Plant Physiol. (1973) 52, 501-507

Purification and Some Properties of Two Polyphenol Oxidasesfrom Bartlett Pearst

Received for publication April 26, 1973

NILO DE JESUS RIVAS2 AND JOHN R. WHITAKERDepartment of Food Science and Technology, University of California, Davis, California 95616

ABSTRACT

Two polyphenol oxidases (enzymes A and B) from Bartlettpear (Pyrus communs) peelings were purified to electropho-retic homogeneity according to polyacrylamide gel by a com-bination of Sephadex gel filtration, diethylaminoethyl cellulosechromatography and hydroxyl apatite chromatography. Whilethe two enzymes differ electrophoretically at pH 9.3, chro-matographically on hydroxyl apatite, and in the effect ofionic strength on activity, they are similar with respect tochromatography on diethylaminoethyl cellulose, substratespecificity, pH activity relations, inhibition by p-coumaric andbenzoic acids, and heat stability. The two enzymes are o-di-phenol oxidases with no detectable monophenolase or laccaseactivities. Pyrocatechol, 4-methyl catechol, chlorogenic acid,and d-catechin are good substrates of the enzymes with Kmvalues in the range of 2 to 20 mM. Dependences of activity onoxygen and chlorogenic acid concentrations indicate a sequen.tial mechanism for binding of these substrates to enzyme B.Vmax and Km values for oxygen and chlorogenic acid were 103umoles 02 uptake per minute per milligram of enzyme, 0.11mM and 7.2 mM, respectively, for enzyme B at pH 4.0. Bothenzymes had maximum activity at pH 4.0 on chlorogenic acid.Km values for chlorogenic acid were independent of pH from3 to 7; the Vmar values for both enzymes gave bell-shapedcurves as a function of pH. p-Coumaric acid is a simple, linearnoncompetitive inhibitor with respect to chlorogenic acid atpH 6.2 with K, values of 0.38 and 0.50 mM for enzymes A andB, respectively. Benzoic acid is a linear competitive inhibitorwith respect to chlorogenic acid at pH 4.0 with K, values of 0.04and 0.11 mM for enzymes A and B, respectively.

Polyphenol oxidase (o-diphenol::2 oxidoreductase, EC1 . 10. 3. 1) is a copper-containing enzyme which catalyzeseither one or two reactions involving molecular oxygen. Thefirst type of reaction is hydroxylation of monophenols lead-ing to formation of o-dihydroxy compounds. The second typeof reaction is oxidation of o-dihydroxy compounds to quinones.

' Taken from the dissertation submitted by Nilo de Jesus Rivasto the Graduate Division, University of California, Davis, inpartial satisfaction of the requirements for the Ph.D. degree. N.J. R. gratefully acknowledges a scholarship from the University ofVenezuela and a grant-in-aid for chemicals from the GraduateDivision, University of California, Davis.

2 Present address: Facultad de Agronomia, University of Vene-zuela, Maracay, Venezuela.

Polyphenol oxidase is also known as phenol oxidase, tyro-sinase, o-diphenol oxidase, catechol oxidase, phenolase, andchlorogenic acid oxidase.Brown discoloration of canned pears and pear concentrate

was found to be related to the enzymatic browning that takesplace before or during processing (18). Since polyphenoloxidase in Bartlett pear has not been adequately investigated,the purpose of this investigation was to purify the polyphenoloxidase complex of Bartlett pear and to determine its kineticproperties. An understanding of the factors which affect ac-tivity of the enzyme may lead to a better understanding of itsfunction in vivo and to its control in the ripe fruit.

MATERIALS AND METHODS

Pears. Bartlett pears (Pyrus communis) at harvest maturity(19 p.s.i.) were obtained from an orchard in Placer County,California and ripened at 20 C and 85% relative humidityto a pressure test of 3 p.s.i. The ripe fruits were peeled, halved,and cored by hand. Peelings and flesh were quick frozenseparately with Freon 12 in a duPont minimark freezer, sealedin No. 2½2 cans under a vacuum of 15 inches Hg and stored at-26 C until used. Under these conditions, the samples showedno darkening after 2 years storage.

Other Reagents. Sephadex G-25, particle size 20 to 80 mi-crons, was from Pharmacia Fine Chemicals, Inc. DEAE3-cellu-lose, medium mesh and ion-exchange capacity of 1.0 meq/gm,was from Sigma Chemical Co. Hydroxyl apatite (BioGelP-15) was from BioRad Laboratories. Polyethylene glycol(average molecular weight of 3000-3700) was from J. T.Baker Chemical Co. Acrylamide, bis-acrylamide, pyrocatechol,and resorcinol were from Eastman Organic Chemicals. Chloro-genic, caffeic, and p-coumaric acids were from Sigma Chem-ical Co. d-Catechin and phloroglucinol were from K and KLaboratories. Ferulic acid was from Nutritional BiochemicalCorporation and 4-methyl catechol was from Aldrich Chem-ical Co. 4-Methyl catechol was recrystallized from toluene andd-catechin was recrystallized from water. All other substratesof reagent grade were used without further purification.

Purification of Enzyme. All steps were carried out at about1 C. Two hundred and fifty grams of frozen pear peelings werehomogenized in a Waring Blendor with 500 ml of ice-cold0.1 M sodium phosphate buffer, pH 6.2, containing 0.03 Mascorbic acid and 1% polyethylene glycol to slow down brown-ing. The homogenate was centrifuged for 30 min at 10,500gin a Sorvall refrigerated centrifuge, and the supernatantliquid was filtered through glass wool.The supernatant liquid in 100-ml aliquots was immediately

placed on a column (4.1 x 37 cm) of Sephadex G-25 equil-

3Abbreviation: DEAE: diethylaminoethyl.501

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RIVAS AND WHITAKER

0.5-

0.4' -v 180- -

0~~~~~~~~~~~~~~~~~~~~~~~~~~(

o\ 140E E

H 0.3 o:0.3 L

2 \ 41~~/- -60~L011 S S < -ol~~~~~~~~~~~~~~~~~10.1-- I -0.1

'L__,y _ 20

L_-r 1 i -20 40 60 80 100 120 140

FRACTION NUMBER (3.OmI)FIG. 1. Analytical DEAE-cellulose chromatography of pear

polyphenol oxidases. The column (1.0 X 45 cm) and extract (fol-lowing gel filtration) were equilibrated at 1 C with 1 mm phosphate,pH 6.2. Elution was with a linear gradient of 200 ml of 1 mm phos-phate, pH 6.2, in mixing chamber and 200 ml of 0.25 M phosphate,pH 6.2, in reservoir. Protein concentration (solid line) was deter-mined by the Lowry method. Enzyme activity was determined in areaction mixture containing 2.8 ml of 10 mM pyrocatechol in 10mm sodium phosphate, pH 6.2, and 0.2 ml of enzyme solution.Activity is expressed as change in absorbance at 420 nm after 10min reaction at 35.0 C.

ibrated with 0.1 M sodium phosphate, pH 6.2. The columnwas eluted with the same buffer, and the eluate was collectedin 20-ml fractions. After each use the column was washedsuccessively with 1 liter of 10% acetic acid and 1 liter of 0.1 Msodium phosphate, pH 6.2.

In preliminary work, fractions from the Sephadex G-25columns with polyphenol oxidase activity were pooled, di-alyzed (with three changes) against 1 mm phosphate, pH 6.2,and chromatographed on DEAE-cellulose columns equilibratedin the same buffer (24). Washing the column with a lineargradient of phosphate (200 ml of 1 mm phosphate, pH 6.2, inmixing chamber and 200 ml of 0.25 M phosphate, pH 6.2,in reservoir) showed that the majority of inactive protein waseluted rapidly from the column, while most of the polyphenoloxidase activity was eluted at 0.15 to 0.20 M phosphate (Fig.1). Consequently, the bulk of the preparations was purifiedwithout prior dialysis on DEAE-cellulose columns (2.1 x 48cm) equilibrated in 0.1 M phosphate, pH 6.2. The majority ofinactive protein was removed by washing the column with 0.1M phosphate, pH 6.2. Two fractions, A' and C, with poly-phenol oxidase activity were eluted with 0.25 M phosphate,pH 6.2, and 0.50 M phosphate, pH 6.2, respectively (Fig. 2).

Electrophoresis on polyacrylamide gel showed that the frac-tion eluted with 0.25 M phosphate (A'), contained two proteinsboth with polyphenol oxidase activity (Fig. 2). Fraction C,eluted with 0.50 M phosphate, gave a single protein band and asingle activity band after electrophoresis; however, these bandswere not coincident (Fig. 2).

Fraction A', which contained more than 90% of the totalactivity, was concentrated by ultrafiltration, using an AmiconModel 401 ultrafiltration cell and a PM 10 membrane, in anatmosphere of nitrogen at 40 p.s.i. The concentrated materialwas dialyzed with three changes against 10 mM phosphatebuffer, pH 6.2, and placed on top of a hydroxyl apatite column(1.2 X 37 cm) equilibrated with the same buffer. The columnwas eluted with a linear gradient of phosphate (200 ml of 10mm phosphate, pH 6.2, in mixing vessel and 200 ml of 0.20 Mphosphate, pH 6.2, in the reservoir). Two well separated frac-

tions with polyphenol oxidase activity were obtained (Fig. 3).These fractions were homogeneous by polyacrylamide gelelectrophoresis as determined both with protein and activitystains which gave coincident bands and were free of peroxidaseactivity.A summary of a typical purification is given in Table I.

From 250 g of pear peelings, we obtained 4.7 mg of enzyme Aand 2.3 mg of enzyme B.

Polyacrylamide Disc-Gel Electrophoresis. Electrophoresiswas performed as described by Davis (3) and Whitaker (22).

F-

Ec

"T 0.4

1 0.2C)

0.25 M PHOSPHATET7

B ALX A ProteinI ~~~BandslZIl WIIIiIII ActivityI _ N I Bands

*1 Z-\ -

1, 0.50 M PHOSPHATE_

ProteinBand

ActivityBand

50 17070 90 150

FRACTION NUMBER (3.Oml)

FIG. 2. Preparative DEAE-cellulose chromatography of pearpolyphenol oxidases. The column (2.1 X 48 cm) and extract (fol-lowing gel filtration) were equilibrated at 1 C with 0.1 M phosphate,pH 6.2. After sample application, the column was washed with 1liter of 0.1 M phosphate, pH 6.2, to remove the bulk of inactiveprotein. Peaks containing polyphenol oxidase activity were elutedwith 0.25 M phosphate and 0.50 M phosphate, pH 6.2, increasedstepwise. Activity was determined as described in Fig. 1. Thehorizontal bars above each peak indicate electrophoretic behavior ofthat material in polyacrylamide gels (see text for procedure).

0.4

E

0cm 0.3

>- 0.2H

V) 0.1

A

r-±

B

+ A

H

10 30 50 70 90 110

FRACTION NUMBER (I.3mI)

FIG. 3. Chromatography of fraction A' (Fig. 2) on hydroxylapatite. The column (1.2 X 37 cm) and fraction A' (concentratedfollowing DEAE-cellulose chromatography) were equilibrated at1 C with 10 mM phosphate, pH 6.2. Elution was with a lineargradient of 200 ml of 10 mm phosphate, pH 6.2, in mixing cham-ber and 200 ml of 0.2 M phosphate, pH 6.2, in the reservoir. En-zyme activity was determined in a reaction mixture containing 2.5ml of 10 mM pyrocatechol in 10 mM phosphate, pH 6.2, and 0.50ml of enzyme solution. Activity is expressed as change in absorb-ance at 420 nm after 10 min reaction at 35.0 C. The horizontalbar above each peak indicates electrophoretic behavior in poly-acrylamide gels as visualized by protein and activity stains.

502 Plant Physiol. Vol. 52, 1973

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POLYPHENOL OXIDASES OF BARTLETT PEARS

The gel tubes, 8.9 cm long and 0.5 cm i.d., contained 5.0 cm ofrunning gel (7%) and 1.0 cm of spacer gel (1.25%). The en-

zyme solution was mixed with 70% sucrose and layered on topof the spacer gel. The starting pH was 8.3, and the running pHwas 9.5. A current of 2 ma/tube was employed at 1 C untilbromophenol blue, used as a reference marker, migrated closeto end of the tube (about 4 hr).

Part of the gels were stained for protein with either 1%Amido black 1OB in 7% acetic acid or with 0.01% Coomassiebrilliant blue in 35% ethanol, 10% acetic acid, 55% water.Other gels, run at the same time as those for protein staining,were developed with one of the following substrate solutions:(a) 30 mm pyrocatechol in 0.2 M phosphate buffer-30%ethanol, pH 6.2; (b) 20 ml d-catechin in 0.2 M phosphate-30%ethanol, pH 6.2; (c) 5 mM pyrocatechol-5 mM L-proline in 0.1M phosphate, pH 6.2.

Protein Estimation. The concentration of protein was deter-mined by the method of Lowry et al. (11) using bovine serum

albumin as standard. The micro-Kjeldahl method of Johnson(9) was used for crude extracts containing phenolic com-

pounds.Enzyme Activity Measurements. Polyphenol oxidase ac-

tivity during purification of enzyme was determined by a mod-ification of the colorimetric method described by Ponting andJoslyn (14). The substrate was 10 mM pyrocatechol in 10 mMsodium phosphate, pH 6.2. The increase in absorbance at 420nm after 10 min incubation at 35 C was determined in a

Beckman DB spectrophotometer.Initially, the colorimetric method was also used in the kinetic

studies. The change in absorbance at 420 nm versus time was

recorded in a thermostatted Beckman DB recording spectro-photometer. Initial velocities were calculated from the slopeof the initial portion of the activity curves. Eventually, thecolorimetric method was abandoned in favor of the polaro-graphic method because (a) better initial rate curves were

obtained, (b) less enzyme per reaction was needed, and (c)the change in oxygen concentration was more comparableamong different substrates than was the colorimetric assaywhere the extinction coefficient of each quinone is different. Ofthe kinetic results reported here, only the Km values in TableII were determined by the colorimetric method.Oxygen uptake during the enzymatic reaction was followed

with a Clark-type oxygen electrode (Yellow Springs InstrumentCo., Yellow Springs, Ohio) coupled to a Model 80A Moseleyautograf recorder and Model 53 Biological oxygen monitor.The electrode was standardized with air-saturated water at30 C by adjusting the recording on the recorder chart to 100.

Table I. Putrificationt of Polyphentol Oxidases from BartlettPear Peelinigs

The starting material was 250 g of peelings.

Procedure ume Activity' Protein Specific Activity Purifi-

,inits/ total |ng/ml units/mng % fotdu?Illnits Iproteini

0fl

1. Gel filtration 800 0.118t 94.4 0.217 0.400 100 1.02. DEAE-cellulose 198 0.78 154 0.119 6.55 164 16.4

column, peakA' (see Fig. 2)

3. Hydroxyl apatitecolumn

Enzyme A 275 0.14 38.5 0.0172 8.15 40.8 20.4

Enzyme B 152 0.22 33.4 0.0150 14.7 35.4 36.7

1 Change in absorbance at 420 nm/min ml enzyme = 1 unit activity.2 Based on activity following gel filtration. Activity measurements could not be

performed on the supernatant liquid from the homogenate because of presence of

ascorbic acid.

Table 11. Substrate Specificity of Bartlett PearPolypheniol Oxidases

Activity was determined as the initial rate of change in absorb-ance at 420 nm (Km determinations) and initial rate of oxygen up-take (Vma. and vs determinations) at 35 C and pH 6.2 (0.1 M sodiumphosphate). Oxygen concentration was 0.24 mM. Pyrocatecholconcentrations ranged from 5 to 30 mM; 4-methyl catechol, d-catechin, and chlorogenic acid concentrations were from 2 to 20mM. vo values were determined at 10 mm concentrations of sub-strate in 0.2 M sodium phosphate, pH 6.2. All solutions for vodeterminations, except those of caffeic acid and dopamine, con-tained 50 AM pyrocatechol.

Enzyme A Enzyme BSubstrate

Km Vmas vo Km Vmax vo

pmmoles 02/min.mg M ymotes 02/tin-protein ~~mg Protein

Pyrocatecholl 20.9 17.2 33.9 88.54-Methyl catechol 8.0 22.7 5.8 64.0Chlorogenic acid 16.1 18.4 11.9 63.5d-Catechin' 2.1 2.6 1.6 6.9Dopamine 3.9 13.8Caffeic acid 1.3 3.9p-Coumaric acid 0 0Ferulic acid 0 0Resorcinol 0 0Phloroglucinol 0 0

'Fraction C (Fig. 2) had Km values of 22.5 and 1.5 mM on pyro-catechol and d-catechin, respectively.

At standard barometric pressure, this reading correspondsto an oxygen concentration of 0.24 mm (7). The reaction vessel,containing 3 ml of buffered substrate solution, was equilibratedin a water bath at 30 C until a steady trace was obtained. Then0.05 ml of enzyme solution was introduced into the reactionvessel through a groove in the electrode by means of a cali-brated syringe (No. 725 SN Hamilton syringe wth a 4-inchneedle). The reaction was allowed to proceed for 5 min. Ini-tial velocities were determined from the initial linear part ofthe curves.

Vmar and Km values were determined from Lineweaver-Burk plots treated by the method of least squares using aPerkin-Elmer desk computer. The lines in Figures 5, 6, and8 are drawn based on the least square treatment.Only observed velocities at a single substrate concentration

(v.) were determined for some substrates (Table II). Theoxygen electrode method was used. The substrates were 10mm in 0.2 M phosphate, pH 6.2. All solutions, except those ofcaffeic acid and dopamine, also contained 50 juM pyrocatecholin order to eliminate any lag period characteristic of the hy-droxylation of monophenols (19).

Most of the kinetic studies were done in 25 mm succinate-25mM pyrophosphate buffers with the ionic strength adjusted to0.22 with NaNO,. These ions are not inhibitory of polyphenoloxidase activity (10).

Effect of Enzyme Concentration. The effect of concentrationof enzymes A and B on measured activity was determined byboth the colorimetric and oxygen electrode methods. Underthe conditions used in this work and over the enzyme concen-tration range used, there was a linear relationship between en-zyme concentration and activity.Oxygen Concentration Effect. Gas mixtures of oxygen and

nitrogen were prepared by means of two Nupro double patternmetering valves (Nupro Co., Cleveland, Ohio) placed at the out-

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RIVAS AND WHITAKER

let of oxygen and nitrogen cylinders. Reaction mixtures wereequilibrated with different oxygen levels by bubbling the appro-priate gas mixture through the reaction solution in the reactionvessel for 10 min before adding the enzyme. Initial oxygenconcentrations were determined with the oxygen electrode.pH Stability of Enzyme B. Aliquots of 0.1 ml of enzyme

solution, previously dialyzed against water, in 10 X 75 mm testtubes were mixed with 0.4 ml of buffer solution ranging in pHfrom 3.0 to 7.0. After incubation for 2 hr at 30 C, the remain-ing activity was determined polarographically by adding 0.2ml of incubated enzyme solution of 2.8 ml of 5 mm chlorogenicacid at pH 4.0. For incubation and activity determinations, thebuffers were 25 mm succinate-25 mm pyrophosphate, ,u = 0.22.

RESULTS AND DISCUSSION

Enzyme Purification. Two proteins with polyphenol oxidaseactivity were purified to homogeneity as determined by poly-acrylamide disc gel electrophoresis. Homogeneity was achievedin just three steps: gel filtration, DEAE-cellulose chromatog-raphy, and hydroxyl apatite chromatography (Table I).

Inclusion of 0.03 M ascorbic acid and 1 % polyethylene gly-col in the extracting fluid slowed down but did not completelyprevent browning following homogenization of the pear peel-ings. Passage of the supernatant liquid through a SephadexG-25 column immediately after homogenization and centrif-ugation separated the enzyme from phenolic compounds andgave a preparation that did not brown even after several daysin the cold.

Pear peelings were used in this work because they containsubstantially more polyphenol oxidase activity than the flesh.The two enzymes combined represented only 7.2 X 10-'% ofthe peel weight (fresh weight basis), although they accountedfor approximately 10% of the extracted protein.The recovery of activity following DEAE-cellulose chroma-

tography was more than 100% even excluding the activitypresent in fraction C (Table I). This is most likely due to re-moval of inhibitory material.About 10% of the polyphenol oxidase activity was found in

a fraction, designated as C, eluted from DEAE-cellulose with0.5 M phosphate (Fig. 2). On electrophoresis, the activity bandmoved in an identical fashion to that of enzyme B. The Kmvalues of this fraction on pyrocatechol and d-catechin weresimilar to those for enzyme B. On standing in the cold fol-lowing elution from the DEAE-cellulose collumn, fraction Cgave a copious, gel-like white precipitate identified as pecticmaterial. Therefore, we conclude that fraction C is proteinfrom fraction A' which had different chromatographic prop-erties on DEAE-cellulose (Fig. 2) because it was complexedwith pectic material.

Substrate Specificity. The results of substrate specificitystudies of pear polyphenol oxidase enzymes A and B are shownin Table II. The two enzymes are o-diphenol oxidases. Nocresolase or laccase activity was present as indicated by lackof activity toward monophenols (p-coumaric and ferulic acids)and in-phenols (resorcinol and phloroglucinol). There was noactivity toward monophenols even in the presence of 50 pAMpyrocatechol (19) or of 1 mm ascorbic acid added to eliminatethe lag phase characteristic of hydroxylation of monophenolsby polyphenol oxidases. Some plant polyphenol oxidases, i.e.mushroom, potato, broad bean, catalyze both the hydroxyla-tion of monophenols and the oxidation of o-diphenols. How-ever, many polyphenol oxidases lack monophenol oxidase ac-tivity (2, 13, 24).

Chlorogenic acid. d-catechin, pyrocatechol. and 4-methylcatechol were all rapidly oxidized. although the last two com-

pounds do not normally occur in pears. The results lend sup-port to earlier suggestions that chlorogenic acid and catechinsare the major substrates for pear polyphenol oxidase (16, 20).Catechins, leucoanthocyanidins, and chlorogenic acid consti-tute about 90% of the total polyphenolic content of Bartlettpears (17).The Vm.x values on all substrates were three to five times

higher for enzyme B than for enzyme A. Whether this meansenzyme B is a more efficient catalyst than enzyme A or whetherthe active site concentration of B is greater than for A cannotbe determined from the data available. The Km values aresimilar to those obtained with other fruit polyphenol oxidases(6, 13, 24).

Effect of Ionic Strength. The activity of enzymes A and Bon pyrocatechol as a function of ionic strength, as adjustedwith NaNO3, was determined (Table III). The activity of en-zyme A was doubled when the ionic strength was increasedfrom 0.012 to 0.20, while there was no effect of ionic strengthon the activity of enzyme B.

Temperature Stability. Temperature stabilities of pear en-zymes A and B are shown in Figure 4. The two enzymes havesimilar heat stability characteristics except for the 10-min lag

Table III. Effect of Ioniic Strenigth onz Pear PolyplhenolOxidases Activity

Activity was determined from the initial rate of oxygen uptake.The reaction conditions were: 10 mm pyrocatechol in 10 mM phos-phate buffer with ionic strength changed by the use of NaNO3,pH 6.2, 30.0 C, and 0.24 mm oxygen.

Specific ActivityIonic Strength

Enzyme A Enzyme B

jAmoles 02/711in-tig protein

0.012 6.95 20.60.10 10.0 19.50.20 12.0 22.60.30 13.6 20.80.40 14.2 27.90.50 14.4 23.2

F-

LLLJ

-j

10 30 50

TIME (MIN)FIG. 4. Heat stabilities of pear enzymes A and B. Enzyme A

(50 )ug/ml) and enzyme B (31 ,ug/ml) in 0.1 M phosphate, pH 6.2,were heated in sealed tubes at various times at either 50 C (0:enzyme A; 0: enzyme B) or 75 C (A: enzyme A; A: enzyme B).Remaining activity was determined with an oxygen electrode atpH 4.0 and 30 C with chlorogenic acid as substrate.

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POLYPHENOL OXIDASES OlF BARTLETT PEARS

in loss of activity of enzyme A at 75 C. Both enzymes lost noactivity over a period of 60 min when incubated at 50 C, pH6.2, and a protein concentration of 50 and 31 ,tg/ml for en-zymes A and B, respectively. After heating for 60 min at 75 C,35 and 27% of the activity of enzymes A and B, respectively,remained. Relatively high heat stabilities are found for mostpolyphenol oxidases.

Effect of Oxygen Concentration. Polyphenol oxidase-cata-lyzed reactions are two substrate reactions with oxygen as thesecond substrate. The effect of systematically varying each sub-strate concentration can give valuable information about themechanism of action of polyphenol oxidase.The effect of chlorogenic acid and oxygen concentrations on

the initial velocity of enzyme B-catalyzed oxidation of cholor-genic acid is shown in Figure 5. Reciprocal plots of substrateconcentration versus initial velocity give a series of lines whichintersect to the left of the vertical axis. Therefore, the experi-mental results suggest a sequential mechanism (23) for thebinding of chlorogenic acid and oxygen to pear polyphenoloxidase. Gregory and Bendall (5) found that with tea poly-phenol oxidase series of parallel lines (indicative of a PingPong mechanism [23]) were obtained for most substrates at dif-ferent oxygen concentrations. The exception was chlorogenicacid, which gave a series of lines converging at the vertical axis.With mushroom polyphenol oxidase, a series of parallel or veryslightly convergent lines was obtained with pyrocatechol,whereas series of convergent lines were obtained in the case ofacetyl catechol and 4-methyl catechol (4). There is no goodexplanation of why the plots should be different with differentsubstrates.

In a sequential mechanism, one of the substrates may be re-quired to bind to the enzyme before the second substrate canbind (ordered mechanism) or the two substrates may bind ran-domly (random mechanism) to the enzyme (23). Ingraham (8)in a study on the mechanism of a polyphenol oxidase fromFrench prunes found his data were consistent with a mecha-nism in which oxygen bound first to the enzyme. Duckworthand Coleman (4) reported that, if the mechanism of mushroompolyphenol oxidase is ordered, oxygen does not bind first. Thedata with tea polyphenol oxidase are consistent with a randommechanism (5). Why such divergent results are found by dif-ferent workers on different polyphenol oxidases must await ad-

6.0 A Bx

0 0.4 0.8 0 4 8 2I/[CHLOROGENIC ACID] ,mM1I /03m~

FIG. 5. Effect of oxygen and chlorogenic acid concentrations oninitial velocity of pear enzyme B-catalyzed reaction. The reactionswere followed with an oxygen electrode at pH 4.0 and 30.0 C. Thebuffer was 25 mM succinate-25 mM pyrophosphate with ionicstrength adjusted to 0.22 with NaNO3. A: the oxygen concentra-tions were: 0: 0.24 mM; A: 0.14 mM; *: 0.101 mM; and X:0.079 mM. B: the chlorogenic acid concentrations were: 0: 5 mM;A: 2 mM; U: 1.3 mM; and X: 1 mM. Activities are expressed per mlof enzyme which contained 31 ,ug/ml of protein.

0 _-0co E

o xE c.

6

>

0.6 0.8 0 0.2 0.6I/[S], mM-, I/[S], mM-'

1.0

FIG. 6. Effect of pH on initial velocity of pear enzyme A- andenzyme B-catalyzed oxidation of chlorogenic acid. The reactionswere followed with an oxygen electrode at 30 C. The buffers were25 mM succinate-25 mM pyrophosphate adjusted to the desired pHwith NaOH and to an ionic strength of 0.22 with NaNO3. Oxygenconcentration was 0.24 mm and chlorogenic acid concentrationsranged from 1 to 5 mm. The pH value is indicated above each line.Activities are per ml of enzyme which contained 50.0 and 31.0,g/ml of protein for enzymes A and B, respectively.

00-J

>

x

2:

zLLJcLr0-

3 4 5 6 7pH

FIG. 7. Effect of pH on Vmax for pear enzyme A- and enzyme B-catalyzed oxidation of chlorogenic acid. The data are taken fromTable IV and normalized relative to the Vmas at pH 4.0 being100%. Symbols used are: 0: enzyme A; 0: enzyme B. The theo-retical curve for enzyme A (solid line) is drawn for pK values of3.5 and 5.2 and adjusted upward to 100% at the maximum value;that for enzyme B (dashed line) is drawn for pK values of 3.2and 6.7.

ditional data on these enzymes. We can make no conclusionsabout the order of addition of substrates to the pear polyphenoloxidases from our data.The following values for the kinetic constants were calcu-

lated from the data of Figure 5: Vma, of 103 /imoles of oxygenuptake/min mg enzyme, Km for oxygen of 0.11 mM and Kmfor chlorogenic acid of 7.2 mM at pH 4.0. Thus, under atmos-pheric oxygen concentrations of 0.24 mm, the enzyme is about68% saturated with oxygen.

Effect of pH on Activity. The effect of pH on V.,., and Kmnfor oxidation of chlorogenic acid by enzymes A and B is shownin Figures 6 and 7 and Table IV. At each pH the initial veloc-ity was determined as a function of chlorogenic acid concen-tration (Fig. 6). The series of lines of the reciprocal plots con-

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RIVAS AND WHITAKER

verge on the X-axis to the left of the vertical axis. Thus, thehydrogen ion is a simple, noncompetitive inhibitor (23) of thepolyphenol oxidases, and Km is independent of hydrogen ionconcentration (Table IV).The Vmax values are quite dependent on pH (Table IV, Fig.

7). In Figure 7, the Vms1 values, relative to the value at pH 4.0,are plotted as a function of pH. The pH-Vmiax profiles for bothenzymes are probably bell-shaped with similar dependencies onpH on the acid side of the pH optimum, but they show differ-ences in their pH dependencies on the alkaline side. The bestvalues for the pK values are: 3.5 + 0.2 and 5.2 ± 0.5 for en-zyme A, and 3.2 + 0.2 and 6.7 + 0.5 for enzyme B. With en-zyme B, the pH activity data do not exclude the possibility oftwo pH optima.

Widely different pH optima have been reported for variousplant polyphenol oxidases. The pH optima of tea polyphenoloxidase on pyrogallol and 4-methyl catechol were found to be5.7 and 5.0, respectively (5). Banana polyphenol oxidase hada pH optimum of 7 on dopamine (13). pH optimum for oxida-tion of chlorogenic acid by potato polyphenol oxidase was 4.3(1), whereas that for apple polyphenol oxidase was 5 (21). ThepH optima for clingstone peach polyphenol oxidases were6.5 to 7.2 on catechol (24). In many of the cases, the relation-ship between substrate concentration and Km as function ofpH is not known. It is of interest that the pH of Bartlett pearsat canning ripeness is pH 4 (18).The polyphenol oxidases were stable for 2 hr at 30 C and

pH 7. Between pH 4 and 7 there was about 20% loss in ac-tivity under these conditions, while the activity loss was 75 and97% at pH 3.5 and 3.0, respectively, after 2 hr. Since initialrates were used in these studies on effect of pH on activity, en-zyme instability is probably not responsible for the effect ofpH on the acid side of the pH optimum and certainly not onthe alkaline side.The data indicate that the prototropic groups of pK 3.5 and

5.2 for enzyme A and pK 3.2 and 6.7 for enzyme B are in-volved in oxidation of chlorogenic acid and not in its bindingto enzyme (pH independence of Kin for chlorogenic acid). Itis not possible to assign specific groups to these pK values atthis time.

Inhibition of Enzymes A and B. Inhibition of pear enzymesA and B by p-coumaric and benzoic acids are shown in Figure8. With p-coumaric acid, the series of lines of the reciprocal

Table IV. Miclzaelis Parameters for Pear Polypheiiol OxidaseOxidationt of Chlorogeniic Acid as Funzctionz ofpH

Activity was determined from the initial rate of oxygen uptake.Substrate solutions ranging from 1 to 5 mm were prepared in25 mM succinate-25 mM pyrophosphate buffer, ionic strength of0.22, at different pH values. Ionic strength was adjusted withNaNO3. Temperature was 30.0 C, and oxygen concentration was0.24 mm. Km and Vma, values were determined from Lineweaver-Burk plots (Fig. 6).

Enzyme A

pH -

3.03.54.05.0 j'6.07.0

Enzyme B

Km max K max

Amoles 02'M inl* ng JA.MOles O2s/min mgproteint protein

9.50 i 1.87 12.9 2.1 8.16 0.79 47.0 4 5.09.10 3.71 24.9 5.4 5.90 1.52 54.0 i 11.411.7 i 3.9 42.6 10.4 8.00 0.91 83.5 i 9.411.4 3.2 22.8 6.1 9.20 1.54 69.0 i 13.18.30 4 0.64 13.6 + 5.6 9.30 0.88 61.4 i 2.1

9.20 1.38 44.2 5.9

OEc12- ENZYME B ENZYME B B

32 12

0 0.2 0.6 1.0 0 0.2 0.6 1.0

I/S,mm-, /ES], mm-'

FIG. 8. Inhibition of pear enzyme A- and enzyme B-catalyzedoxidation of chlorogenic acid by p-coumaric and benzoic acids.The reactions were followed with an oxygen electrode at 30.0 Cand pH 6.2 (p-coumaric acid) or pH 4.0 (benzoic acid). The buff-ers were 25 mM succinate-25 mM pyrophosphate with ionic strengthadjusted to 0.22 with NaNO,. Activities are expressed per ml ofenzyme which contained 50.0 and 31.0 ,ug/ml of protein forenzymes A and B, respectively. Chlorogenic acid concentrationsranged from 1 to 5 mM. Concentrations of p-coumaric acid were:*: no inhibitor; A: 0.1 and 0.2 mM for enzymes A and B, re-spectively; *: 0.4 mM; X: 0.75 mm. Concentrations of benzoic acidwere: *: no inhibitor; A: 0.1 mM; *: 0.25 mM; X: 0.366 mM.

plot converge on the X-axis to the left of the vertical axis. Re-plots of the slopes and intercepts as a function of inhibitor con-centration gave straight lines. Thus, p-coumaric acid givessimple, linear noncompetitive inhibition (23) of both enzymeswhich indicates that the substrate, chlorogenic acid, and p-coumaric acid bind at different sites on the enzyme. Ki valuesfor p-coumaric acid were 0.38 and 0.50 mm for enzymes A andB, respectively.A number of monophenols are inhibitory of polyphenol ox-

idase activities (12, 15). p-Coumaric acid was found to be anoncompetitive inhibitor of potato tuber polyphenol oxidase-catalyzed oxidation of chlorogenic acid (12) with Ki of 5.2 mM;however, the series of lines of the reciprocal plot convergedabove the X-axis.

Inhibition of pear enzymes A and B by benzoic acid, withchlorogenic acid as substrate, gave a series of lines which con-verged on the Y-axis. Plots of slopes of the lines as a functionof benzoic acid concentration gave straight lines. Thus, benzoicacid is a linear competitive inhibitor (23) of these enzymeswhich indicates that it and chlorogenic acid bind at the samesite or overlapping sites on the enzymes. The Ki values were0.04 and 0.11 mM for enzymes A and B, respectively, based onthe unionized benzoic acid concentration at pH 4.0 (15).

Benzoic acid has been found to be a competitive inhibitorof other polyphenol oxidases (4, 10, 13). The Ki value for ben-zoic acid inhibition of mLshroom polyphenol oxidase-catalyzedoxidation of catechol was reported to be 1.02 /.tM (4); this valueis much lower than the values found for pear polyphenol oxi-dases and most other polyphenol oxidases.

LITERATURE CITED

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2. CLAYTON, R. A. 1959. Pr-opereties of tobacco polyphenol oxidase. Arcb.Bioclito1m. Bophxs-s. 81: 404 -417.

506 Plant Physiol. Vol. 52, 1973

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POLYPHENOL OXIDASES

3. DAVIS, B. J. 1964. Disc electrophoresis. II. Metlhod of application to humanserum proteins. Ann. N. Y. Acad. Sci. 121: 404-427.

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5. GREGORY, R. P. F. A.ND D. S. BENDALL. 1966. The purification and someproperties of the polyphenol oxidase from tea (Camellia sinensis L.). Bio-chem. J. 101: 569-581.

6. HAREL, E., A. M. MI.AYER, A-ND Y. SHAIN-. 1964. Catechol oxidases from apples,their properties, subcellular location, and inhibition. Physiol. Plant. 17: 921-930.

7. HODGEMAN, C. D. 1957-58. Handbook of Chemistry and Physics, Ed. 39.Chemical Rubber Co., Cleveland, Ohio. p. 1607.

8. INGRAHAM, L. L. 1957. Variation of the Michaelis constant in polyphenoloxidase catalyzed oxidations: substrate structure and concentration. J.Amer. Chem. Soc. 79: 666-669.

9. JOHNSON, M. J. 1941. Isolation and properties of a pure yeast polypeptidase.J. Biol. Chem. 137: 575-586.

10. KRUEGER, R. C. 1955. The inhibition of tyrosinase. Arch. Biochem. Biophys.57: 52-60.

11. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951.

Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

12. MACRAE, A. R. AND R. G. DUGGLEBY. 1968. Substrates and inhibitors ofpotato tuber phenolase. Phytochemistry 7: 855-861.

13. PALMER, J. K. 1963. Banana polyphenol oxidase. Preparation and properties.Plant Physiol. 38: 508-513.

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15. ROBB, D. A., T. SWAIN, AND L. W. NIAPSON. 1966. Substrates and inhibitorsof the activated tyrosinase of broad bean (Vicia faba L.). Phytochemistry5: 665-675.

16. SIEGELMAN, H. W. 1955. Detection and identification of polyphenol oxidasesubstrates in apple and pear skins. Arch. Biochem. Biophys. 56: 97-102.

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Food Technol. 20: 534-538.18. TATE, J. N., B. S. LUH, AND G. K. YORK. 1964. Polyphenol oxidase in Bartlett

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in the hydroxylation of p-coumaric acid by a phenolase from leaves of

spinach beet (Beta vulgaris L.). Biochem. J. 119: 89-94.20. WALXER, J. R. L. 1964. The polyphenol oxidase of pear fruit. Aust. J. Biol.

Sci. 17: 575-576.21. WALKER, J. R. L. 1964. Enzymic browning of apples. II. Properties of apple

polyphenol oxidase. Aust. J. Biol. Sci. 17: 360-371.22. WHITAKER, J. R. 1967. Paper Chromatography and Electrophoresis. I. Elec-

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