PURIFICATION OF PEACH POLYPHENOL OXIDASE IN

THE PRESENCE OF ADDED PROTEASE INHIBITORS1

WILLIAM H. FLURKEY2
Departments of Food Science and Biochemistry
Clemson University
Clemson, SC 29631
and
JOSEPH J. JEN3
Department of Food Science and Human Nutrition
Michigan State University
East Lansing, M I 48824
Received for Publication February 1, 1980

ABSTRACT

Crude preparations o f peach fruit (Prunus persica Batsch cv. Redskin)

polyphenol oxidase (PPO) showed many apparent isoenzyme forms.
Some o f these forms were probably the result o f proteolytic action o f
peach proteases while other forms were the result o f association o f PPO
with carbohydrate materials.
In the presence of protease inhibitors, Trasylol and phenylmethylsul-
f o n y l fluoride, three apparent isoenzyme forms of PPO were purified to
homogeneity. The purification scheme included hydrophobic chroma-
tography on phenyl sepharose CL-4B, hydroxylapatite chromatography,
DEAE cellulose chromatography, and gel filtration on Ultrogel A c A 34.
Minor contaminants remaining after these steps were separated from PPO
by gel electrophoresis.
The major PPO isoenzyme form ( A )was purified 44 fold with an over-
all yield of 5.6% and contained no detectable carbohydrates. lsoenzyme
forms A ' and A" were purified 104 and 67 fold respectively, but still
were associated with carbohydrate material. Cesium chloride centrifuga-
tion partially removed the carbohydrates associated with PPO A' and A".
Purified peach PPO A showed greater activity toward D-catechin

'Contribution No. 1774 from South Carolina Agricultural Experiment Station,

Cle2mson, SC 29631
Current address: Department of Agricultural Chemistry, Washington State Uni-
ver$ty, Pullman, W A 99164
To whom correspondence should be addressed. Current address: Campbell
Institute for Research and Technology, Campbell Place, Camden, NJ 08101

Journal of Food Biochemistry 4 (1980) 29-41. At! Righis Reserved.

@Copyright 1980 b y Food & Nutrition Press, Inc., Westport, Connecticut 29
30 WILLIAM H. FLURKEY AND JOSEPH J. JEN

(539%)and pyrogallol(l82%) than to catechol (100%). A n apparent

K , of 4,0 . 3 , and 2 mM was obtained with D-catechin, pyrogallol and
catechol, respectively. The enzyme was severely inhibited by 10 pM
2,3-naphthalenediol (91%) and b y 1 0 pM diethyl dithiocarbamate (100%).

INTRODUCTION

Purification and characterization of homogeneous polyphenol oxidase

(PPO) appears frequently in the literature. Most purification and charac-
terizations have been confined to fungi (Gutteridge and Robb 1975;
Jolley et al. 1969; Bull and Carter 1973). In higher plants, the enzyme
has been purified to apparent homogeneity from spinach (Vaughan et al.
1975), potatoes (Balasingam and Ferdinand 1970; Patil and Zucker 1965),
and tea leaves (Gregory and Bendall 1966). Polyphenol oxidase in fruits
has been characterized from many sources (Benjamin and Montgomery
1973; Kahn 1977; Palmer 1963; Stelzig et al. 1977; Wong et al. 1971a),
but has been purified to apparent homogeneity in only a few instances
(Kidron et al. 1977; Rivas and Whitaker 1973; Wong et al. 1971a). Many
of the problems associated with the isolation of PPO from fruit tissues
are due t o the existence of high concentrations of phenolic materials,
pectic substances and proteases.
Enzymatic browning of peaches in food processing is believed to be
related to PPO (Luh and Phithakpoll972; Reyes and Luh 1960). The
enzyme has been studied with regard t o its development patterns
(Flurkey and Jen 1978; Flurkey 1979; Harel et al. 1970; Jen and Kahler
1974),laccase-like activity (Mayer and Harel 1968), plant hormone and
hormone analogs (Knapp et al. 1970), and isoenzyme forms (Wong et al.
1971a; Flurkey and Jen 1978; Jen and Flurkey 1979. Wong et al.
(1971a) reported the partial purification and characterization of 4 PPO
isoenzymes. However, accurate assessment of the characteristics and
functions of the enzyme requires the isolation of all isoenzyme forms to
homogeneity. In addition, care must be taken to limit generation of arti-
facts during the isolation of PPO. This report describes the purification
of peach PPO t o apparent homogeneity in Redskin peaches, a freestone
variety of peach, in the presence of added protease inhibitors.

MATERIALS AND METHODS

Enzyme Assay
Polyphenol oxidase was assayed as described previously with catechol
 PURIFICATION OF PEACH PPO 31

as substrate (Flurkey and Jen 1978). Column eluates were monitored for
PPO activity by incubating 50 p1 of each fraction with 5 ml of 10 mM
catechol for 15 min and the absorbance at 420 nm was read, Using this
quick assay method, all column fractions could be monitored for PPO
activities within a 30 min period. Polyphenol oxidase was also assayed
with such substrates as D-catechin, D,Lcatechin, pyrogallol, dopamine,
4-methyl catechol, L d o p a , protocatechuic acid, D-dopa, caffeic acid,
gallic acid and o-phenylenediamine. Lineweaver-Burk plots of some sub-
strates were constructed and used to determine apparent KM's. Poly-
phenol oxidase activities were determined in the presence of such inhi-
bitors as EDTA, KCN, diethyldithiocarbamate (DIECA), naphthalenediol
at 1and 10 pM with catechol as the substrate.
Trypsin, chymotrypsin, pepsin and papain-like protease activities were
assayed as described in the Worthington Enzyme Manual (1976). The
Bio-Rad protease detection kit (Bio-Rad Laboratories, Richmond, CA)
was used for qualitative determination of protease activity. Protein con-
tent was determined either by the Lowry method (1951) or by the Bio-
Rad Protein assay (Bio-Rad Laboratory, Richmond, CA).

Enzyme Extraction
Peaches (Prunus persica Batsch cv. Redskin) were harvested at the
yellow green stage of maturity as described by Jen and Graham (1975).
Acetone powders of the peaches were prepared and the powders (lOOg/
batch) were extracted with 3 liter of 50 mM phosphate buffer, pH 6.2,
containing 1M KC1, 0.5 mM phenylmethylsulfonyl flouride (PMSF,
Sigma Chemical Company, St. Louis) and 10 ml of Trasylol (10,000
Kallikrein inactivator units, Mobay Chem. Company, NY) for 2 h at
room temperature. The slurry was centrifuged at 15,000 X g for 1 5 min
at 4C. Unless mentioned, 0.5 mM PMSF was included in all subsequent
steps. Calcium acetate (0.5 g/20 ml extract) was added t o the supernatant
t o remove pectic substance and the mixture was stirred for 4 h at 4C.
The suspension was centrifuged and ammonium sulfate was added to the
supernatant t o a final concentration of 1M. The suspension was centri-
fuged and the supernatant was used as crude extract of PPO for further
purification of the enzyme.

Purification Scheme
The purification scheme is presented in Table 1. Column conditions
are described as follows. All sample applications and development of
columns were regulated at a rate of 1-1.5 ml/min by a peristaltic pump.
(1)Phenyl Sepharose (PS) chromatography: A 5 X 28.5 cm column of
32 WILLIAM H. FLURKEY AND JOSEPH J. JEN

Table 1. Summary of purification of peach polyphenol oxidase

PPO
Volume Protein Activity Specific Yield Purification
Fraction (ml) (mg) (units) Activity (%) (fold)

Crude extract 2625 903 11248 12.45 100 1

PS column 560 237 7951 34.0 70.7 2.70
HA column
PPO A 250 30 2970 99 26.0 8.0
PPO A' 66 46.7 2880 62 25.6 59.6' 5.0
PPO A" 47 43.2 902 21 8 .O 1.7
DEAE column
PPO A 46 5.7 1022 179 9.1 14.4
PPO A' 57 0.68 880 1294 7.8 18.7' 103.9
PPO A'' 40 0.25 208 832 1.8 66.8
AcA 34
PPO A 32.5 1.16 632 545 5.6 43.8
~~~

'
Number following the bracket represent the total yield of the 3 isoenzyme forms added to-
gether t o reflect the yield of the column

PS was packed and equilibrated with 50 mM phosphate buffer, pH 6.2,

containing 1M KC1 and 1M (NH,), SO4 (designated as EB). The sample
(2625 ml, 14,060 units PPO, 903 mg protein) was applied and the column
was first washed with EB. Enzyme was eluted from the column with 5
mM phosphate buffer, pH 6.2, and dialyzed overnight vs 2 X 20 liter of
1mM phosphate buffer, pH 6.2.
(2) Hydroxylapatite (HA) chromatograph: A 2.5 X 16 cm column was
packed and equilibrated with 1mM phosphate buffer, pH 6.2. The sam-
ple (560 ml, 7950 units PPO, 237 mg protein) was applied, the column
was washed with the same buffer followed by a linear gradient of 1t o
50 mM phosphate containing 1M KC1. A second linear gradient from 0
to 1M ammonium sulfate in 50 mM phosphate containing 1M KCl was
followed. Fractions 38-65 (PPO A), 66-72 (PPO A'), and 73-79
(PPO A") were pooled respectively, and each dialyzed vs 2 X 20 liter of
1mM phosphate at pH 8.3.
(3) DEAE cellulose chromatography: A 0.5 X 15 cm column was pre-
pared and equilibrated with 1mM phosphate buffer at pH 8.3. The PPO
A sample (227 ml, 1386 units PPO, 30 mg protein) was applied and
washed with the same buffer. The column was developed with a linear
gradient of 1-20 mM phosphate buffer and finally washed with 50 mM
phosphate buffer containing 1M KCl. The PPO A fractions were collected,
concentrated with Aquacide IIA (Calbioch-Behring Corp., La Jolla, CA),
and used for gel filtration step. The PPO A' and PPO A" fractions were
 PURIFICATION OF PEACH PPO 33

similarly chromatographed.
(4) Gel filtration chromatography: A 2.6 X 34 cm Ultrogel AcA 3 4
(LKB Instruments, Rockville, MD) column was prepared and equilibrated
with 100 mM phosphate buffer at pH 6.2. The PPO A sample (5.6 ml,
646 units PPO, 5.7 mg protein) was applied and eluted by gravity filtra-
tion with the same buffer.
(5) Disc gel electrophoresis: In some cases, minor contaminants were
present after the gel filtration step. These contaminants were removed
by disc gel electrophoresis as described by Davis (1964) and by Jen et al.
(1980). Area of gels with PPO activity were excised after staining a du-
plicate gel with catechol, then homogenized and centrifuged. The super-
natant served as the source of purified PPO A.
(6) Cesium chloride centrifugation: The PPO A' and PPO A", contain-
ing high amount of 260 nm absorbing materials, were purified after the
DEAE-cellulose step by ultracentrifugation in a linear CsCl gradient
(1g/ml of CsCl in 50 mM TES buffer a t pH 8.0 containing 50 mM NaCl
and 5 mM EDTA). The samples were centrifuged a t 45,000 rpm for 48 h
in a TI-75 rotor in a Beckman L5-65 centrifuge at 4C. The gradients were
fractionated. The PPO A' or PPO A" fractions were collected, concen-
trated and dialyzed before analyses.

RESULTS AND DISCUSSION

Preliminary Observations
The purification of Redskin peach PPO was exceedingly difficult due
t o the presence of pectic substances, low protein content, the existence
of multiple isoenzyme forms and of proteases. A number of active PPO
forms were observed by disc gel electrophoresis in crude extracts and in
PS column and HA column fractions. Preparation and extraction of the
acetone powders, with or without protease inhibitors, influenced the in-
tensities of the isoenzyme forms observed on disc gels and on isoelectric
focusing (Flurkey 1979). These forms of PPO isoenzymes were partially
eliminated with the use of protease inhibitors in the initial extraction
step. Complete elimination of these forms was achieved when PMSF was
included in all purification steps as described in methods section (Fig. 1).
Assays for trypsin, chymotrypsin, pepsin and papain-like protease acti-
vities in crude extracts were inconclusive; however, minor amounts of
protease activity were detected with the Bio-Rad protease detection kit.
In addition, use of protease inhibitors was found to change the elution
profiles of proteins on PS and HA columns. Use of PMSF appeared t o
decrease the present of active enzyme fragments which clustered with
34 WILLIAM H. FLURKEY AND JOSEPH J. JEN

.1

.2

.3

.4

Rf .5

.6

.7

.8

.9

1.0
A B

FIG. 1. TOTAL NUMBERS OF PPO ISOENZYME FORMS OBSERVED

FROM ALL FRACTIONS OF HA COLUMNS AS SHOWN ON DISC GELS
WITH OR WITHOUT THE USE OF PROTEASE INHIBITORS IN ENZYME
PREPARATIONS
Each line represents an enzyme band which appeared after staining with
catechol. A. Without protease inhibitors. B. With protease inhibitors during
the initial extraction of the acetone powders only. C. With protease inhibitors
at each step in the purification scheme.

the native enzyme. These fragments showed much different pH optima,

K, 's, molecular weights, substrate specificities and electrophoretic
characteristics compared t o the native enzyme (Flurkey 1979). In grape
catechol oxidase, Hare1 et al. (1973) observed that conversion of fast
moving enzymes t o slower moving enzymes could be produced with the
use of certain proteases such as trypsin. Wong et al. (1971a) separated
and partially purified 4 PPO isoenzyme forms from clingstone peaches.
Many of the characteristics reported by them were similar to the char-
acteristics of Redskin PPO when purification was carried out without
the use of protease inhibitors. However, clingstone peaches may not con-
tain as many proteases as Redskin peaches.

Purification
As shown in previous reports (Flurkey and Jen 1978; Jen and Flurkey
 PURIFICATION OF PEACH PPO 35

1979), a considerable amount of 280 nm absorbing materials did not

bind to PS columns. The enzyme eluted from these columns was usually
purified 2-3 fold with yields greater than 50%.The yields seemed t o be
dependent upon or related t o the age of the acetone powder material. In
a earlier study of Redhaven peaches, when freshly prepared acetone pow-
der was used, the yield of PS column was always over 100% showing re-
moval of inhibitory substances (Jen and Flurkey 1979).
Several peaks of PPO activity were separated on HA columns (Fig. 2).
One form of PPO, designated PPO A, showed a low affinity toward the
resin. The second and third form of PPO, designated PPO A' and A"
respectively, exhibited greater affinity toward the resin than PPO A. All
forms of the enzyme showed the same electrophoretic mobility on native
disc gels.

I
m
2
.s
Gl G2
1 700

500

.4

300

100

Fraction number

FIG. 2. HYDROXYLAPATITE COLUMN CHROMATOGRAPHY O F

REDSKIN PPO
Sample ( 5 6 0 ml) equilibrated in 1 mM phosphate buffer, pH 6.2 o n t o
column (2.5 x 1 6 cm) a t a flow rate of 90 ml/h. Each fraction was 10 ml.
S represents where t h e sample was applied. B indicates where buffer was
applied. G I indicated application of 200 ml of linear gradient of 1-50
mM phosphate containing 1 M KCI. Gz indicated application of 200 mi
of linear gradient of 0-1 M ammonium sulfate in the same buffer system.

When further purified on DEAE-cellulose column, PPO A, A' and A"

each showed one peak of PPO activity which was eluted from the gradi-
ent at approximately 8 mM phosphate buffer. The difference being that
36 WILLIAM H. FLURKEY AND JOSEPH J. JEN

PPO A' and PPO A" showed larger amounts of proteins which did not
bind to the resin (Fig. 3). Chromatography of PPO A on Ultrogel AcA
34 showed that the major contaminants were of lower molecular weight
species than PPO (Fig. 4). The enzyme and protein profiles for PPO A
appeared symmetrical, indicating purification may have been completed.
A t this stage, PPO A showed a single protein band on native disc gels. In
some preparations, minor contaminating proteins still exist after the gel
filtration step. This enzyme fraction was further Durified through pre-
parative gel electrophoresis. The final purified PPO A showed one band
on several kinds of electrophoresis such as acetate strips, acidic gels, etc.
(Flurkey 1979).

I
(D

qN

1"
10 IA1

10 20 30 40 SO
Fraction number
60
I
70

FIG. 3. DEAE CELLULOSE COLUMN CHROMATOGRAPHY O F RED-

1: J 30

20

10

SKIN PPO
A ) for PPO A, B) for PPO A' (PPO A" profile was similar t o PPO A').
Columns equilibrated at pH 8.5, 1 mM phosphate buffer, flow rate at 60
ml/h. Each fraction was 6 ml. S represents where sample was applied and
B represents where buffer was applied. G represents application of 200
ml of linear gradient of 1-20 mM phosphate buffer. H represents appli-
cation of high salt wash ( 1 M KCI + 1 M Ammonium sulfate).

A summary of the purification of PPO from Redskin peaches is shown

in Table 1. The PPO A was purified 44 fold with a yield of 5.6%relative
to the crude extract.
 PURIFICATION OF PEACH PPO 37

0
QD
I
E
aN *E
.
In
rl

r 0
N
4
.3

10 20
Fraction number
30

FIG. 4. ULTROGEL AcA 34 COLUMN CHROMATOGRAPHY OF

40
1 .2

.1

PPO A.
Column ( 2 . 6 x 34 cm) equilibrated with 100 mM phosphate buffer at
pH 6 . 2 , at a flow rate of 60 ml/h. Each fraction was 5 ml. Elution was
with equilibration buffer.

The A’ and A” forms of PPO appeared to be nonspecifically associated

with carbohydrate materials. Centrifugation in a CsCl gradient removed
much but probably not all of the contaminating material (Fig. 5 ) . The
amount of carbohydrates present in each preparation was variable and
was probably responsible for the varied affinities of PPO toward hydroxy-
laptatite. Some RNA and polysaccaride-like material were identified with
certain staining methods on disc gels and by chemical analysis to be as-
sociated with PPO A’ and A” (Flurkey 1979). Apparently, PPO A, A’
and A” differ in their carbohydrate contents but not the protein content.
The association of carbohydrates with PPO in other species has been re-
ported by several workers (Ben-Shalom et al. 1977). Bull and Carter (1973)
showed that RNA was found to associate with tyrosinase and was neces-
sary for enzyme activity in Asperigillus. In apples, the carbohydrate moi-
ety of PPO was RNAse insensitive but reacted positively with orcinol
(Stelzig et al. 1972). Absorption profiles of PPO A’ and A” were more
similar to reported absorption profiles from carbohydrate-associated
PPO than to noncarbohydrate associated PPO.
Substrates and Inhibitors
Compared to catechol, PPO A was most active with catechin and
38 WILLIAM H. FLURKEY AND JOSEPH J. JEN

P
P

Bottom Fraction number

FIG. 5. CESIUM CHLORIDE GRADIENT CENTRIFUGATION

OF PPO A‘ AND A”
A) for PPO A’, B) for PPO A“. Sample (1.8ml) was brought t o
full tube volume (10.25 ml) with 10%CsCl in TES buffer (50 mM,
pH 8.2, 2.5 mM EDTA). Run was performed in Beckman L5-65
at 45,000 rpm for 48 h at 2OoC in a TI-75 rotor. A constant vol-
ume of water was added to each tube after density and PPO acti-
vity measurements to obtain A2 6 0 and A2 8 0 readings. Density
was determined by filling a 20 ml capillary tube t o the mark and
weighing on a analytical balance and expressed as mass/volume.
Symbols 0 __ 0, density; - 0, PPO activity; 0------
0,A2 8 0 ;
a ---_-a, A ~ ~ ~ -

pyrogallol as substrates (Table 2). Less activity was observed with L-dopa,
protocatechuic acid, D-dopa, caffeic acid, gallic acid, or o-phenylene-
diamine. No activity was observed using L-tyrosine, phenol, p-cresol,
o-coumaric acid or ferulic acid as substrates. Luh and Phithakpol (1972)
reported that PPO in Halford clingstone peaches was more active towards
D-catechin in comparison with catechol. Jen and Kahler (1974) reported
the most active substrates in Redhaven peaches were catechol, 4-methyl-
catechol, and pyrogallol. The enzyme apparently lacked a cresolase-like
activity. Apparent K, values of 2 mM, 4 mM, and 0.3 mM were observed
using catechol, D-catechin and pyrogallol as substrates, respectively. Not
 PURIFICATION OF PEACH PPO 39

Table 2. Relative activity of PPO A toward vari-

ous substrates and inhibitors

Relative
Activity (%)
Substrate (1mM) o r Inhibitors Compared to
( 1 o r 10 p m ) Catechol

D-catechin 5 39
D, L-catechin 531
pyrogallol 182
dopamine 103
4-methyl catechol 103
catechol -
100
L-DOPA 23
protacatechuic acid 15
D-DOPA 13
caffeic acid 7
gallic acid 5
o-phenylenediamine 3
EDTA 1 p M 103
rn
10 102
KCN 1pM 95
10 pM 32
DIECA 1 p M 80
10 pM 0
2,3-naphthalenediol 1pM 17
10 pM 9

all Lineweaver-Burk plots were linear and the K, values represented an

approximation of those points which determined a straight line. These
results were in good agreement with those obtained by Wong et al.
(1971b) with regard to substrate specificity and K, values. A K, value
for catechol of 2 mM was obtained for Redskin peach PPO A in this
study. This value was similar to the 4.2 mM for PPO isoenzyme B re-
ported by Wong et al. (1971b). In contrast, Luh and Phithakpol(l972)
showed a K, of 1 5 mM in clingstone peaches and Jen and Kahler (1974)
showed a K, of 29 mM in Redhaven peaches. It seems that the K, may
vary with variety of peaches studied or it may be related t o the purity of
the preparation.
In Table 2, the greatest inhibition of Redskin peach PPO was observed
with 1 0 pM DIECA (100%). No inhibition was observed with EDTA a t
either 1 or 10 pM level. A small amount of inhibition was observed wi-ih
1pM KCN, while 68% inhibition was present with 10 pM KCN. Wong
et al. (1971b) showed their isoenzyme C was inhibited 37% a t 10 pM
KCN, while isoenzyme D was inhibited 57% a t 33 pM. In general, Redskin
40 WILLIAM H. FLURKEY AND JOSEPH J. JEN

PPO showed a much lower affinity for catechol and was inhibited to
greater extents than PPO isoenzymes from the clingstone peaches re-
ported by Wong et al. (1971b). It is therefore difficult to extrapolate
data from different varieties of peaches for direct composition.
Interestingly enough, the enzyme isolated here shared similar proper-
ties to a laccase-like enzyme found in peaches grown in Israel by Mayer
and Hare1 (1968). The laccase-like enzyme did not stick to DEAE column
at pH 7.2 and showed a pH optimum around 6.0.
The results obtained in this study reflected some of the problems which
arise when investigating plant PPO enzymes. The generation of artifacts
from protease and nonspecific carbohydrate associations is a major con-
cern. One must use caution when interpreting information about PPO
isoenzyme forms and functions since these artifact abberations can show
much different characteristics than the native enzyme.

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