at least one U.V.-absorbing intermediate has been trapped, converted to its trimethylsilyl derivative and identified by gas chromatography-mass spectrometry. This intermediate is l-carbohydroxy-2,4,6,8-tetraza-3,7-dioxo-4-ene-bicyclo-(3,3, O)-octane. At pHa7 the product is allantoin while at lower pH S-hydroxyhydantoin-5-carboxamide is also formed as a major product. The intermediates and products formed and spectral and kinetic measurements observed during and after peroxidase-catalyzed oxidation of uric acid are virtually identical to those noted upon electrochemical oxidation. It has thus been concluded that the mechanisms of electrochemical and enzymic oxidation of uric acid are, in a chemical sense, indentical.
INTRODUCTION
In recent reports from this laboratory it has been demonstrated that the electrochemical and peroxidase-cat,alyzed oxidation of uric acid [ 1,2] and various N-methylated uric acids [2-41 appear to proceed, in a chemical sense, by very similar if not identical mechanisms at around physiological pH. We have recently investigated the electrochemical oxidation of uric acid over a wide pH range (pH 1.5-9.5) in phosphate buffers using thin-layer spectroelectrochemistry to generate and study intermediates [5,6]. Some intermediates and all major products were separated and analyzed by gas chromatography-mass spectrometry (GC-MS). On the basis of these studies it has been concluded that the electrooxidation of uric acid is significantly more complex than was originally reported. At pH values > pK, for uric acid the electrochemical mechanism seems to be the most straightforward. Thus, the monoanion of uric acid [I, equation (l)] is electro-
l l l
Permanent address: Department of Chemistry, Roorkee University, Roorkee, India. * Current address: Department of Chemistry, University of Maine, Orono, Maine, U.S.A. ** To whom correspondence and reprint requests should be directed.
0302-4598/82/0000-OOOOo/$
02.75 0 1982 Elsevier Sequoia
S.A.
Peak
I,
-2 e-- 2 Hz +2P+2H+ Peak
Ic
I:
1
4 H,O,H+
(HOC)
1-co* 0 Allantoin
oxidized in a quasi-reversible 2e--2H+ reaction to a very unstable quinonoid-diimine (II, equation l), having a maximal half-life at around pH 8 (t,,, = 22 ms), which rapidly disappears in a pseudo first order hydration reaction [l] to give an anionic imine-alcohol [III, equation (l)]. This intermediate undergoes a ring contraction reaction to give l-carbohydroxy-2,4,6,8-tetraaza-3,7-dioxo-4-ene-bicyclo-(3,3,0)octane [BCA, equation (l)]. The intermediate III and BCA [equation (l)] may be inferred by analysis of absorbance versus time (A verms f) curves for decay of intermediate species generated upon electrooxidation of uric acid in a thin-layer cell containing an optically-transparent reticulated vitreous carbon (RVC) electrode. BCA decomposes to a single organic product, allantoin [equation (l)]. At pH a 6 phosphate appears to have no significant effect on the basic mechanism shown in equation (1). Between pH 3 and 5.6 a rather more complex reaction scheme appears to occur. In the presence of very low concentrations of phosphate (H,POi) neutral uric acid [l, equation (2)] is electrooxidized in a quasi-reversible 2e--2H+ reaction to the neutral quinonoid-diimine [II, equation (2)] which is very rapidly hydrated to the two isomeric imine-alcohols III, and III, [equation (2)]. Hydration of imine-alcohol III, [equation (2)] leads to the diol [VIII, equation (2)] and hence to 5-hydroxy-hydantoin-5-carboxamide [equation (2)] and, at pH 3 and below, alloxan [equation (2)]. Imine-alcohol III, [equation (2)] undergoes a ring contraction to give BCA [equation (2)] and hence allantoin. The values of k, and k, shown in equation (2) were determined by analysis of A versus t curves obtained following electrooxidation of uric acid in a thin-layer cell. In the presence of high concentrations of phosphate between pH 3 and 5.6 the putative quinonoid diimine [II, equation (3)] seems to be
-2
Ht-2
e-
I
H2O
(Illb)
1
1
-H+
“20
0
(VIII)
(2) H2O
k2
+ COOH
0 OH OH 0 Alloxan
(VI)
1
H,O 402 -NH3
“2$ f=O WJ H2N
1
-cop
0
5 Hydroxyh&antoin -5-carboxamide
(VIII
Allanloin
42
;2H+-2e-.
xkNho
+2Ht+2e-
“z-
o
,
;~‘f’-&>~
t OH-
u
(II) H20
In
HzP04
(Ilk)
H2Po4k,
H20 1
OH-
+
0 0 (Illb)
H2P04 (IV)
H2P04-
“20 i
(3) 0 0 (BCA)
Alloxan H20
H$
1 k,
t=o H&
H2N
0
5-Hydroxyh&ntoin -5-carboxamide 1 -co2
0 Allantoin
43
preferentially attacked by H,PO; rather than by water to give the phosphorylated intermediates III, and III, [equation (3)]. Intermediate III, [equation (3)] is further attacked by H,PO; (or water) to give VIII [equation (3)] and hence 5-hydroxyhydantoin-5-carboxamide or, at pH 3, small amounts of alloxan [equation (3)]. Analysis of A versus t curves indicates that three distinct intermediates are involved. This has been interpreted as shown in equation (3). Thus, intermediate III, [equation (3)] is rapidly hydrated to give IV [equation (3)] which undergoes a ring contraction to V [equation (3)J characterized by the pseudo first order rate constant k,. Intermediate V [equation (3)] then loses phosphate to give BCA [k,, equation (3)] which hydrates [k3, equation (3)] leading ultimately to allantoin. The work reported here was undertaken to investigate the enzymic oxidation of uric acid catalyzed by peroxidase. In particular, we were interested to find out if the detailed mechanisms elucidated from electrochemical studies were applicable to the enzyme catalyzed reactions. This study was undertaken not only to obtain fundamental information about the enzymic reaction but also to further justify our long-held contention that electrochemical studies of the redox mechanisms of biomolecules can provide uniquely valuable guidance and insights into the overall chemical pathways followed by substrate molecules in enzymic redox reactions. EXPERIMENTAL
Chemicals
Uric acid (Calbiochem) was used without additional purification. Type VIII peroxidase (R, = 2.8), isolated from horseradish peroxidase, and catalase (activity of 2000 units mg-‘) were obtained from Sigma. High phosphate buffers were prepared from reagent grade chemicals (NaH,PO, and Na,HPO,) and had an ionic strength of 0.5 M [7]. Low phosphate buffers used for trapping intermediates and for some kinetic studies were 0.5 M in NaCl plus 5 mM Na,HPO,, the pH being adjusted to the desired value by addition of HCl or NaOH. N,O-bis-trimethylsilylacetamide (BSA), N,N-trimethylsilyltrifluoroacetamide (BSTFA) and silylation grade pyridine and acetonitrile were obtained from Pierce Chemical Co. Deuterated BSA (d,-BSA) was obtained from Merck (St. Louis). Apparatus
Conventional electrochemical equipment was used [5,6]. U.V. studies of the enzymic oxidation of uric acid utilized either a Hitachi 100-80 microprocessor-controlled spectrophotometer or a Harrick Rapid Scanning Spectrophotometer. GC-MS studies employed a Hewlett-Packard Model 5985B instrument using packed columns (1.8 m X 2 mm i.d., SE-30). Details of the GC-MS procedure employed have been described elsewhere [5].
44
Procedure for enzymic oxidation
of uric acid
Stock solutions of type VIII peroxidase (0.4 pLM*), H,O, (600 pLM) and uric acid (600 pLM) were prepared fresh each day in an appropriate buffer. Normally, 0.7 cm3 each of the uric acid and peroxidase solutions were mixed in a 1.0 cm quartz spectrophotometer cell. The enzymic reaction was initiated by addition of 0.7 cm” of the H,O, stock solution. The course of the oxidation was monitored by repetitively recording spectra of the solution. When approximately 95% of the uric acid had been oxidized the reaction was terminated by addition of 0.7 cm3 of buffer solution containing 0.7 mg catalase. The reference cell for spectrophotometric studies contained all buffers, reagents and enzymes contained in the sample cell but did not contain uric acid. Isolation and identification
of intermediates
and products
The procedures used and information obtained to isolate and identify intermediates and products formed on thin-layer electrochemical oxidation of uric acid have been described in detail [5,6] and will not be repeated here. In order to identify some intermediates and the major organic products formed on enzymic oxidation of uric acid low phosphate buffers at pH 5.2 and 7.5 were used. The U.V.-absorbing intermediate was trapped in the following way: 0.04 cm3 of 3 mM uric acid in one of the latter buffers was placed in a small beaker and to it was added 0.04 cm3 of 4 PM peroxidase in the same buffer. The oxidation was initiated by addition of 0.05 cm3 of 6mM H,O,, again in the same buffer. After time periods ranging from 30 s to 2 min the solution was transferred from the beaker into a small vial (Reacti-vial, 2cm3 volume, Pierce Chemical Co.) maintained at -78°C (dry ice-acetone). The sudden and rapid decrease in the temperature of the reaction mixture served to quench the oxidation reaction and to prevent extensive decomposition of the U.V.-absorbing intermediate. The frozen solution was then dried by lyophilization with the vial maintained at temperatures below 0°C at all times. The dried mixture was then derivatized with BSA (10 mm3) in pyridine (100 mm3) in a sealed vial at 12O’C for 15 min. The derivatized mixture was then analyzed by GC-MS using, typically, a 5 mm3 sample injection. GC-MS studies revealed that maximal intermediate could be trapped when the enzyme reaction was quenched after 60-90 s. This observation was in accord with spectral (U.V.) studies of the oxidation reaction under the reaction conditions used to trap the U.V.-absorbing intermediate. A similar procedure was employed to identify the products of the enzymic reaction except that the oxidation was allowed to proceed for 15-20 minutes. The solution was then frozen and dried by lyophilization. Silylation of the product mixture was carried out with BSTFA (100 mm3) in acetonitrile (100 mm3) for 15 min
l
Based on a molecular
weight of 40,000
at 12O’C. Typically, GC-MS.
5 mm3 of the resulting
derivatized
mixture
was analyzed
by
RESULTS
The peroxidase-catalyzed oxidation of uric acid by H,O, was studied both in the low and high phosphate buffers. In low phosphate buffers the enzymic oxidation may be studied at pH between about 5 and 9 while in the high phosphate buffers it may be studied at pH between 5 and 8. At higher and lower pH values the enzymic oxidation proceeds very slowly. The electrochemical oxidation of uric acid has been studied in detail at pH between about 1.5 and 10 and has been reported elsewhere [6]. These results will not be described in detail here but for reference purposes typical cyclic voltammograms obtained at pH 7.5 in low (Fig. 1A) and high (Fig. 1B) phosphate buffers are shown in Fig. 1. Peak I, is the 2e--2H + electrooxidation of uric acid to a diimine and peak I, corresponds to the reverse reaction. Peak II, is thought to be due to reduction of one or more of the U.V.-absorbing intermediates formed by hydration of the latter species, i.e., intermediates III and/or BCA [(equation (l)]. Apart from the slight
-1.0
I
-0.5 I
0 I
I/ (V vs. s.c.e )
0.5 I 1
Fig. I. Cyclic voltammograms at the PGE of I m M uric acid at pH 7.5. Voltammogram (A) was obtained in 0.5 M NaCl plus 5 mM Na,HPO, and voltammogram (B) in phosphate buffer (NaH2P04-Na2HP0,) having an ionic strength of 0.5 M. Sweep rate: 200 mV s ‘_
46
broadening of peaks I, and I, in the low phosphate buffers and the appearance of a small adsorption post-peak after peak I, it appears that phosphate has only a minor effect on the electrochemical behavior [6]. The primary oxidation peak I, of uric acid is pH dependent with the peak potential, U, in high phosphate buffers being given by the expression: U, (pH 4-9) = [ 1.030 - 0.086 pH]V [4]. Considerable information concerning both the electrochemical and enzymic oxidations of uric acid was obtained from spectral studies of the reactions. Electrochemical oxidations were carried out in a thin-layer cell containing an opticallytransparent reticulated vitreous carbon (RVC) electrode. Enzymic oxidations were carried out in a conventional 1 cm quartz spectrophotometric cell. Some typical spectra obtained upon electrochemical and enzymic oxidations of uric acid at pH 7.5 in high phosphate buffer having an ionic strength of 0.5 M are presented in Fig. 2. Uric acid exhibits two U.V. absorption bands at A,,, = 290 nm and 230 nm (curves 1, Fig. 2A and 2C). Initiation of the enzymic and electrochemical oxidations causes the band at 290 nm to decrease and, correspondingly, the absorbance at 315-330 nm increases. The band at 230 nm, however, shifts to shorter wavelengths and grows. Curves 2 in Fig. 2 show the spectra when there is about maximal concentration of the U.V.-absorbing intermediate in the solution. After scanning curve2 in Figs. 2A and 2C the oxidation is terminated. In the electrochemical experiment this is done simply by open-circuiting the RVC electrode. In the enzymic experiment catalase is added (both to sample and reference cells). In both cases the intermediate bands decrease as noted between curves 2 and 3 in Figs. 2B and 2D. In the electrochemical experiment A versus t curves monitored at either the long or short wavelength band of the intermediate increase for a few seconds after termination of the electrolysis before starting to decrease (Fig. 3). This effect could not be observed in the enzyme system because of the time required to add and disperse the catalase necessary to terminate the reaction. Above pH 7.5, in high phosphate buffers, the enzymic oxidation of uric acid proceeded extremely slowly. However, in low phosphate buffers (0.5 M NaCl + 5 mM Na,HPO, adjusted to required pH with NaOH or HCl) the enzymic oxidation proceeded quite rapidly at pH values up to ca. 9.0. In both the high and low phosphate buffers at pH 2 7 within the latter restrictions, the thin-layer spectroelectrochemical and enzymic oxidations exhibited spectral behavior similar to that shown in Figs. 2 and 3. At pH values below the pK, of uric acid (5.75 [S]) somewhat different spectral behavior was noted. Upon electrochemical oxidation in high phosphate buffer at pH 5.2, for example, the U.V. bands of uric acid (X,, = 286 nm, 231 nm and 198 nm) decrease. The spectral changes shown between curves 1 and 2 in Fig. 4A occur over the course of 76 s electrolysis. If the electrolysis is terminated after scanning curve 2 in Fig. 4A then the changes shown in Fig. 4B occur. Thus, between about 330 nm and about 205 nm the absorbance first increases. Curve3 in Fig. 4B represents the spectrum of the solution at the point when this absorbance has reached its maximal value. The entire spectrum then decreases and curve 4 in Fig. 4B shows the spectrum 20 min after curve 3 was recorded. A clearer picture of the A versus t changes which occur following termination of the electrolysis is seen in Fig. 5. Thus, at 280 nm,
I
I
325
340
300
260
220
340
275
300
260
225
220
Fig. 2. Oxidation of uric acid in phosphate buffer (NaH,PO,-Na,HPO,) pH 7.5 having an ionic strength of 0.5 M. A, B: 200 PM uric acid, 200 uLM H,O, and 0.133 gM peroxidase. C, D: 1 mM uric acid undergoing electrooxidation at 0.8 V in a thin-layer cell containing an optically-transparent reticulated vitreous carbon electrode. In A and B each spectral sweep had a duration of 60 s with a 15 s gap between each spectral scan. In C and D each spectral sweep had a duration of 19 s and 38 s, respectively, with no significant time interval between each scan. Curves (1) are the initial spectrum of uric acid. Curves (2) represent the spectrum of the solution containing approximately maximal concentration of the U.V-absorbing intermediate. Curves (3) show the decay of the intermediate after termination of the electrochemical or enzymic oxidation.
where the changes are quite small (Fig. 5A) then upon terminating the electrolysis an initial decrease of absorbance occurs followed by an increase and then a further decrease. At 220 nm a sigmoidal increase of absorbance first occurs followed by a decrease (Fig. 5B). The spectral behavior observed upon enzymic oxidation of uric acid (Figs. 6A and 6B) was essentially identical to that observed on electrochemical oxidation (Fig. 4A, 4B). The spectra shown in Fig. 6B, particularly at short wavelengths, are somewhat distorted compared to those shown in Fig. 4B because of
400
600
Fig. 3. Absorbance at 308 nm nersu.s time curve obtained during and after electrochemical oxidation of I m M uric acid at 0.8 V in phosphate buffer pH 7.0 having an ionic strength of 0.5 M. The potential was applied at time zero and turned off after 50 s (arrow).
320
260
200
3zo
260
200
Fig. 4. Spectra observed on electrochemical oxidation of 1 mM uric acid in phosphate buffer pH 5.2 having an ionic strength of 0.5 M at 0.8 V in a thin-layer cell containing an optically transparent reticulated vitreous carbon electrode. Curves 1 are the initial spectrum of uric acid. (A) Spectra observed during the electrooxidation; after scanning curve 2 the electrolysis was terminated. Spectral sweeps of 19 s are shown. (B) Spectra observed after terminating the electrolysis. The changes observed between curves 2 and 3 first occur (sweeps of 38 s are shown). After scanning curve 3 approximately IS min elapsed before curve 4 was recorded.
49
Fig. 5. Absorbance at (A) 280 nm and (B) 220 nm oerswl time curves observed during and after electrochemical oxidation of I m M uric acid in phosphate buffer pH 5.2 (ionic strength=0.5 M) at 1.O V in a thin-layer cell. The potential was applied at time zero and then turned off after 50 s (arrow).
300
260
200
I
I
300
260
h(nm)
I 200
Fig. 6. Spectra observed upon enzymic oxidation of uric acid in phosphate buffer pH 5.2 (ionic strength=05 M). Initial solution contained 200 pM uric acid, 200 pM H20z and 0.13 PM type VIII peroxidase in a total volume of 2.1 cm3. (A) Spectra observed during the oxidation. Curve I is the initial spectrum of uric acid and spectral sweeps of 19 s duration are shown. After scanning curve 2 the oxidation was terminated by addition of catalase (0.7 cm3 having a concentration of I mg/cm3). (B) Spectra observed after termination of the electrolysis. Curve 2 is the first spectrum with the absorbance increasing to curve 3 (I9 s repetitive sweeps). Curve 4 was recorded approximately 10 min after curve 3.
50
the absorbance of catalase. A ~er~u.s t curves observed following termination of the enzymic oxidation of uric acid at pH 5.2 were similar to those shown in Figs. 5A and 5B. However, electrolyses may be stopped simply by open-circuiting the RVC working electrode so that A versus t curves (Figs. 5A, 5B) may be observed immediately following termination of the oxidation. In the enzyme-catalyzed reaction the oxidation is terminated by adding catalase and then stirring the solution to disperse the latter enzyme and to dislodge bubbles that form. This typically takes about 30 s. Thus, it was not possible to observe the initial decrease of absorbance noted, for example at 280 nm, following termination of the electrochemical oxidation. Thus, at pH 5.2 in high phosphate buffers at both 280 nm and 220 nm only an increase followed by a decrease in absorbance could be observed. In low phosphate buffers at pH 5.2 somewhat different spectral behavior was noted (Fig. 7A, B). Thus, upon initiation of both the electrochemical (Fig. 7A) and enzymic (Fig. 7B) oxidations the uric acid band at 283 nm begins to decrease. The band at 226 nm, however, immediately begins to grow and to shift to shorter wavelengths. Upon terminating the oxidations the absorbance between about 300 nm and 200 nm decreases. However, A versus t curves monitored at 220 nm revealed that following termination of the electrochemical oxidation the absorbance continued to increase for about 5-10 s and then decreases. Because of the time required to stop the enzymic reaction the latter phenomena could not be observed in the enzymic process. Following complete decay of the intermediate species generated
320
260
200
350
300
250
200
Fig. 7. Oxidation of uric acid in low phosphate buffer (0.5 M NaCl plus 5 m M Na,HPO,) pH 5.2. (A): I mM uric acid undergoing electrochemical oxidation at 0.8 V in a thin-layer cell containing an optically-transparent reticulated vitreous carbon electrode. Each spectral scan took 19 s with no significant time lapse between sweeps. Curve I is the initial spectrum of uric acid, the electrolysis being initiated following completion of this scan. (B): 200 p M uric acid, 200 p M HzO, and 0. I33 P M peroxidase. Each spectral scan took 60 s with a 15 s interval between scans. Curve I is the spectrum taken immediately after initiation of the enzymic oxidation.
electrochemically or enzymically the end product exhibited an U.V. band at X,,, = 204 nm. The behavior noted at pH 5.2 in low and high phosphate buffers upon both electrochemical and enzymic oxidation of uric acid was also observed at pH 5.6. The A versus t curves observed following electrochemical oxidation of uric acid in high phosphate buffers in a thin-layer cell containing an optically-transparent RVC electrode at pH 5.2 and 5.6 (Fig. 5) are indicative of a three-step process of the type shown in equation (4). At 220 nm (Fig. 5A), for example,
A must react to give a less strongly absorbing intermediate, B, which subsequently reacts to give a more strongly absorbing intermediate C. The latter then decomposes to weakly absorbing D. The scheme shown in equation (4) should result in A versus t behavior predicted by equation (5) [9] assuming that all steps are first order reactions. The (Y’Sin equation (5) are constants dependent upon the initial A = a, exp( -k,t)
+ (Y*exp( -k2t)
+ a3 exp( -k3t)
(5)
concentration and molar absorptivities of the various intermediates [9]. In low phosphate buffers at pH 5.2 and 5.6 and in both high and low phosphate buffers at pH 2 6 A versus t curves are indicative of only a two-step reaction. Analysis of the A versus t curves obtained following electrochemical oxidation of uric acid to obtain the first order rate constants shown in equations (l-5) was carried out using a non-linear least squares computational method which has been described elsewhere [6]. A portion of these results relevant to this report is presented in Table 1. The results obtained by a similar analysis of A versus t curves obtained following enzymic oxidation of uric acid are also shown in Table 1. In many instances the values of k, (Table 1) could not be evaluated in the enzymically oxidized solutions because of the time required to add and diperse the catalase required to terminate the reaction. Furthermore, analysis of A versus t curves recorded following electrochemical oxidation of uric acid generally gave considerable uncertainties in the rate constant values for the faster processes (Table 1). Such uncertainties were also noted in analyses of A versus t curves following enzymic oxidation. However, the rate constants measured for the last or slowest kinetic step appear to be less uncertain. A comparison of the latter rate constants measured following both electrochemical and enzymic oxidation reveals that they are very similar. The minor differences noted between these rate constants measured following electrochemical and enzymic oxidation of uric acid are probably related to the fact that the composition of the solutions are somewhat different. For example, the enzymically oxidized solution contains significant amounts of both peroxidase and catalase and any inorganic/organic contaminants present in these enzymes. However, it is possible to conclude from the spectral and kinetic studies that following both electrochemical and enzymic oxidation of uric acid, intermediates are formed which exhibit virtually identical spectral behavior and qualitatively similar kinetic behavior. A direct
-t-lPJln 8888 dddd tlulj 8888 6666
s&m~~
dddddddd $1 tl tl $1 $1 +I +I $1
ssssss~~ ombb~v,
dddddddd
53
comparison of all first order rate constants kinetic step which may be observed exhibits Characterization
of intermediates
is not possible but the last or slowest the same rate constant.
and products
Some intermediates and all major organic products formed on electrochemical and enzymic oxidation of uric acid were characterized by carrying out the reactions in the low phosphate buffers. Electrochemical oxidations were performed in thin-layer cells, enzymic oxidations were carried out with relatively high concentrations of uric acid, H,O, and peroxidase (see EXPERIMENTAL). Intermediates were trapped by allowing the oxidations to proceed until maximal concentrations of U.V.-absorbing intermediates were present. Then, the reactions were quenched by rapidly cooling the reaction solutions to -78°C (dry ice-acetone). The samples were then lyophilized and, when dry, derivatized (silylated) with BSA in pyridine at 120°C. Since large excesses of phosphate interfere with the silylation reaction only the low phosphate buffers could be employed. A gas chromatogram (GC) of the intermediate trapped upon peroxidase oxidation of uric acid at pH 7.5 is shown in Fig. 8A. The GC shown in Fig. 8B is that obtained when a blank mixture (i.e., containing no uric acid) is carried through the same procedure. The multiple peaks noted in Fig. 8B are undoubtedly due to the silylated derivatives of the degradation products of peroxidase. No attempts were made to identify these peaks. The most noticeable difference between the GC in Fig. 8A and that in Fig. 8B is the peak which occurs at a retention time (t,<) of 28.7 min. Electron impact (EI) and chemical ionization (CI)* mass spectra of the species responsible for this peak are shown in Figs. 9A and 9B, respectively. The EI mass spectrum (Fig. 9A) shows a very small ion at m/e = 472 and a larger ion at m/e = 457 indicating loss of CH,. The latter suggests that the ion at m/e I= 472 is the molecular ion [9]. This conclusion was confirmed by the CI mass spectrum shown in Fig. 9B which exhibits a large ion at m/e= 473 corresponding to the expected (M + H)+ peak. In addition an ion at m/e = 501 corresponding to the (M + C,Hs)+ species characteristically formed in CI mass spectrometry using methane as the reactant gas. The base peak (m/e = 457) corresponds to the (M-CHs)+ species. The number of trimethylsilyl groups substituted onto the compound of molecular weight 472 was determined by derivatizing the enzymically generated and trapped intermediate with deuterated BSA (d,-BSA). The EI mass spectrum of the species responsible for the GC peak at t, - 28.7 min (Fig. 8A) exhibited a small peak at m/e= 508 (M+) and a larger peak at m/e= 490 (M-CD3)+. That the peak at m/e = 508 was the molecular ion was confirmed by CI mass spectrometry where a large peak at m/e = 509 (M + H)+ was formed (Fig. 9C) along with characteristic peaks at m/e = 490 (M-CD,)+, 537 (M + C,HS)+ and 549 (M t- C,H,)+. The difference in mass between the deuterated silyl derivative (MW = 508) and the normal silyl derivative (MW = 472) corresponds to 36 amu and proves that the *Using
methane
as the reactant
gas.
B
,,,,
~__ 17
19
21
23
25
27
29
31
33
35
17
19
21
23
25
27
29
31
33
35
Fig. 8. Total ion current chromatograms obtained intermediate formed upon peroxidase oxidation of sample prepared in an identical fashion to (A) except SE-30 on chromosorb W was used with He as the spectrometer was in the electron impact mode using
upon GC-MS of (A) the trapped U.V.-absorbing uric acid in low phosphate buffer pH 7.5 and (B) without uric acid. A 1.8 mX2 mm i.d. column of 3X carrier gas at a flow rate of 10 cm3/min. The mass an electron beam energy of 70 eV.
trapped intermediate species is substituted with four trimethylsilyl groups [lo] and hence has a molecular weight of 184. The various mass spectra reported above for the derivatized intermediate generated upon peroxidase-catalyzed oxidation of uric acid agree with those obtained for the intermediate generated by electrochemical oxidation of uric acid [5,6]. If the U.V.-absorbing intermediate(s) generated upon peroxidase oxidation of uric acid at pH 7.5 was allowed to decompose and then the solution was lyophilized, derivatized and analyzed by GC-MS, the chromatogram shown in Fig. 10A was obtained. The EI mass spectrum of the component eluted under peak 1 (Fig. 10A) is shown in Fig. 10B. This spectrum agrees with that observed for pentasilylated allantoin. GC peak 2 in Fig. 10A is due to the tetrasilylated derivative of uric acid. The U.V.-absorbing intermediate formed on peroxidase-catalyzed oxidation of uric acid in low phosphate buffer pH 5.2 was trapped and derivatized as described above. GC-MS of the silylated mixture gave the results shown in Fig. 11. Peak 1 in the gas chromatogram shown in Fig. 1lA gave a mass spectrum which was identical
55
A
280
300
1: t
320
340
360
380
400
420
440
480
460
500
1
057 I+-CH3 .J
80
60
0
IM+HI+ 473
4
J
5 %
40
t
2
280
300
320
340
360
360
400
420
440
460
480
500
540
560
100--
490
L'lc"'+
80 -
C
IMtHl+
20 -
I 340
360
380
400
1I
I 420
440
460
480
500
520
Fig. 9. (A) EI mass spectrum, (B) CI mass spectrum (methane reagent gas) of silylated intermediate formed upon peroxidase catalyzed oxidation of uric acid in low phosphate buffer pH 7.5 (peak under arrow in Fig. 8A). (C) CI mass spectrum of same intermediate derivatized by deuterated d,-BSA.
to that of authentic Shydroxyhydantoin-Scarboxamide (Fig. 11 B). Peak 2 gave the mass spectrum expected for allantoin (Fig. 11C) and peak 3, the major peak, gave a mass spectrum (Fig. 11 D) essentially identical to that observed for the tetrasilylated intermediate species observed at pH 7.5 (Fig. 9B). If the intermediate is allowed to decompose before lyophilization and derivatization peak 3 shown in Fig. 11A
56
I,
I
20
24
I
I
I
I
28
I,
32
1
I
I,
40
36
44
B 60 -
Me3SI,
s 40 - : 8
Me&f&0
‘0 5 %
:IMe3
SiMeS SiMe,
M+ 318
20 -z a
I 320
II
I 340
360
I 380
I
400
I,
I
420
m/e
II
1
440
460
M-CH, 503
I 480
I IL 500
1 520
Fig. IO. (A) Total ion current chromatogram of the silylated product obtained after peroxidase-catalyzed oxidation of uric acid in low phosphate buffer pH 7.5. (B) EI (70 eV) mass spectrum of component eluted under peak 1 in (A).
disappears and the peaks due to 5-hydroxyhydantoin-5-carboxamide and allantoin grow. These GC-MS results clearly indicate the formation of an U.V.-absorbing intermediate at both pH 5.2 and 7.5 having a molecular weight of 184 and possessing four silylatable sites. Both electrochemical and peroxidase-catalyzed oxidation of uric acid gives rise to intermediates which have identical U.V. spectra (Figs. 2,4,6,7) and a molecular weight of 184. The mass spectra of the silylated intermediate trapped following both the electrochemical and enzymic oxidation of uric acid are also identical. On the basis of the kinetic analysis of A versus t curves and the time required to generate and trap the intermediate we have concluded that the intermediate identified is BCA [equations (1) and (2)]. However, BCA and its precursor imine-alcohol [III, equation 1 and III,, equation (2)J have exactly the same molecular weight and molecular formula and possess four silylatable positions. Thus, it is possible that a mixture of imine-alcohol and BCA are in fact trapped. These would be expected to exhibit very similar chromatographic behavior and mass spectra. The ultimate end products of both enzymic and electrochemical oxidation of uric acid oxidation in low phosphate buffers at pH > and < pK, of uric acid are also identical. In addition the spectral and kinetic behaviors observed in high phosphate buffers upon electrochemical and enzymic oxidation of uric acid are virtually identical.
A
16 loo-
aO-s
20
24
28
32
36
40
44
432
M-CH3 /
B
60-2 z 40-z
C
Fig. I I. (A) Total ion current chromatogram of the silylated mixture obtained after trapping the U.V.-absorbing intermediate(s) generated upon peroxidase-catalyzed oxidation of uric acid in low phosphate buffer pH 5.2; (B) CI mass spectrum of GC peak I (5-hydroxyhydantoin5carboxamide; (C) CI mass spectrum of GC peak 2 (allantoin) and (D) CI mass spectrum of GC peak 3 (intermediate of molecular weight 184).
58
A further piece of evidence that the enzymic reaction parallels the electrochemical oxidation is obtained from cyclic voltammetric experiments. Voltammogram A in Fig. 12 was obtained with uric acid in low phosphate buffer pH 7.5. Peak 1, corresponds to the 2e--2H + electrooxidation of uric acid to the quinonoid-diimine, peak I, to the reverse reaction. Peak II, is due to reduction of the putative imine-alcohol [III, equation (I)] and/or BCA. That this is so has been previously demonstrated by Wrona et al. [4] who found in high phosphate buffers that if the U.V.-absorbing intermediate is generated in a thin-layer cell then application of a potential corresponding to reduction peak II, causes a significant increase in the rate intermediate or of disappearance of the latter intermediate, i.e., the U.V.-absorbing intermediates
-1.5
I
-1.0
are reducible
-0.5
0.0
I I I u (V/S s CC)
in the peak II, reaction.
0.5
I
The voltammogram
shown
in
10
1
la
a.
A- A
04 1
::5 %-0
“C
‘c
-S B
Fig. 12. Cyclic voltammograms at the PGE in low phosphate buffer (0.5 M NaCl+ 5 m M Na, HPO,) pH 7.5. (A) 0.6 m M uric acid, initial sweep towards positive potentials. (B) 0.56 PM type VIII peroxidase. 0.6 m M H,O, initial sweep towards positive potentials. (C) 1 m M uric acid, 0.56 PM peroxidase. 0.6 m M H,O,, 30 s after initiation of the enzymic oxidation: initial sweep towards negative potentials. Sweep rate: 200 mV s-‘.
59
Fig. 12B is for a mixture of peroxidase and H,O, and clearly no significant oxidation or reduction peaks may be observed. Figure 12C is a voltammogram taken about 30 s after initiation of the enzymic oxidation of uric acid. Reduction peak II, is clearly present without having electrochemically oxidized uric acid. Thus, the intermediate species responsible for peak II, is clearly generated in the enzymic reaction. When the enzymic reaction is complete and sufficient time has elapsed for BCA to decompose to allantoin then peak II, disappears. CONCLUSIONS
It appears to be quite evident from this investigation between about pH 5.2 and 8 in buffers containing both low and high concentrations of phosphate that the enzymic oxidation of uric acid by H,O, catalyzed by type VIII peroxidase and the electrochemical oxidation yields intermediates that are spectrally, kinetically and analytically identical. These intermediates then decompose to identical end-products. Peroxidase catalysis is generally characterized by a one-electron oxidation of substrate molecules [11] and indeed Howell and Wyngaarden [ 121 have proposed that the oxidation of uric acid with methemoglobin and hydrogen peroxide leads to formation of a radical intermediate. This conclusion was based largely on the fact that uric acid methylated at the N(7) or N(9) position may be oxidized in the presence of methemoglobin/H,O, whereas 1,3,7,9_tetramethyluric acid is not. Thus, it was concluded that an unsubstituted N(7) or N(9) position is necessary to permit oxidation to occur and that the oxidation proceeded via a dehydrogenation reaction to give a radical. However, recent results from this laboratory [2] reveal that 7,9-dimethyluric acid may be oxidized by peroxidase/H,O, and hence the premise upon which the latter mechanism was proposed cannot be correct. The electrochemical studies carried out on uric acid reveal that it is oxidized in a quasi-reversible 2e‘--2H+ reaction. Unfortunately, it is not possible to decide whether this process in fact occurs via two very fast sequential 1 e‘ processes. The electrochemical and enzyrnic studies described in this and earlier reports from this laboratory certainly support the conclusion that both types of oxidations lead to the same intermediates and endlproducts. Thus, although the electrochemical investigations do not provide direct information concerning the mechanism of the initial enzyme oxidation they do provide considerable insights into the initial, unstable products of the biological process, i.e. diimine [II, equations (1), (2) and (3)], imine-alcohol [III, equations (1) and (2)] and BCA (equations (1), (2) and (3)] species. The mechanisms presented in equations (I)-(3), which were deduced on the basis of electrochemical experiments [I-6], are clearly quite complex. There are undoubtedly modified forms of these mechanisms which could be advanced to account for the observed spectral, kinetic and analytical information. However, at this time these mechanisms appear to be quite plausible and account well for the experimental observations. It is our view that such detailed mechanisms would be very difficult to elucidate based solely upon studies of the enzymic reactions and support our contention that electrochemical and related methodologies can provide
60
uniquely valuable insights and guidance nisms of enzyme processes.
into chemical
aspects
of the redox mecha-
ACKNOWLEDGEMENTS
This work was supported by the National Institutes of Health through Grant No. GM-21034-07. Additional support was provided by the Research Council of the University of Oklahoma. REFERECES 1 2 3 4 5 6 7 8 9 10
H.A. Marsh and G. Dryhurst, J. Electroanal. Chem., 95 (1979) 81. M.Z. Wrona and G. Dryhurst, Biochim. Biophys. Acta, 570 (1979) 371. R.N. Goyal, M.Z. Wrona and G. Dryhurst, Bioelectrcchem. Bioenerg., 7 (1980) 433. M.Z. Wrona, J.L. Owens and G. Dryhurst, J. Electroanal. Chem., 105 (1979) 295. A. Brajter-Toth and G. Dryhurst, J. Electroanal. Chem., 122 (1981) 205. R.N. Goyal, A. Brajter-Toth and G. Dryhurst, J. Electroanal. Chem., 131 (1981) 181. G.D. Christian and W.C. Purdy, J. Electroanal. Chem., 3 (1962) 363. F. Bergman and S. Dikstein, J. Amer. Chem. Sot., 77 (1955) 691. S.W. Benson, The Foundations of Chemical Kinetics, McGraw-Hill, New York, 1960, Chap. 3. J.A. McCloskey in Basic Principles in Nucleic Acid Chemistry, P.O.P. T’so (Editor), Academic Press, New York, 1974, Vol. I, p. 209. 11 I. Yamazaki in Molecular Mechanisms of Oxygen Activation, 0. Hayaishi (Editor), Academic Press, New York, 1974, Chap. 13. 12 R.R. Howell and J.B. Wyngaarden, J. Biol. Chem., 235 (1960) 3544.