mechanism of the chlorination reaction catalyzed by horseradish

Vol. 254. No. 9. lswe of May 10. pp. 3175-3181, 1979 Prrnted in U.S.A.

Mechanism of the Chlorination Reaction Catalyzed by Horseradish Peroxidase with Chlorite*

(Received for publication, June 23, 1978)

W. Donald Hewson and Lowell P. Hager From the Roger Adams Laboratory, Department of Biochemistry, University of Illinois, Urbana, Illinois 61801

Horseradish peroxidase and chlorite, NaC102, are able to catalyze chlorination of monochlorodimedone to form dichlorodimedone. Catalytic amounts of horseradish peroxidase act to disproportionate chlorite forming chlorine dioxide and chloride ion. The chlorine dioxide thus formed is responsible for the chlorination of monochlorodimedone. It was previously thought (Chiang, R., Rand-Meir, T., Makino, R., and Hager, L. P. (1976) J. Biol. Chem 251, 6340-6346) that the initial transient intermediate formed between horseradish peroxidase and chlorite was a halogenating intermediate and was called Compound X. However, complete transient state spectra of the enzyme species formed directly from horseradish peroxidase with chlorite shows this intermediate to be Compound II. This result is the first recognized example of direct oxidation of native horseradish peroxidase to Compound II. The disproportionation of chlorite by horseradish peroxidase was investigated in the absence of a halogen ac- ceptor. At pH 5.05, 5 mol of chlorite are disproportionated to 4 mol of chlorine dioxide and 1 mol of chloride ion. Chlorine dioxide yields are limited by the inactivation of the enzyme by chlorine dioxide. A maximum yield of 2.5 x lo3 nmol of chlorine dioxide/nmol of horseradish peroxidase was obtained by optimizing the pH and optimizing the enzyme/chlorite ratio. Titration of horseradish peroxidase with chlorite showed a 1:l molar stoichiometry for the formation of Compound I. The reaction between the enzyme and excess chlorite failed to produce measurable quantities of hydrogen peroxide. The reaction of chlorine dioxide with monochlorodimedone was also studied in the absence of horseradish peroxidase and chlorite. Chlorine dioxide and monochlorodimedone react with a 1:l molar stoichiometry to form 1 mol of protons, 0.4 mol of dichlorodimedone, and 0.6 mol of chlorite.

Several categories of oxidants have been used to form the oxidized intermediates, Compound I and Compound II, of horseradish peroxidase (EC 1.11.1.7, donor: H202 oxidoreduc- tase). Compound I and Compound II are known to be 2 and 1 oxidation eq, respectively, above the oxidation state of the native enzyme. In addition to hydrogen peroxide, alkyl hydro- peroxides (l-6), substituted perbenzoic acids (1, 3, 6-8), and oxyhalogen acids (5, 9, 10) have been employed to form Compounds I and II. In particular, George (5) discovered that hypochlorite, ClO-, and chlorite, CIOZ-, were capable of form-

* This work was supported by grants from the National Institute of General Medical Sciences (GM 07768) and the National Science Foundation (PCM 76-12547). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ing these oxidized enzyme intermediates. Chlorate, C103-, did not oxidize the enzyme (5), at least at an appreciable rate. Shortly thereafter, Chance (9) studied the chlorite reaction with horseradish peroxidase to determine whether Compound I formation preceded Compound II formation. In addition Chance posed the question of whether or not Compound II which is formed by the oxyhalogen acids has the same reactivity as the intermediate formed from peroxide substrates. It was concluded (9) that chlorite initiates the same sequence of reactions as peroxide, and that Compound II formed from either chlorite or peroxide has the same reactivity with nitrite.

Interest in the reaction between horseradish peroxidase and chlorite was recently renewed by the discovery that this system could perform a chlorination reaction (11). Although the horseradish peroxidase and hydrogen peroxide system is able to utilize iodide ion to perform iodination reactions, this system is not able to chlorinate by using hydrogen peroxide and chloride ion as substrate (12). However, chloroperoxidase and hydrogen peroxide can utilize chloride ion to catalyze electrophihc chlorination (11, 13). When horseradish peroxidase and chlorite were mixed in the presence of 5,5-dimethyl- 2-cNoro-1,3cyclohexanedione (monochlorodimedone or MCD)’ the monochlorodimedone was chlorinated to form 5,5- dimethyl-2,2-dichloro-1,3cyclohexanedione (dichlorodimedone or DCD) (11). Radioactive labeling experiments using ?l demonstrated that the chlorine atom in chlorite was incorporated directly into the dichlorodimedone, even in the presence of a large excess of chloride ion. This result ruled out chemical halogenation via a hypochiorite intermediate and led to the hypothesis of an intermediate chlorinated enzymatic species (14). Spectral evidence for what was believed to be the new chlorinated intermediate was obtained with a rapid scanning spectrophotometer in combination with a stopped flow apparatus (15,16). This technique showed the direct conversion of native horseradish peroxidase to a species with an absorption maximum near 417 nm. Although it was realized that Compound II had an absorption peak near 417 nm, it was concluded that the 417 nm absorbing species was a new oxidized, chlorinated enzyme intermediate and was named Compound X (14, 16). Compound II was originally ruled out because early experiments indicated that Compound I formation always preceded the appearance of Compound II.

This paper describes further studies on the nature of the chlorination of monochlorodimedone using horseradish peroxidase and chlorite.

EXPERIMENTAL PROCEDURES

Horseradish peroxidase was purchased from Sigma Chemical Co. as a salt-free powder. The RZ value, the ratio of absorbances at 403

’ The abbreviations and trivial names used are: MCD, monochlorodimedone, 5,5-dimethyl-2-chloro-1,3-cyclohexanedione; DCD, dichlorodimedone, 5,5-dimethyl-2,2-dichloro-1,3-cyclohexanedione.

3175

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3176 The Reaction of Chlorite with Horseradish Peroxidase

and 280 nm, of the commercial sample was 1.38. Isozyme C, according to the notation of Shannon et al. (17) and of Paul and Stigbrand (18), was purified as previously reported (11). Purified isozyme C had an RZ value of 3.37 (19). Enzyme was stored at 4°C in a 5 mM Tris/ nitrate buffer, pH 8.40. The concentration of horseradish peroxidase was measured spectrophotometrically at 403 nm using a molar absorptivity Of 1.02 X 10” Mm’ Cm- (3).

Sodium chlorite from Matheson, Coleman and Bell was shown to be of high purity, >98%, by iodometry with standard sodium thiosul- fate solution. Stock solutions of NaC102 were freshly prepared gravimetrically each day and were contained in a black-painted flask to minimize photodecomposition (20). Since the pK, of chlorous acid, HC101, is 1.8 at an ionic strength of 0.1 (21), the pH of the NaC102 stock solutions, which were approximately 0.1 M, in distilled water was about 7.4. Thus, the acid-catalyzed disproportionation of chlorous acid which occurs at low pH was avoided (21).

tions used (24,25,30-32), but with the larger quantities of horseradish peroxidase the enzyme did contribute significantly to the absorbance increase. To correct for the enzyme absorbance, the Cl02 was degassed with nitrogen from a control solution; and the spectrum of the enzyme, which was denatured, was recorded. A molar absorptivity for denatured horseradish peroxidase at 359 nm of 2.1 x lo4 M-l cm-’ was calculated from the known amount of enzyme. Depending upon the amount of enzyme used, this correction factor varied from about 1 to 18% of the absorbance change.

Chlorine dioxide, ClOz, was prepared by either of two chemical reactions: the disproportionation of chlorous acid in strongly acid aqueous medium (21-26), or the oxidation of chlorite by potassium persulfate (27). In either case the gaseous Cl02 remained dissolved in the aqueous reactant solution. The Cl02 was displaced from the reactant solution with air. Cl02 is highly reactive and potentially explosive when not largely diluted with air. A vacuum was used to draw air through the reactant solution and the air stream now containing some CI02 passed through water in a receiver flask. This Cl02 solution was used as the stock solution. Between the reactant and receiver flasks the mixture of air and Cl02 was drawn through a U-tube containing solid granular NaC102 to remove by chemical reaction any traces of chlorine gas or hypochlorous acid (22,27). Glass wool dust traps before and after the U-tube filtered out airborn droplets and particulate NaC102. The entire Cl02 generation apparatus was operated at 4°C in subdued light (22). By painting the reactant flask black, a reserve of Cl02 could last about a week. Due to the unstable nature of C102, fresh stock solutions were prepared immediately before an experiment, kept in the dark, and used for no longer than 3 h. The concentration of Cl02 was determined spectrophotometrically at 359 nm using a molar absorptivity of 1.2 x 19’ Mm’ cm-’ (22, 25, 28).

The synthesis of mono- and dichlorodimedone have been described (29). The pH measurement were made with a Brinkmann Metrohm model 103 pH meter outfitted with a combination glass electrode. Fresh commercial standard buffers were used to calibrate the meter to f0.02 pH unit. Glass-distilled water was used for all aqueous solutions, and buffers were prepared from reagent grade materials. Absorbance measurements and spectra were routinely obtained with a Cary 15 or 219 spectrophotometer. For the determination of oxygen concentrations in aqueous solutions, an oxygen electrode, model 5331, from Yellow Springs Instrument Co. was used in combination with a Gilson model K oxygraph. The oxygen electrode was calibrated by adding known amounts of hydrogen peroxide to the solution containing catalytic amounts of beef liver catalase, Sigma Chemical Co., twice crystallized. Before mixing, all solutions were allowed to stand at ambient temperature in open containers for at least 30 min. This was done to avoid anomalies due to different levels of air saturation. The concentration of hydrogen peroxide was determined as previously reported (1). A stopped flow apparatus (16) was used for fast kinetic measurements, and electron paramagnetic resonance spectra were recorded with a Varian E9 spectrometer with the samples at -196°C.

Ascending thin layer chromatography of the reaction products of monochlorodimedone and C102 was carried out with commercial plates, Eastman 6060, or E. Merck F-254 silica gel. Both thin layer adsorbents contained fluorescent indicators. However, a combination of the iodine vapor and fluorescent indication techniques proved to be the most satisfactory method of visualizing compounds on the chromatogram. The ultraviolet transparency of dichlorodimedone required the combined techniques. Of many solvent systems tried, including those already published for these compounds (ll), I-butanol provided the best separation. 1-Butanol, however, required about 1 h to fully develop a chromatogram (5 X 10 cm). Column chromatography with silica gel, 0.05 to 0.2 mm, grade II to III, was used to separate large amounts of products. A column (1.3 x 30 cm) was eluted with ethyl acetate.

The horseradish peroxidase-catalyzed evolution of Cl02 from NaC102 solutions was studied spectrophotometricaly. The quantity of Cl02 evolved was calculated from the absorbance increase at 359 nm. To initiate C102 evolution, microliter volumes of horseradish peroxidase were added to 2.0 ml of buffer, p = 0.1, containing NaC102. Chlorite anion absorbs insignificantly at 359 nm at all the concentra-

RESULTS

Chlorination of Monochlorodimedone with Horseradish Peroxidase and Chlorite-The chlorination of monochlorodimedone by horseradish peroxidase and NaClOa was con- firmed by isolating dichlorodimedone from the following reaction mixture. Monochlorodimedone (0.5 g, 2.87 mmol) was dissolved in 10 ml of 95% ethanol and then added to 200 ml of 12 mM acetate buffer, pH 5.40, p = 0.01. The buffer also contained NaClOa (0.26 g, 2.87 mmol). Over a period of 15 min a Hamilton microliter syringe was used to add three loo-p.1 aliquots of 67.4 PM horseradish peroxidase (20.2 nmol, total). The solution was continuously stirred. With each aliquot of horseradish peroxidase the transient appearance of the yellow- green color of ClOa could be detected by the unaided eye. Ethyl ether extracts of the reaction mixture were dried with anhydrous sodium sulfate, rotary-evaporated to a yellow oil, and ehromatographed on a column. Dichlorodimedone was the major product, 10% yield, and was identified by compari- son with authentic dichlorodimedone using thin layer chromatography and mass spectroscopy. Yields of dichlorodimedone could have been increased by the addition of further aliquots of horseradish peroxidase. A control experiment at pH 5.40 showed that chlorite alone does not react with monochlorodimedone which agrees with previous work (11). How- ever, at lower pH values for long periods of time, about 3 to 4 h, chlorite and monochlorodimedone do appear to react. This is due to the acid-catalyzed disproportionation of chlorous acid which forms ClOa, and it is Cl02 which then reacts with monochlorodimedone.

Stopped Flow Transient State Spectra-The stopped flow apparatus was used to obtain transient state spectra for the reaction of native horseradish peroxidase with chlorite. Fig. 1 presents the transient state spectra of horseradish peroxidase while reacting with chlorite in 15 mM acetate buffer, pH 5.05, p = 0.01. Absorbance changes were monitored at 5 nm inter- vals, and spectra were calculated at dead time, 100, 200, and 500 ms. For measurements in the Soret region 1.0 pM horseradish peroxidase was reacted with 10 pM chlorite, whereas in the visible region where the molar absorptivity of the enzyme is considerably smaller, 5.0 pM horseradish peroxidase was

022- .

-011

OX- -009

8 6 014- -007 z + 6 54 O.lO- -0.05 g

s :: ocm- -003 Q

ooz- -001 II I I 1 I” 1 1

400 420 440 460” / 1 1 1,

380 460 480 500 520 540 560 580 600 Nanometers

FIG. 1. Soret and visible transient state spectra of the direct conversion of horseradish peroxidase to Compound II with chlorite. Spectra were obtained at pH 5.05 using a stopped flow apparatus at dead time, 100, 200, and 500 ms represented by the symbols 0, 0, A, and 0, respectively. Within spectral resolution isosbestic points near 411,455, and 422 nm indicate the conversion of horseradish peroxidase directly to Compound II.


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The Reaction of Chlorite with Horseradish Peroxidase

reacted with 10 pM chlorite. Approximate isosbestic points at 411, 455, and 522 nm indicate interconversion of two species. The isosbestic points are slightly imperfect since the spectral resolution is only every 5 nm. Absorbance maxima at 420,525, and 551 nm in the relatively stable spectrum at 500 ms identify this intermediate as Compound II (33). This demonstrates that chlorite is able to oxidize native horseradish peroxidase directly to Compound II sans the intermediate formation of Compound I (34). Given sufficient time at this pH of 5.05, Compound II will be oxidized to Compound I (34).

Chlorination of Monochlorodimedone with Chlorine Diox- ide-The ability of chlorine dioxide to chlorinate monochlorodimedone to form dichlorodimedone was demonstrated by isolating dichlorodimedone from a reaction mixture of C102 and monochlorodimedone. Monochlorodimedone (0.204 g, 1.17 mmol) was dissolved in 200 ml of distilled water, and a solution of Cl02 was added dropwise until the perceptible color of C102 persisted. Dichlorodimedone precipitated from the reaction mixture and was recrystallized from hexane (29) with an overall yield of 25% of the theoretical. In order to correct for the dichlorodimedone remaining in the mother liquor a known amount of authentic dichlorodimedone was recrystallized under identical conditions with a 60% yield. When this correction factor is applied to the chlorine dioxide- monochlorodimedone reaction, the 25% yield of dichlorodimedone rises to 35 to 45% of the theoretical.

Chlorine Dioxide Evolution from Horseradish Peroxidase and Excess Chlorite-If CIOZ is responsible for chlorination of monochlorodimedone in the chlorite/monochlorodimedone/horseradish peroxidase mixture; then horseradish peroxidase must be able to react with chlorite to form C102. The formation of C102 was easily verified by adding a catalytic amount of horseradish peroxidase to a solution of 1 mM

chlorite in a 0.11 M acetate buffer, pH 5.5, p = 0.1, and recording the characteristic absorption spectrum of Cl02 (24, 25). Fig. 2, A and B, shows the nanomoles of Cl02 evolved in the pH 5.55 acetate buffer with various reactant concentrations. At certain points along the curves the amounts of Cl02 (in nanomoles) evolved per nmol of horseradish peroxidase are given. Fig. 2A illustrates the effect of increasing the amount of horseradish peroxidase while the chlorite concentration is constant at 1 mM, and, in Fig. 2B, the chlorite is varied while the concentration of horseradish peroxidase is constant at 0.163 pM. In Fig. 2A, the ratio of nanomoles of C102 evolved/nanomoles horseradish peroxidase added increases with decreasing amounts of horseradish peroxidase. Fig. 2B shows that this ratio increases with increasing amounts of chlorite. The extent of Cl02 evolution was also studied as a function of pH (Fig. 3). To initiate the reaction, horseradish peroxidase (0.326 nmol) was added to 2.0 ml of various buffers, p = 0.1, containing 1 mM chlorite. The quantity of Cl02 increases with decreasing pH for a constant ratio of horseradish peroxidase and chlorite as reactants. The nanomoles of C102 formed per nmol of horseradish peroxidase are written along the curve. Glycine nitrate buffers at low pH produced an anomalous effect. At pH values less than about 2.5 the acid-catalyzed disproportionation reaction of chlorous acid became significant and added to the extent of Cl02 formation.

Figs. 2, A and B, and 3 show that not all of the chlorite is converted into chlorine dioxide. There is evidence that the yield of CIOZ is limited by the denaturing effect of excess amounts of CIOZ toward horseradish peroxidase. To test the effect of excess C102 on horseradish peroxidase, 1.63 nmol of horseradish peroxidase were added to 2.0 ml of 0.11 M acetate buffer, pH 5.60, p = 0.1, containing 0.7 pmol of Clot. After standing for 2 to 3 min the solution was thoroughly swept with nitrogen to remove the Cl02 which required about 15

I ( 3.6 42 48

MIcromoles NoCIO,

FIG. 2. A, extent of chlorine dioxide formation with increasing amounts of horseradish peroxidase. Microliter quantities of horseradish peroxidase were added to a cuvette containing 2.0 ml of 0.11 M acetate buffer, pH 5.5, pg = 0.1, with 1 mM NaClOa. Cl02 formation was rapid, and the amount formed was measured at 359 nm. The numbers of nanomoles of Cl02 formed per nmol of horseradish peroxidase are listed along the curve and increased with decreasing amounts of horseradish peroxidase. B, extent of Cl02 formation with increasing amounts of NaC102. The same as A except horseradish peroxidase, 2.0 ~1 containing 0.326 nmol, was added to the cuvette containing varying amounts of NaC102. The numbers of nanomoles of Cl02 formed per nmol of horseradish peroxidase (HRP) increased with increasing amounts of chlorite.

min. When 2.0 ,smol of chlorite were added to this solution there was no increase in absorbance at 359 nm showing that no C102 was evolved. Chlorine dioxide had denatured the enzyme.

Quantitative Product Analysis for the Reaction of Horse- radish Peroxidase with Excess Chlorite-When horseradish peroxidase in catalytic amount reacts with chlorite to form CIOZ an oxidation of ClOz- to C102 occurs. The oxidation of chlorite, Cl(III), to chlorine dioxide, Cl(IV), is a l-electron oxidation. Therefore, there must be some reduction of chlorite occurring as well. Since horseradish peroxidase was present in typically one-thousandth of the chlorite amount, very few oxidation-reduction equivalents could be consumed by the enzyme. A complete study of the products formed when horseradish peroxidase disproportionates chlorite was made more convenient when horseradish peroxidase was added in sufficient amount to consume all the chlorite. Such a method required physically removing the Cl02 since chemical removal of CIOZ would leave chlorine-containing products which could interfere in the subsequent analysis. The reaction was taken to completion by adding small aliquots of horseradish peroxidase to the chlorite solution. Between the addition of the aliquots, the solution was flushed with nitrogen to carry off the CIOZ. Consequently, the next horseradish peroxidase aliquot was not quickly rendered inactive by the Cl02 produced by the previous aliquot. The amount of CIOZ evolved per aliquot was calculated from the absorbance increase at 359 nm. For example, 2.0 ml of 2.0 mM NaCIOz (4 pmol) in 7.3 mu acetate buffer, pH 5.05, p = 0.005, was reacted with a 2-~1 aliquot of 163 pM horseradish peroxidase (0.326 nmol) and the


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3178 The Reaction of Chlorite with Horseradish Peroxidase

0.7-

SOS-

0

cJ7 O5- a,

2 $2 04-

2 03- 8=0

OE- Cl

011 ’ / I 1 I I 1 1 1 I 2.6 30 34 3.0 4.2 46 5.0 5.4 5.8 6.2 6.6

PH FIG. 3. Extent of C102 formation as a function of pH. The reaction

was initiated by the addition of horseradish peroxidase, 2.0 ~1, 0.326 nmol, to 2.0 ml of various buffers, p = 0.1, containing 1 mM NaC102. The numbers of nanomoles of C102 formed per nmol of horseradish peroxidase are listed along the curve and increased with decreasing pH. The buffers are phosphate, 0; acetate, 0; citrate, A; and glycine nitrate, 0. Glycine nitrate buffers at low pH produced an anomalously low extent of C102 formation.

final absorbance increase at 359 nm recorded. C102 was swept from the solution with a fine stream of nitrogen bubbles until the 359 nm absorbance indicated no residual ClO*. Each degassing step required about 15 min. Another 2-~1 aliquot of horseradish peroxidase began another cycle. Ten 2-~1 aliquots of horseradish peroxidase were required in this example to consume all the chlorite. The chlorite was consumed when the increase in absorbance at 359 nm was about 0.007, attrib- utable to the added horseradish peroxidase. The total absorbance increase at 359 nm, the sum of the 10 individual changes, of 1.82 was used to calculate the moles of Cl02 evolved. Beginning with 4.0 pmol of chlorite, a total of 3.1 prnol of Cl02 were measured.

A spot test (35) was used to determine that chloride was also a product formed in the enzyme-catalyzed disproportionation of chlorite. The reduction of chlorite, Cl(III), to chloride (X(-I), involves a 4-electron reduction. The quantity of chloride was determined by the mercuric thiocyanate method essentially as described by Vogel (35). From 4.0 pmol of chlorite, 0.92 pmol of chloride were measured. A control experiment with just the enzyme solution required an adjust- ment from 0.92 to 0.87 pmol of chloride. A very sensitive spot test for chlorate (36) was negative to several observers.

Reaction of Equimolar Amounts of Horseradish Peroni- dase and Chlorite-The reaction between equimolar amounts of horseradish peroxidase and chlorite, eventually resulting in the formation of Compound I (34), has a well defined stoichiometry. Titration of horseradish peroxidase with increments of chlorite in 0.083 M acetate buffer, pH 3.80, p = 0.01, showed a 1:l molar stoichiometry (see Fig. 4). The average of three such titrations showed that 8 nmol of horseradish peroxidase forms 8 nmol of Compound I with 8.5 nmol of chlorite.

Absence of H20z in the Horseradish Peroxidase Reaction with Excess Chlorite-Present attempts failed to detect hydrogen peroxide as a product when horseradish peroxidase reacts with excess chlorite. The same method of detection and measurement of H20, using catalase and an oxygen electrode was used as previously described (16). Standard H202 solutions indicated that the oxygen electrode was operating lin- early and with sufficient sensitivity to detect easily the levels of Hz02 previously reported (16) to be formed in the horserad-

ish peroxidase-chlorite reaction. Over the span of several experiments the concentrations of H+, catalase, and chlorite were varied almost an order of magnitude from the published solution specifications (16) without detectable amounts of H202 formed.

Reaction of Chlorine Dioxide with Monochlorodimedone- The reaction between monochlorodimedone and C102, in the absence of horseradish peroxidase and chlorite, was investigated in more detail. Cl02 and monochlorodimedone react quickly and quantitatively with a 1:l molar stoichiometry as determined using a spectrophotometric titration technique at 290 nm where monochlorodimedone absorbs. In 2.0 ml of 0.11 M acetate buffer, pH 5.55, p = 0.1, 65.8 nmol of Cl02 were added to 101 nmol of monochlorodimedone. The final absorbance at 290 nm was used to calculate that 36.0 nmol of monochlorodimedone remained unreacted. Therefore, 65.8 nmol of CIOZ reacted with 65.0 nmol of monochlorodimedone. This spectrophotometric method of determining stoichiometry is based on the fact that the reaction products do not absorb appreciably at 290 nm (29). The conjugated system present in the enol or enolate anion of monochlorodimedone is the chromophore responsible for the large ultraviolet absorbance at 290 nm. When the possibility of enol or enolate formation is eliminated by chlorination or other reactions, this chromophore is destroyed. This is the basis for the monochlorodimedone assay of chloroperoxidase (29).

The 1:l stoichiometry for the reaction of monochlorodimedone and CIOZ was also determined using EPR spectroscopy. C102 is a paramagnetic molecule, and its EPR spectrum has been recorded (37). In 0.20 ml of 2.8 XnM acetate buffer, pH 5.55, p = 0.025, it was found that 8 nmol of monochlorodimedone were required to eliminate the EPR signal due to 8 nmol of ClOz.

The glass electrode was used to discover that 1 proton was released for every molecule of monochlorodimedone and Cl02 that reacted. Monochlorodimedone (10 pmol in 8.0 ml) in distilled water had a pH of 3.15, and addition of Cl02 (10 pmol in 2.0 ml) for a final volume of 10.0 ml gave a pH of 2.99. This corresponds to the release of 10 pmol of protons.

Since it was estimated that only 35 to 45% of monochlorodimedone is chlorinated to form dichlorodimedone by C102, yet monochlorodimedone and C102 react with a 1:l stoichiometry, it is clear. there must be other chlorine-containing products. Such products may be inorganic or organic. Thin layer chromatograms of the ether-extractable products showed at least one other major spot, RF = 0.03, other than monochlorodimedone or dichlorodimedone, RF = 0.31, 0.70, when developed with l-butanol. Lyophilization of the aqueous reactant mixture gave an identical chromatogram.

,7.5 J 8 10 12 14 16 10 20

Nanomoles NaCIO,

FIG. 4. Spectrophotometric titration of horseradish peroxidase with chlorite. The AA,“:, was recorded for microliter additions of chlorite solution. In 0.083 M acetate buffer, pH 3.80, p = 0.01, 8 nmol of horseradish peroxidase formed the steady absorbance of Compound 1 after 7.5 nmol of chlorite were added.


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After the reaction of equimolar amounts of monochlorodimedone and C102 in 0.083 M acetate buffer, pH 3.80, p = 0.1, the solution still had considerable oxidizing capabilities. This was demonstrated by addition of excess potassium iodide which resulted in triiodide ion formation. The absorbance of triiodide at 353 nm, 6 = 2.55 X lo4 M-’ cm-’ (l), permitted spectrophotometric monitoring of this reaction. It was found that the products of the reaction between monochlorodimedone and CIOz could oxidize iodide at pH 3.80 but not at pH 5.60. A control experiment demonstrated that chlorite could easily oxidize iodide at pH 3.80 but not at pH 5.60. For the oxidizing product in the monochlorodimedone and C102 reaction, a pseudo-first order rate constant of 0.13 min-’ at pH 3.80 for triiodide formation was obtained at 4 mM iodide. With chlorite, 30 pM, and iodide 4 mM, at pH 3.80, the rate of Is- formation obeyed pseudo-first order kinetics and yielded a pseudo-fast order rate constant of 0.18 min-‘. (Under pseudo- first order conditions, a first order rate constant is only de- pendent upon the concentration of the reactant in excess amount (38).) These facts suggest that chlorite may be a product in the reaction between monochlorodimedone and C102. Spectroscopic identification of chlorite as a product was not possible due to its very small absorbance at the concentration levels of the reactants (24, 25, 30-32).

Since 1 mol of chlorite oxidizes 4 mol of iodide to form 2 mol of molecular iodine, Equation 1

ClOz- + 41- + 4H+ + Cl- + 212 + 2H20 (1)

then 2 mol of triiodide can form. The amount of triiodide formed was used to calculate the amount of chlorite formed in the monochlorodimedone and C102 reaction. When 80 nmol of monochlorodimedone and C102 react, 47 nmol of chlorite are formed representing 59% or about three-fifths of the total chlorine in the system. Overall, the reaction between monochlorodimedone and C102 can be represented as in Equation 2.

HC Cl C1+5C10,-+5H++-2 + -3c10,- (2)

H.,C Cl

0

An oxygen electrode revealed that oxygen or hydrogen peroxide was not a detectable product of this reaction.

Dissociation Constant of Monochlorodimedone-For the reactions of monochlorodimedone, it was of interest to know the state of ionization of monochlorodimedone, an acidic molecule. Monochlorodimedone can exist in three forms: the keto, enol, and enolate anion forms. See Equation 3.

,O P PH

A spectrophotometric titration curve; Fig. 5, was con- structed from pH 0.75 to 9.30. The total concentration of monochlorodimedone in all three forms was 48.4 pM, and the ionic strength of the buffers was 0.1. The total absorbance of all three forms at 290 nm, Azw, was measured. Equation 4 was derived for a monoprotic acid and was used with the data in Fig. 5 for

Am = [monochlorodimedonelo

r W’l Emo”ochlorcdlmedoner, + hwlatr 1

1 fiWP

W’l (4)

r+1 WP

PH FIG. 5. Spectrophotometric titration curve for the ionization of

monochlorodimedone. The total absorbance at 290 nm, Am, was measured as a function of pH in HNOJ, glycine nitrate, acetate, phosphate, Tris/nitrate, and carbonate buffers with an ionic strength of 0.1 at 20°C. The total concentration of monochlorodimedone was 48.4 PM. The smooth curve was drawn using the parameters in Table I with Equation 4.

TABLE I Values ofparameters in Equation 4 as computed by nonlinear

least squares analysis of the data in Fip. 5

PK.,, 2.99 + 0.02 hn"nuchlorodunedone (4.76 f 0.08) X 10’ Mm’ cm-’ Eenolate (1.99 + 0.02) X lo4 Mm’ Cmm’

a nonlinear least squares analysis to determine the best fit values of K app, %&dorodlmedane, an d &n&k ’ In Equation 4, Kapp is the apparent acid dissociation constant for monochlorodimedone; and &wnochlorodimedone and l enolate are the molar absorp- tivities for monochlorodimedone and its enolate anion. Values of these parameters are given in Table I. Kapp is an apparent dissociation constant since this experiment does not distinguish between the ionization of the keto or enol forms of monochlorodimedone (39). As Equation 3 demonstrates, ionization of either the keto or enol forms yields a common anion. Previous reports suggest that the enolic form vastly predom- inates the keto form of monochlorodimedone (40). The relatively large absorbance at 290 nm at low pH where monochlorodimedone is un-ionized indicates the presence of the conjugated system and hence the presence of the enol. The spectrophotometrically determined value of pK,,, agrees very well with the value determined using the glass electrode (41). As mentioned, 10 pmol of monochlorodimedone in 8.0 ml of distilled water had a pH of 3.15, and this corresponds to a PK.,, of 3.03 for monochlorodimedone.

DISCUSSION

In aqueous solution, monochlorodimedone and chlorite are involved in acid-base equilibria. Since most of the buffers used in the present work had pH values greater than the pK,, 1.8, of chlorous acid, solutions of NaClO* contain largely the chlorite anion with only a small fraction of chlorous acid. Such solutions have been referred to as chlorite solutions since this is the most abundant form. However, the most abundant form is not necessarily the reactive form. Only the diffusion-controlled limit rate for biomolecular reactions is able to distinguish which of two species in acid-base equilibrium is the reactive species (42). In 0.15 M acetate buffer, pH 5.05, p = 0.1,

’ I. Ralston performed the computation. Department of Chemistry, University of Alberta, Edmonton, Alberta.


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the apparent second order rate constant for the formation of Compound II from horseradish peroxidase and chlorite is Kapp = (4.6 f 0.3) X lo5 M-’ s-l. If chlorous acid were the reactive species the true second order rate constant would be K = 8 X 10’ M-’ s-l (42). The magnitude of K may or may not exceed the diffusion-controlled limit for the reaction of an enzyme with a small molecule (7). The nature of the reactive form of chlorite is unresolved. However, the increased reactivity of chlorite solutions which are acidic tend to suggest that chlorous acid is the reactive form. A similar situation exists for monochlorodimedone where it is unresolved whether monochlorodimedone in the keto, enol, or enolate anion form is the reactive form. Again, however, the increased reactivity of the enolic forms of ketones suggests the enol to be the reactive form.

The transient state formation of Compound II prior to Compound I is of great significance in peroxidase chemistry. This reaction between horseradish peroxidase and chlorite is the first recognized example of the direct oxidation of native horseradish peroxidase to Compound II. Within the time scale of the stopped flow instrument, Compound II is the first product to appear in the oxidation of horseradish peroxidase with chlorite. However, these experiments do not absolutely rule out the presence of short lived intermediates which might transiently appear within the dead time of the instrument, about 4 ms. The oxidation of horseradish peroxidase to Com- pound II is a l-electron oxidation. Since horseradish peroxidase and chlorite eventually form Compound I, an oxidation of Compound II to Compound I must also occur. The oxidation of Compound II to Compound I is also a l-electron oxidation. These reactions are investigated in more detail in the following paper (34). Chance (9) fist noted that Com- pound I was not observed in the transient state when horseradish peroxidase and chlorite were reacted in a stopped flow apparatus. The absence of Compound I was explained by the assumption that conversion of Compound I to II was much faster than the formation of Compound I. On the basis of this assumption it was concluded that Compound I formation preceded the appearance of Compound II. More recently a rapid scanning spectrophotometer was used to show the direct conversion of horseradish peroxidase to an enzymatic species absorbing maximally near 417 nm (14, 16). This wavelength is essentially the same as the 418 to 420 nm absorbance maximum for horseradish peroxidase Compound II; however, the species was formed directly from horseradish peroxidase and was believed to be a new enzymatic species responsible for the horseradish peroxidase-catalyzed chlorination reaction of monochlorodimedone (11, 14, 16). This supposedly new intermediate received the name Compound X (14, 16). The species formed directly from horseradish peroxidase by chlorite might have been easily recognized as Compound II if it were not for the dogma in the literature which has a long history of promulgating the compulsory order of appearance of the oxidized intermediates. In fact, Compounds I and II derive their name, in part, from their order of appearance in the usual steady state cycle with hydrogen peroxide.

It remains to explain the observed chlorination reaction of monochlorodimedone (11, 14, 16). Prior research demonstrated that Na%102- reacted with horseradish peroxidase in the presence of monochlorodimedone and high concentrations of chloride ion to yield [36Cl]dichlorodimedone (11). This eliminated the possibility that 3”C102- was converted to la- beled hypochlorite, 3”C10-, which . bl t hl IS a e 0 c orinate monochlorodimedone. Chloride rapidly exchanges with 36C10- (11); yet, in the presence of an excess of chloride, the amount of 3fiC1 incorporated into dichlorodimedone was not diluted.

Before Compound X was identified as Compound II, an

attempt to record the EPR spectrum of Compound X revealed the formation of the paramagnetic molecule, chlorine dioxide. Chlorination of monochlorodimedone to form dichlorodimedone by C102 was demonstrated by reacting C102 and monochlorodimedone independently of the horseradish peroxidase reaction with chlorite. The transient presence of Cl02 can actually be detected visually under conditions described under “Experimental Procedures.” The transient nature of Cl02 is explained by its rapid formation from horseradish peroxidase and chlorite and its rapid disappearance when it reacts with monochlorodimedone. Under the conditions described, the transient appearance of Cl02 lasted about 2 or 3 s.

In the absence of monochlorodimedone, the extent of C102 formation was investigated at pH 5.60 by varying the enzyme/ chlorite ratio. A maximum amount of about 1600 nmol of CIOZ are formed/nmol of horseradish peroxidase when the amount of horseradish peroxidase was small. The pH dependence of the extent of C102 formation showed about 2500 nmol of C102 were formed/nmol of horseradish peroxidase at low pH. In all cases, formation of Cl02 ceased before all the chlorite was consumed. The build-up of Cl02 as a product soon destroyed the enzyme. It appears that the varying extent of Cl02 formation per nmol of horseradish peroxidase is a function in- volving a trade off between the velocity of C102 formation and the velocity of horseradish peroxidase inactivation by C102. When the formation of Cl02 is fast as it is at low pH, more CIOZ is formed in the time it takes for enzyme inactivation. With a constant amount of horseradish peroxidase and increasing amounts of chlorite, the velocity of Cl02 formation increases resulting in more C102.

There is an anomalous effect on the extent of Cl02 formation by the glycine nitrate buffer. Two glycine nitrate buffers were used at pH 2.98; a freshly prepared buffer and a 4-week- old buffer. Both fresh and old buffers gave the same result. The glycine nitrate buffers appear to either inhibit the rate of CIOZ formation or enhance the rate of horseradish peroxidase inactivation by ClOz.

The enzymatic disproportionation of chlorite at pH 5.05 showed that 4 pmol of chlorite was converted to 3.1 pmol of C102 and 0.87 pmol of chloride for a total of 3.97 pmol of chlorine. This figure indicates that the horseradish peroxidase may be catalyzing the disproportionation with either of two stoichiometries. The accuracy of the measured quantities of Cl02 or chloride cannot distinguish between Equations 5 and 6.

4HClOz horseradish peroxidase a 3ClOr + Cl- + 2HzO (5)

5HC102 horseradish peroxidase t 4C102 + Cl- + 2HzO + H+ (6)

However, Equation 5 is not electronically balanced. According to the stoichiometry of Equation 6, 4 pmol of chlorite would yield 3.2 pmol of C102 and 0.8 pmol of Cl-. This fits the measured quantities well especially considering that the amount of C102 measured would be expected to be slightly low since CIOZ exchanges with atmospheric gases during the reaction and measurement times. Equation 6 describes a disproportionation reaction in which 4 mol of chlorous acid, Cl(III), are oxidized to 4 mol of chlorine dioxide, Cl(IV); and 1 mol of chlorous acid is reduced to chloride Cl(-I). Qualita- tive analysis for chlorate repeatedly proved negative.

Attempts were made to record the spectrum of the enzyme in the steady state reaction with chlorite as in Equation 6. This would determine the predominant form of the enzyme during the reaction. At low pH the steady state was too short lived, and at high pH the unreactivity of Compound II pre- vented the steady state reaction. At intermediate pH values the spectrum indicated the presence of the native enzyme,


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The Reaction of Chlorite with Horseradish Peroxidase 3181

Compound I, and Compound II; none of which were clearly predominant. No definitive statement could be made about the predominant form of the enzyme during the steady state.

At pH 3.80 horseradish peroxidase and chlorite react with a 1:l molar stoichiometry to form Compound I. This result contrasts with earlier work (11) where 0.5 mol of chlorite reportedly formed 1 mol of Compound I from horseradish peroxidase. The earlier titration (11) was performed at pH 6, but present titrations at this pH still indicate a 1:l stoichiometry. Therefore, pH 3.80 was chosen for the titration since at lower pH there is a minimal interference by stable Com- pound II (34).

Also in contrast to a previous report (16), no significant amount of hydrogen peroxide was formed as a product of the reaction between horseradish peroxidase and excess chlorite. It was reported (16) that 1 mol of hydrogen peroxide was formed for every mole of horseradish peroxidase that reacted with chlorite, and the formation of H202 was used to explain the previous report (11) of a 2:l stoichiometry for the formation of Compound I from horseradish peroxidase and chlorite. Now that this stoichiometry has been correctly established to be l:l, there is no need to invoke Hz02 formation.

The reaction between monochlorodimedone and C102 and the release of 1 proton occur with 1:l:l stoichiometries. The spectrophotometric, EPR, and glass electrode techniques used to determine these stoichiometries give no information con- cerning the amount of dichlorodimedone formed. The formation of dichlorodimedone is only one reaction which eliminates the conjugated chromophore in the enolic form of monochlorodimedone. There may be reactions other than chlorination which eliminate the possibility of conjugation and thus eliminate the absorbance at 290 nm. The per cent yield of dichlorodimedone prior to recrystallization was estimated to be between 35 and 45%. Thin layer chromatography revealed the presence of other organic reaction products. However, most of the chlorine can be accounted for, 35 to 45% in dichlorodimedone and 59% as chlorite. If dichlorodimedone and chlorite were the only chlorine-containing products, then a more ac- curate yield of 41% for dichlorodimedone can be calculated. An example of the reduction of chlorine dioxide to chlorite by an enolic compound has been previously reported (43).

Only the enolic form of a ketone is easily oxidized, and since Cl02 is a strong oxidant, perhaps part of the reaction between monochlorodimedone and Cl02 could be represented by Equa- tion 7.

/OH /O.

Cl + ClOL + cl + ClOl- + H’ (7)

This accounts for the formation of chlorite, a proton, and a free radical which may explain the variety of reaction products detected by thin layer chromatography. The mechanism of chlorination of monochlorodimedone forming dichlorodimedone by C102 is not clear.

In summary, the chlorination of monochlorodimedone to form dichlorodimedone as carried out by horseradish peroxidase and chlorite, proceeds via a chemical intermediate rather than a chlorinated, oxidized, enzymatic intermediate known as Compound X. Chlorite, or chlorous acid depending upon the reactive form, is disproportionated catalytically by horseradish peroxidase according to Equation 6; and the product chlorine dioxide is responsible for the chlorination of monochlorodimedone. The Soret and visible optical spectrum of what had been known as Compound X revealed this intermediate to be Compound II. This is the first recognized example of the direct oxidation of native horseradish peroxidase to Compound II. The oxidation of horseradish peroxidase

to Compound II, and the oxidation of Compound II to Com- pound I by chlorite are described in the following paper (34).

1.

2. 3.

4. 5. 6.

7.

8.

9. 10. 11.

12.

13.

14.

15.

16.

17.

18.

19. 20. 21.

22.

23. 24.

25. 26. 27.

28.

29.

30. 31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42. 43.

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W D Hewson and L P Hagerwith chlorite.

Mechanism of the chlorination reaction catalyzed by horseradish peroxidase

1979, 254:3175-3181.J. Biol. Chem.

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