W‘M Ad‘v-vv w————

 

.- u—uu...._|_

Enzymnc sm‘ms AND SUBSTRATE!» mvowzb.
m magma; PEACH snowmua '

Thesis 6m fha- Dogma 04‘ Ph. D. ,
M‘CHlGAN STATE UNIVERSWY
Kamai EE-Dén Hussien Matawi‘
19.65

 

LIBRARY

Michigan State
University

 

This is to certify that the
thesis entitled

ENZYMATIC SYSTEMS AND SUBSTRATES INVOLVED
IN FREESTONE PEACH BRCMNING

presented by

Kamal El-Din Hussien Motawi

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Food Science

(5- % fizzféé 1
Major prof sor )7”
Date __JLZQ_,__l9_6.6__Ma

0-169

 

 

 

 

 

 

 

 

 

 

AB STRAC T

ENZYMATIC SYSTEMS AND SUBSTRATES INVOLVED
IN FREESTONE PEACH BROWNING

by Kamal El-Din Hussien Motawi

The browning of peaches during preparation for processing
and frozen storage is a serious and costly problem. The process is
considered to be enzymatic in nature and due to the oxidation of
phenolic compounds by oxidizing enzymes in the presence of molecu—
lar oxygen.

This study was conducted to investigate the relationships
between the enzyme system and the substrates responsible for bring-
ing about brown discoloration in freestone peaches. The enzyme,
polyphenol oxidase (PPO) , was prepared by acetone precipitation
and purified by ammonium sulfate fractionation and Sephadex G-100.
The enzyme solution was not stable at pH 8. 0 at all the temperatures
used, from 220 to ~31. 70 C. The enzyme was not very stable at
room temperature, 220 C. , losing 93. 3% of its activity in 6 days.
The frozen solutions stored for 12 weeks at -17.80 C. and -3l.70 C.
showed a loss in activity of 20 and 16. 6%, respectively. The enzyme

showed an optimum pH of 6. 4 with citrate-phosphate buffer and

 

 

 

 

Kamal El-Din Hus sien Motawi

catechol as substrate and an optimum temperature of 430 C. The
enzyme from the eight varieties studied showed the same substrate
specificity. Elberta peaches had the highest enzyme activity while
Redskin showed the lowest level of activity.

The phenolic constituents of peach extract in order of
prominence were leucoanthocyanins, 35. 6 to 39.4%, chlorogenic
acid, 22.8 to 25. 9%. flavonols, 18.8 to 19.4% and catechin, 12.0
to 14. 8%. Richhaven peaches had the highest total phenolic content,
173. 2 mg./lOO g. and Sunhaven the lowest, 85. 4 mg. /100 g. Catechin
exhibited the highest reactivity with the enzyme followed by chlorogenic
acid, leucoanthocyanins, and flavonols. The degree of discoloration
of peaches during preparation for processing, frozen storage or on
thawing is primarily attributed to the original activity of the enzyme
in the varieties that have from moderate to high total phenolic con-
tent. Paper chromatographic and spectrophotometric techniques
were used for isolation and identification of the phenolic compounds

in peach extract.

w
. 1.8.
m3! .3 '1

i'

4—-
v"

 

t‘w

 

 

 

 

 

ENZ YMATIC SYSTEMS AND SUBSTRATES INVOLVED

IN FREESTONE PEACH BROWNING

BY

Kamal El -Din Hus sien Motawi

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science

1966

 

 

 

 

 

 

DEDICATION

3‘s:

To my Mother and my Wife

 

 

 

 

 

 

ACKNOWLEDGMENTS

The author is deeply indebted to Dr. C. L. Bedford for
his assistance in conducting this study and his counsel in the prepa-
ration of the manuscript. The author wishes to express his sincere
appreciation for the assistance given him by Dr. P. Markakis who
provided laboratory facilities and contributed valuable discussions;
and to Dr. R. C. Nicholas for his encouragement throughout this
study.

Appreciation is also expressed to the Michigan Agricultural
Station for financial support of the project and to the United Arab
Republic Ministry of Education for the financial support which pro-

vided this opportunity for study.

 

 

 

 

TABLE OF CONTENTS

ACKNOWLEDGMENTS . .
LIST OF TABLES . . . . . . . . .

LIST OF FIGURES

INTRODUCTION . . .
REVIEW OF LITERATURE
b/IATERIALS AND METHODS .

Enzyme Extraction .
Fractionation by Ammonium Sulfate .
Enzyme Purification.

Protein Determination . . . . . . .
Assay Procedure.

Effect of pH on Enzyme Activity .
Effect of Temperature on Enzyme Activity
Enzyme Stability . . . . . . .
Substrate Specificity . . . .
Extraction of Phenolic Compounds . . . .
Total Phenolic Determination

Phenolic Oxidation . . .
Spectrophotometric Analysis

Paper Chromatography .

RESULTS AND DISCUSSION .. . . . . . . .

Effect of pH and Temperature on Enzyme Activity .

Stability of the Enzyme . . . . . . .
Substrate Specificity . . . . . . . .
Total Phenolic Compounds . . . . .
Detection of Enzyme Substrates in Peach Extract

iv

Vi

viii

17

17
18
19
19
20
20
21
21
22
22
23
25
26
27

31

33
37
41
42
45

 

 

 

 

 

 

 

TABLE OF CONTENTS-- (Continued)

Total Phenolic Oxidation by Endogenous Enzymes

Isolation and Identification of Peach Phenolics .

Enzymatic Oxidation of the Phenolic Components
CONCLUSIONS . . . . . . .

REFERENCES . . . . . . . . . . . .

Page
47

50
60

68

 

 

 

 

 

Table

10.

ll.

 

LIST OF TAB LES

Fractionation of the enzyme solution with
ammonium sulfate 0 O O O O O O O 0

Comparison of enzyme activity of the different
varieties of peaches . . . . . . . . .

Effect of storage on enzyme activity at 12 weeks .

Michaelis constant and maximum velocities for
purified peach polyphenol oxidase . . . . .

Total extractable phenolics of the different vari-
ties of peaches for the 1964 and 1965 seasons

Oxidation of extractable phenolics of 100 g. of
peach tissue from different varieties, blended
with 100 ml. of distilled water for 15 minutes

Description of the phenolic components of Elberta
peach extract as separated on one dimensional

paper chromatograms . . . . . . . . . .

Effect of blending heated and unheated samples of
peach tissue on total phenolic content . . . .

Phenolic oxidation of peach tissue of different
varieties by endogenous enzymes . . . . . .

Color reaction of spots 1 to 8 with different spray
reagents and the absorbance of their elutes . .

Concentration of the identified phenolic components
in four peach varieties . . . . . . . . . .

vi

 

4O

42

44

44

47

48

51

53

60

 

 

LIST OF TAB LES- -( Continued)

Table Page
12. Progress of oxidation of the different phenolic
components of Elberta peaches by endogenous
enzyme................ 64
13. Progress of oxidation of the phenolic components
of Richhaven peaches by endogenous enzyme . . 64
14. Progress of oxidation of the phenolic components
of Sunhaven peaches by endogenous enzyme . . 65
15. Progress of oxidation of the phenolic components
of Redskin peaches with endogenous enzyme . . 65

 

 

Figure

10.

ll.

12.

 

 

LIST OF FIGURES

Folin-Denis pigment formation by catechin in a
standard curve for phenolic determination . . . . .

The chromatography of partly purified peach poly-
phenol oxidase on Sephadex G-100 column
( 1. 5 x 70 cm. ) ....................

Enzyme activity of the crude enzyme preparations
(0.001 M catechol, 0.1 m1. enzyme and citrate-
phosphate buffer, pH 6.4) ..............

Influence of pH on peach PPO activity ........

Influence of temperature on peach PPO activity

Stability of crude peach PPO at different storage
temperatures, and pH 8. 0 .............

A reaction curve of peach PPO with catechol by
using the Lineweaver and Burk procedure ( 1934)

Paper chromatograms of Elberta peach extract,
a - Treated with FeCl -K Fe(CN)
b - Sprayed with partly purified peach PPO . . . .

Ultraviolet spectra of peach phenolic extract before
and after oxidation with endogenous PPO. . . . . .

Ultraviolet spectra of a-the combined elute of spots
1, 2, 3 and 4; and b-the individual spots . . . . . .

Ultraviolet spectra of a-the elutes from spots 5 and
6; and b-pure chlorogenic acid ...........

Ultraviolet spectra of a-the elute of spot 7; and
b-commercial catechin .......... . . . . .

Page

24

32

35

36

38

39

43

46

49

54

55

56

 

 

 

 

Figure
13.

14.

15.

16.

 

 

LIST OF FIGURES--(Continued)

Ultraviolet spectrum of the elute of spot 8 .....

Two -dimensiona1 paper chromatography of peach
ethyl acetate extract ....... . ........

Phenolic components of Elberta peach extract as
separated on one-dimensional paper chromato-
grams. 1 and 2 leucoanthocyanins, 3 and 4
flavonols, 5 and 6 chlorogenic acid, 7 catechin
and 8 flavonol ................. . . .

Peach polyphenol oxidase activity toward the dif-
ferent components of Elberta peach extract . . . .

ix

Page

58

59

61

62

 

 

 

INTRODUCTION

The enzymatic discoloration of plant tissues following
cell damage is a serious and long standing problem. Many fruits
undergo rapid change in color after mechanical or physiological
injury during harvesting and storage. Such color change in fruit
products is accentuated during preparation for processing by can-
ning, dehydration or freezing and continues during freezing storage
and subsequent defrosting of frozen fruits. The natural color of
the product may be destroyed or masked by formation of dark brown
pigments. This browning is generally accompanied by change in
color, flavor and nutritive value which greatly detract from the
quality of the finished product.

The problem of browning in peaches during processing
and frozen storage has been the subject of numerous articles and
reports. The process is considered to be enzymatic in nature and
due to oxidation of the so called catechol tannins by oxidizing enzymes
in the presence of molecular oxygen.

The purpose of this study is to investigate the relation-
ship between the enzyme system and the substrates responsible for

bringing about brown discoloration in frozen freestone peaches.

 

 

REVIEW OF LITERATURE

Enzyme catalyzed oxidative browning was recognized by
Kastle ( 1910). Lindet ( 1895) concluded that the changes in color
occuring in fresh cider are due to oxidation of tannin by a laccase-
like enzyme contained in the tissues of apples.

Most of the early research on enzymatic oxidation of
fruit products has been directed to qualitative characterization of
the oxidizing enzyme present and methods of inactivation or control
of the enzyme involved in browning (Basset and Thompson, 1911;
Onslow, 1920; Overholser and Cruess, 1923; Cruess and Fong, 1926,
1929a, 1929b, Joslyn, 1941; and Guadagni, Sorber and Wilbur, 1949).

Quantitative methods in studying plant oxidases were
first used in the United States by Bunzell (1912a, 1912b, 1916a,
1916b, 1916c) . Nelson and his coworkers investigated the tyrosinase
of the common edible and wild mushroom during the period of 1938-
1941 and made many contributions to quantitative techniques reviewed
by Nelson and Dawson ( 1944) .

Quantitative investigations of enzymatic oxidation of fruit
products were carried out by Samisch, 1935a and 1937; Hussien and

Cruess, 1940; Jimenez, 1947; Cruess and Sugihara, 1948; Ponting

 

 

and Joslyn, 1948; Cruess, 1948; El-Tabey and Cruess, 1949 and

Tauber, 1949.

There are many different enzymes having the ability to
catalyze the oxidation of phenols by molecular oxygen.
Several theories have been proposed for the nature and

course of enzymatic browning differing considerably in nomenclature

 

as well as in mechanism. It was believed that plant tissues which

darken on injury contain a substance called oxygenase which in the

_ 1

presence of air undergoes autooxidation, yielding a peroxide. This
peroxide, activated by the enzyme peroxidase present in most plants,
then brings about the oxidation of the natural phenolic substances.
Kastle and Lovenhart (1901) and Overholser and Cruess (1923)
ascribed discoloration to such a system.

As early as 1920 Onslow showed that peaches contain
both oxidizing enzymes and certain phenolic compounds, the latter
being converted to brown-black substances of unknown constitution
during the oxidation. She modified the existing concept of oxygenase
by considering it as an enzyme which in the presence of air catalyzed
the oxidation of o-dihydric phenols yielding as one of the oxidation
products a peroxide or hydrogen peroxide. The latter, in the presence
of peroxidase was activated and oxidized a suitable chromogen. She
believed that this secondary oxidation involved mono-, di-, and poly-
hydroxyphenolic compounds, including tannins which after oxidation

transferred into the characteristic colored pigments .

 

 

Onslow ( 1931) systematically investigated the oxidizing
enzymes present in higher plants and segregated them into two
groups, those which contain oxygenase and catechol compounds and
those in which oxygenase and catechol compounds were absent, the
peroxidase plants. The first group of plants discolors rapidly on
injury and includes apple, apricot, banana, cherry, fig, grape,
peach, pear, and strawberry. The second group of plants which
does not discolor on injury includes citrus fruit (lemon, orange,
lime, and grapefruit), red currants, melon, pineapple and tomato.

Szent ~Gyorgyi ( 1925) succeeded in showing that darken-
ing of plant tissues could take place in the absence of peroxidase.

Balls and Hale ( 1935) reported that pigment formation
in injured apple tissue was accelerated by the addition of horse-
radish peroxidase and inhibited by the addition of direct inhibitors
of peroxidase. They concluded that the darkening of freshly cut
surfaces of apples is a reaction catalyzed by peroxidase and that
the formation of hydrogen peroxide from molecular oxygen by a
respiratory enzyme is a necessary preliminary step.

Raper and co -workers ( 1932-1938) investigated the
intermediate reactions involved in the conversion of tyrosine into
melanin by tyrosinase without the intervention of peroxidase.

Shortly afterward, with the improvement of methods of
enzyme separation and assaying techniques, the existence of copper-

containing oxidases catalyzing the oxidation of phenols by molecular

_: W, 3:4. -. as- , ~ -~

a

 

 

 

 

oxygen, as distinct from the iron porphyrin peroxidases, catalyzing

the oxidation of phenols by hydrogen peroxide was established through
the classical work of Keilin and Mann ( 1938) and Parkinson and
Nelson (1940).

Nelson and Dawson ( 1944) used the term tyrosinase for

the preparation they obtained from mushrooms and found that it had

 

the ability to catalyze the oxidation of monophenols in addition to
catalyzing the oxidation of polyphenols. The ratio of rate of oxida- in
tion of monophenol oxidation to that of polyphenol oxidation, however,
varied considerably in preparations from different mushroom sources.
Keilin and Mann ( 1938) purified polyphenol oxidase from
mushroom and found it did not oxidize monophenols appreciably.
Cruess and Sugihara ( 1948) used the term oxidase for
the mixture of polyphenol oxidases and peroxidases in extracts of
crude enzyme preparations from fruit tissues. The phenolase in
apricot extract prepared by extraction of frozen ground tissue by
Samisch and Cruess ( 1934) was able to catalyze the oxidation of
catechol and pyrogallol and not that of phenol, resorcinol, quinol,
phloroglucinol or tyrosine. Samisch ( 1927) also reported that the
extracts of avocado behaved the same as that of apicot and Jimenez
(1947) reported similar results with guava extract. Cruess and
Sugihara ( 1948) extracted olive tissue with cold acetone and found

that it catalyzed only the oxidation of o-dihydroxyphenols.

 

 

Kertesz ( 1933) showed qualitatively that the oxidizing

enzymes of Sunbeam peaches were similar to those of other peach
varieties. He concluded that the low concentration of catechol tannins
in this variety was responsible for the failure of the sliced fruit to
darken.

Adams and Nelson ( 1938) reported that the phenol oxidi-
zing enzyme in aqueous extract of sweet potato possesses the ability
to catalyze the aerobic oxidation of o-dihydric phenols but cannot bring
about the oxidation of monohydric phenols such as p—cresol and tyro-
sine. As a result the enzyme has been called catecholase by Graubard
( 1939) and orthophenolase by Somner and Somner ( 1943) .

Eiger and Dawson ( 1949) found that purified sweet potato
phenolase oxidizes only polyphenols with the ortho-dihydroxy grouping,
although sweet potatoe slices will oxidize p-cresol also.

El-Tabey and Cruess ( 1949) also reported that apricot
oxidase containing phenolase and peroxidase catalyzed the oxidation
of catechol, protocathuic acid, caffeic acid, and digallic acid.

Ponting and Joslyn ( 1948) prepared polyphenolase from
apples mainly by acetone precipitation. Freezing in dry ice and ace-
tone caused no 1055, but subsequent storage at -34.60 C. caused a
loss of 80%of the activity in three months. Freeze-drying the enzyme
solution rendered the enzyme completely insoluble. The loss in solu-

. 0 .
t1on at 0 C. was about as low as in the frozen storage. They reported

 

 

 

 

an optimum temperature of 400 C. and optimum pH of 7. 0. It was
also noted that polyphenolase is quite sensitive to heat. Catechol,
resorcinol, p-cresol, quiacol and phenol were tested as substrates.
Only catechol was oxidized by the enzyme.

Studying the characteristics of browning enzymes in Fay
Elberta freestone peaches, Reyes and Luh ( 1960) reported an opti-
mum pH of 5. 9 to 6. 3 for the polyphenolase with citrate-phosphate
buffer and catechol as substrate. Michaelis constant of 0. 12 M cate-
chol was also reported. They found that peach peroxidase and poly-
phenolase act primarily on the ortho -dihydroxy configuration. The
para-dihydroxy structure was also oxidized by both enzymes but at
a slower rate. Neither enzyme exhibited significant activity with
mono -hydroxy and meta -dihydroxy compounds as substrates.

Palmer ( 1963) isolated a polyphenoloxidase from banana
and showed that it catalyzed the oxidation of o-dihydric phenols, but
not that of monophenols such as tyrosine, tyramine, ortho- and para-
cresol. He noted also that addition of catechol or dopamine in an
attempt to eliminate any induction period had no effect on the oxidation
of tyrosinase.

Most of the investigations of the natural substrates in
fruits have been qualitative in nature and largely based on the tech-
nique introduced by Onslow ( 1931) for the domonstration of the presence

of catechol compounds. Flavones and flavonols have long been suspected

 

 

 

of being involved in discoloration. Nagi (1921) reported that certain

anthocyanins were completely decolorized by the action of oxidizing
enzymes which also caused certain flavones, flavonols and their glu-
cosides to yield characteristic oxidation colors. He showed that the
color of aqueous extract of many plant tissues rich in flavones changes
to brown or reddish-brown when treated with fresh plant juices con-
taining oxidizing enzymes. He reported also that quercetin and quer-
citrin were oxidized by plant oxidases into deep colored pigments
which rapidly changed to brown.

Sando ( 1924) isolated and identified quercetin from the
peels of the McIntosh apple and postulated that it may be the chromo-
gen involved in storage scald.

Cruess and Alsberg (1934) made an extensive study on
the bitter principle of the olive, oleuropein. This substance was
later found by Cruess and Sugihara (1948) to be a substrate for poly-
phenolase, proved to be a glucoside of caffeic acid, the latter being
esterified with a phenol. Only the caffeic acid portion of this com-
pound contains the ortho-dihydroxy grouping necessary for polypheno-
lase oxidation. They reported that natural substrate should not be
called a tannin, since when purified it was not precipitated by gelatin
or absorbed by hide powder, reactions typical of tannins. Thus the
statement commonlymade, that the darkening of fruit is caused by
oxidation of tannins, does not apply in the case of the only well

investigated fruit and it probably does not apply to any other fruit.

 

 

. 9
(

Blake and Davidson (1941) classified a number of peach

varieties on the basis of the color reaction between 4 ml. of peach
juice and 3 drops of 6% FeCl3 solution. They estimated the tannins
by the color and thickness of the discolored layer formed by adding

the FeCl3 to the peach juice. On this basis they found that varieties

high in acidity as well as in tannin were objectionably astringent and

 

more susceptible to browning.

 

Guadagni et__a_l. (1949) studied enzymatic oxidation of J u
phenolic compounds in frozen peaches. They found fairly good cor-
relation between the oxidation of phenolic compounds and the changes
in the optical density of aqueous extracts of peach tissue using Folin-
Denis reagent. It was found that only a fraction of the total phenolic
substances in peaches is of the oxidizable type. The susceptibility
of enzymatic browning in several peach varieties was found to be
directly related to the amount of oxidizable tannin present. Initial
browning tendency, however, was also governed by the original
activity of the enzymes.

Johnson et_a_l_. ( 1950) reported using synthetic anion ex-
change resin for the isolation of peach tannins and obtained the latter
in relatively pure form by this method. They used paper chroma-
tography and ultraviolet absorption techniques in the characterization

and identification of the phenolic substances in the isolated material.

The isolated tannins contained D-catechin, a reddish colored substance,

 

 

 

10

probably polymeric derivitives of catechin, chlorogenic acid and
anthocyanin pigment.

Guadagni and Nimmo ( 1952) studied the effect of growing
area on tannin and its relation to astringency in frozen Elberta peaches.
Peaches representing three areas of different climatic conditions
were analyzed for tannin content and subjectively compared for dif-
ference in astringency. They found the area having the warmest and
clearest weather produced peaches of lowest tannin content while the
cooler, cloudy area produced fruit of highest tannin content. The
area having climate considered to be intermediate between these two
extremes produced peaches with intermediate tannin content. The
ability to detect differences in astringency due to the tannins found
in these peaches appears to depend on the magnitude of the total
amount present as well as the magnitude of the differences between
samples. It was concluded that tannins appear to be correlated
fairly well with relative astringency determined by organoleptic
means.

Bate-Smith ( 1954) , in a study on astringency in foods,
paid considerable attention to astringency in peaches. In peaches,
as in many other fruits, a certain degree of astringency is desirable,
otherwise the fruit is insipid, but the astringency must not be exces-
sive. Some varieties, in particular one named "Sunbeam, " are well

known for the complete absence of tannin from their flesh, but they

.f' Y

,. f/ w ‘x
I, I} \ / '/
‘I

I
2‘ ‘5 _ clan-n- ' -.

l
I
,i
(I
f
\/
r
«no . . . _-. _

 

 

11

have the advantage of not becoming brown by phenolase action during
processing.

Siegelman ( 1954) reported on a modified paper chromato-
graphic method for detection and identification of polyphenoloxidase
substrates in apple and pear skins. The apple polyphenoloxidase
enzyme was used as a chromatographic spray for detecting the pos-
sible endogenous substrates of the browning reaction. In the tests
described, the browning reaction of chlorogenic acids on the chro-
matograms after it was sprayed with PPO was not nearly as intense
as that of catechin. Three additional unidentified compounds were
found which darkened after treatment with PPO, but none corres-
ponds to the already known catechins. He emphasized that a browning
reaction on the chromatogram is not conclusive evidence of the par-
ticipation by that substance in the browning reaction.

Swain and Hillis ( 1959) critically examined and made
some modifications of existing methods of quantitative analysis of
anthocyanins, leucoanthocyanins, flavones and total phenols in plant
tissue extract. They concluded that all the procedures for their
estimation are necessarily empirical. For total phenol determina-
tion the methods based on the use of oxidizing agents are the most
useful. The more modified method of Folin and Denis has proved
to be more convenient and consistent. All the phenolic compounds

tested showed an almost linear relationship between optical density

 

   

 

 

 

 

 

12

and concentration, although the slopes of the curves were slightly
different.

Corse ( 1953) isolated a new isomer of chlorogenic acid,
”neochlorogenic acid, " from Elberta and Halford peaches by Counter-
Current distribution. The ultraviolet absorption spectrum is typical
of the other isomers, but the rotation, melting point, and the crystal-
line nature were different.

The polyphenolic compounds in Elberta peaches during
storage and ripening were studied by Craft ( 1961) . He found that
the principal polyphenols separated from ethanolic extracts of im-
mature peaches were in order of prominence on the paper chromato-
grams; leucoanthocyanins, chlorogenic acids, catechin, and flavonols.
There was no qualitative change in this polyphenolic pattern during
ripening, and the relative proportions of the individual components
remained fairly constant. Total phenolic compounds were estimated
by the Folin-Denis reagent method and leuco -anthocyanin by hydroly-
sis to anthocyanidin.

A long term study of the discoloration of canned peaches
was carried out by Claypool ( 1962) . He reported that the most pro-
nounced change in maturing peaches is the appearance of anthocyanins
and a decrease in leucoanthocyanins. Other compounds of phenolic
origin which have been tentatively identified on chromatograms are

L-epicatechin, D-catechin and four isomers of chlorogenic acids.

 

 

 

13

Present information shows a negative correlation between discoloration
and the total tannins present in the fruit even though these compounds
are susceptible to enzymatic browning. He concluded that two factors,
advanced maturity and nutritional status of the trees, contribute to
susceptibility to discoloration of canned freestone peaches.

Luh fl. ( 1962) reported that discoloration of canned
freestone peaches seems to be related to the chelation of anthocyanin
with tin lining to form a stannous -anthocyanin complex which is blue
to purple in color. The formation of such a complex causes the dis-
coloration of the syrup and the peach tissue. During storage the
anthocyanin fades away, while brown colored oxidation and polymeri-
zation products are formed.

Hsia gt_a_l_. ( 1964) isolated leucoanthocyanidin from im-
mature Elberta peaches by Counter-Current extraction. Cleavage
with hydrochloric acid yielded cyanidin chloride and catechin. Alkali
degradation of the resultant cyanidin chloride to protocatechuic acid
and phloroglucinol further substantiates the cyanidin structure. They
reported also that the peach leucocyan does not have a glycoside
linkage, which was proven by a negative reaction with the glucostat
enzymatic system, after leucocyan was hydrolyzed with dilute hydro-
chloric acid. They confirmed the presence of catechin and certain
chlorogenic acids and their isomers in Elberta peaches. Traces of
closely related but unidentified companion leucocyanidin compounds

were found on chromatograms.

 

 

 

14

Hsia 31311: ( 1965) studied anthocyanin from Elberta
freestone peaches. The pigment was characterized by its absorption
in the visible and infrared regions. Acid hydrolysis of the pigment
yielded glucose and cyanidin, present in equal amounts on a molar
basis. No intermediate glycoside was found when the pigment was
hydrolyzed under mild conditions, indicating a monoglucoside struc-
ture. Alkali degradation of the aglycone yielded phyloroglucinol and
protocatechuic acid, showing the presence of cyanidin in the pig-
ment. The absorption peak of the pigment in a 0. 01% methanolic
HCl solution shifted from 525 to 568 mH when aluminum chloride
was added to form a chelate, indicating the presence of ortho -phenolic
groups in the molecule. The peach anthocyanin is identified as a 3-
monoglucoside of cyanidin.

Griffith ( 1959) found that the principle browning sub-
strate in edible banana is 3, 4-dihydroxyphenylethylamine. It was
oxidized rapidly with banana PPO resulting in dark brown color. He
reported also that the enzyme failed to oxidize the leucoanthocyanin
present in banana.

Patil e_t_a_l_. ( 1956) reported that crude extracts of potato
tubers contain three electrophoretically different polyphenol oxidases.
One fraction had high cerolase activity and the other two showed
catecholase activity, though they oxidized chlorogenic acid faster

than catechol .

 

 

 

15

Bauchilloux 3L3]: ( 1963) showed multiple forms of mush-
room tyrosinase in homogenous state by a process involving preparative
electrophoresis and chromatography on hydroxylapatite. Four enzymes,
q,fl ,‘6‘ , and 6 tyrosinase were obtained, the last three essentially
pure. Although these enzymes possessed partially different activities
toward mono- and o-diphenols, the three homogenous ones had very
similar amino acid composition. Both cuprous and cupric copper
were present in each enzyme.

Joslyn and Goldstein ( 1964) studied the changes in pheno-
lic content of persimmons during ripening and processing with rela-
tion to astringency. They observed changes in astringency with
changes in concentration of phenolic substances extractable with
methanol and aqueous methanolic solution. Oxidation was found to
be responsible for loss in astringency on air drying of sliced per-
simmons or high speed blending. Pureeing and freezing of astringent
tissue resulted in loss of astringency and decrease in phenolics even
in the presence of added ascorbic acid or sulfite. The leucoantho-
cyanin isolated from methanolic extracts of astringent tissue con-
tained both leucodelphinidin and leucocyanidin, apparently being
present together in the molecule.

A widely held view of the function of polyphenol oxidase
in plants is that it functions as the terminal oxidase in respiration.

This was proposed by Boswell and Whiting ( 1938) . They added

 

0- q--

 

 

16

catechol to respiring potato slices and found a sharp increase in the
rate of oxygen uptake followed by a gradual decrease to 33% of that
with potato slices alone. Further addition of catechol caused no
response, indicating that the polyphenol oxidase was inactive.
Robinson and Nelson ( 1944) and Nelson ( 1945) reported that the
enzyme tyrosinase is involved in plant respiration. They concluded
that the 3-, 4-dihydroxyphenylalanine is the hydrogen carrier func-
tioning adjacent to the terminal oxidase tyrosinase in a respiration
chain present in sweet and common potato tubers.

Lamb and Sreerangachar ( 1940b) noted that a particular
tea bush has leaves which did not undergo the typical polyphenolase
catalyzed fermentation when crushed. They showed that this was
due to the absence of polyphenolase; fermentation proceeded when
an enzyme preparation from other tea leaves was added. Thus
respiration is not necessarily related to polyphenolase in tea leaves.

In view of the contradicting reports about the role of
polyphenol oxidase in plant respiration, it can only be stated that

the role of the enzyme in plants remains unknown at present.

 

 

 

MATERIALS AND METHODS

Eight freestone peach varieties were obtained during the
1964 and 1965 seasons from orchards in the South Haven area. They
were harvested at the firm ripe stage of maturity. The peaches were
halved, pitted, packed in polyethylene bags under partial vacuum,
sealed and stored at -17.7OC. , 0° F. In preparation for analysis,
20 peaches of each varietywere cut into thin slices and ground with
a meat grinder at -l7.7°C. , 00 F. The ground frozen material was
thoroughly mixed and stored at -31. 60 C. , -250 F. in polyethylene

bags until used.

Enzyme Extraction

The crude enzyme solution was prepared by blending the
peach tissue with cold acetone. Although initial studies indicated that
the acetone extraction procedure resulted in about 15% more enzyme
inactivation than the extraction with citrate-phosphate buffer, pH 7. 0,
acetone had the advantage of eliminating most of the phenolic sub-
stances that resulted in browning in the buffer extract. A11 extrac-

tions were made in a cold room at 1.10 C. , 340 F.

17

 

 

 

 

 

 

 

 

       
 

y "H
/ ~ / k/

l/

{I
I
' ,1
' .
'- ii"- . ..
' " I)!“ 1 _
r ‘-.._ -..-l.'.m ‘ E ' 1" 4 .._.I—_I. - _ ‘I' .

_I--IE-——____a -_ -———n——-—:_—-

 
   
 

 

18

One hundred grams of frozen peach tissue were blended
for 5 minutes in a Waring blender with 200 ml. of -31. 6° (2., -25°F.
acetone and centrifuged at 2200 rpm for 20 minutes. The supernatant
liquid was discraded and the precipitate was reblended with 200 ml.
1. l0 C. , 340 F. distilled water for 3 minutes and centrifuged at
2200 rpm for 20 minutes. The supernatant liquid was removed,
filtered, and 2. 5 volumes of -23. 30 C. , -10°F. acetone were added.
The mixture was allowed to stand for 5 minutes, then centrifuged at
2200 rpm for 15 minutes, and the supernatant liquid was discarded.
The precipitated enzyme was dissolved in 30 m1. of 0. l M citrate
-0. 2 M phosphate buffer pH 7. 0, centrifuged at 10, 000 rpm for 20
minutes to remove debris, and the supernatant liquid containing the
crude polyphenol oxidase enzyme was filtered. The enzyme sclution was

pale, yellow and clear and had little or no peroxidase activity.

Several preparations were pooled for further purification.

Fractionation by Ammonium Sulfate

Five -hundred ml. of crude enzyme solution were fractionated
with ammonium sulfate. Sufficient solid ammonium sulfate was added
to the enzyme solution with continuous mechanical stirring to make it
40% saturation. After 30 minutes the protein precipitate was removed
by centrifugation at 10, 000 rpm for 20 minutes and dissolved in cold

citrate-phosphate buffer, pH 7. 0 Successive additions of ammonium

V: b
’15...

' r:
.l‘* ,

 

,7

_ f , /
_ ‘ x'
/
f . 17/

_...--_...,r _'

     

 

19

sulfate, collections of precipitate, and dissolving in cold buffer were
carried out in the same way for 50, 60, 70, 80, and 90% saturation.
The resulting enzyme solutions were desalted by passing over a
column of Sephadex G-25, concentrated by dialysis against sucrose

powder, and assayed for protein content and enzyme activity.

Enzyme Purification

The fraction with the highest specific enzyme activity
from the ammonium sulfate fractionation was used for further puri-
fication. Sephadex G-100 was prepared by soaking in excess citrate-
phosphate buffer, pH 7. 0, for three days with occasional stirring.

It was packed in a glass column (1. 5 cm. X 85 cm.) to a height of

70 cm. , equilibrated and washed with buffer for 72 hours at 34 de-
grees F. Two ml. of concentrated enzyme solution, having a protein
content of 3. 71 mg./m1. and a specific activity of 1600 units per mg. ,
was charged gently at the top of the gel bed. It was eluted with
citrate-phosphate buffer pH 7. 0. Three ml. fractions were collected
and assayed for protein content and polyphenol oxidase activity. The
flow rate of the column was 13 ml. per hour for the buffer and 8 ml.

per hour for the enzyme solution.

Protein Determination

The spectrophotometric method of Warburg and Christian

( 1942) was used and the protein content calculated by the Kalckar

 

_ '17:: gas -

 

 

 

 

 

20

( 1947) formula:

1.45 absorbance at 280 mM - 0.74 absorbance at 260 mad

= mg . protein/ml.

As say Procedure

The enzyme activity was determined by a modification of
the tyrosinase assay method of Fox and Burnett ( 1958) . The reactions
were carried out in small, thin-walled test tubes (1 cm. x 7 cm.) in
a 300 C. , 860 F. water bath. The reaction mixture contained 1 ml.
0. 03 M catechol (final concentration of 0.01 M substrate), 1.0 m1.
0. 1 M citrate - 0. 2 M phosphate buffer, 0. 8 ml. distilled water and
0. 2 m1. enzyme solution. The reaction was stopped by the addition
of 0. 2 m1. 0. 001 M potassium cyanide. In all cases a blank of all
reagents with inactive enzymes (potassium cyanide added to the en-
zyme) was used. Within one or two minutes, the tube contents were
transferred to the 1 cm. light path spectrophotometer cells and ab-
sorbance at 420 mil was determined.

One unit of enzyme activity represented the amount of
enzyme that causes an increase of 0. 001 in absorbance at 420 me“

per minute under the assay conditions.

Effect of pH on Enzyme Activity

The rate of catechol oxidation was determined over the

pH range of 4.0 to 10 at 0.5 intervals at 30° c. A blank determination

   
 

,
I... ’
M - e... .

  

l
, ‘11-. "I g. .. I . -ail-
"'-— 77' - 0H .'

'J-a... '.

l I
, fl . 'I /
4“" ‘ Ag; ‘3,“ . :-

.7 ...-W-=i- W“ - - -./

 

 

21

was made at each pH to compensate for any autooxidation. The re-

action mixture was the same as described in the assay procedure.

Effect of Temperature on Enzyme Activity

The enzyme activity was determined at temperatures
from 150 to 700 C. at 50 intervals, using catechol as the substrate.
The reactions were carried out in small, thin-walled (1 cm. x 7 cm.)
test tubes in a thermostatically controlled water bath. The enzyme
was added after the tube contents had attained the bath temperature.
The reaction was stopped after 2 minutes with 0. 2 ml. of 0.001 M

pota s sium cyanide .

Enzyme Stability

Aliquots of the enzyme solution were adjusted to pH
values from pH 4. 0 to 10 at 0. 5 intervals with citrate—phosphate
buffer. The final volume at each pH was 60 ml. , with protein con-
tent of 0.42 mg. /ml. and specific activity of 750 units/mg. 15 ml.
aliquots at each pH were transferred to rubber stoppered tubes and
stored at -31.6°, -17.7°, 1.1° and 21° C., -25°, 0°, 34°, and 70°
F. Enzyme activity determination was made at regular intervals for
three months. To determine activity of these solutions they were

thawed at 1. 10 C. , 340 F. and then refrozen. Some solutions were

also stored at -31.60 and -17. 70 C., -250 and 00 F. for three months

22

without thawing to compare-with the other solutions for the effect of

repeated thawing and freezing on enzyme activity.

Substrate Specificity

Monophenols as 0-, m-, and p-cresol, 2, 4-dimethylphenol,
phenol, tyrosine, hydroquinone, resorcinol, orcinol, catechol, caffeic
acid, D, L-dopa, dopamine and pyrogallol and polyphenols as chloro-
genic acid, catechin, quercetin, quercitrin and rutin were used to
determine their suitability as substrates for purified peach polyphenol
oxidase. In the cases where no oxidation was immediately apparent,
the reaction mixture was held for 2 hours to insure the absence of a
long induction period. In addition a further study was made using
larger amounts of enzyme and maximum substrate concentration.

The Michaelis constants and maximum velocities were
calculated for all enzymatic reactions, plotting llactivity against

l/substrate concentration (Lineweaver and Burk, 1934) .

Extraction of Phenolic Compounds

One -hundred g. ground frozen peach tissue were dropped

into 300 ml. of boiling 95% ethanol and boiled for 5 minutes to in-

activate the enzyme systems. The mixture was transferred to a

Waring blender and sufficient 95% ethanol added to obtain a final

concentration of 70% ethanol. It was blended for 5 minutes, centri-

fuged 15 minutes at 2000 rpm and the supernatant decanted off. The

 

 

23

precipitate was suspended in 200 m1. of 95% ethanol, blended for 5
minutes, filtered and the filtrate pooled with previous extract. The
ethanolic extract was concentrated under reduced pressure at 400 C.
to about 50 m1. This concentrate was washed three times with 100
ml. petroleum ether to remove chlorophylls and carotenes and then
extracted with 500 ml. of ethyl acetate. The ethyl acetate extract
was concentrated under reduced pressure in a flash evaporator at
400 C. This extract was used for identification of the phenolic com-

pounds by chromatography and spectrophotometric methods.

Total Phenolic Determination

Total phenolic concentration was determined by the Folin-
Denis procedure (1915) as modified by Swain and Hillis (1959) . A
standard curve was prepared for catechin (Figure 1) and the results
are reported as catechin equivalent. A suitable aliquot of the solution
was diluted with distilled water to 8. 5 ml. in a test tube. Five -tenths
ml. Folin-Denis reagent was added and the contents were thoroughly
mixed. Exactly three minutes later, 1. 0 ml. of saturated sodium
carbonate sulution was added and well mixed. After one hour the
optical density was determined by a Beckman DU spectrophotometer
at 675 my“ using a blank of water and reagents only. The solutions

were filtered before optical density determination.

Absorbance at 675 Millimicrons

 

 

 

 

 

 

0'8 ' '7 1 I ' I ' I
0.7 L- -
0.6 " .4
0.5 b ..
0.4 - a
0.3 - -
0.2 " ..
0.1 L- '1
0 w1 7 17 l L we l

0 20 40 60 80 100 120

Catechin Concentration, Micro gr ams

Figure 1. -- Folin-Denis pigment formation by catechin in a standard curve
for phenolic determination.

 

 

 

 

 

 

25

Phenolic Oxidation

 

Enzymatic oxidation of phenolic substrates in peaches
was achieved by blending peach tissue with citrate-phosphate buffer,
pH 6. 4, in a Waring blender.

Four 100 g. samples of ground frozen peach tissue were
used for this experiment. One sample was extracted for total phenolic
content as previously described and served as a control. The second
sample was dropped into 100 m1. of boiling citrate-phosphate buffer,
pH 6.4, and boiled for 5 minutes to inactivate enzymes. It was then
transferred into a Waring blender and blended for an hour. Sufficient
boiling 95% ethanol was added to obtain a final ethanol concentration
of 70% . It was blended at high speed for 5 minutes, centrifuged at
2000 rpm for 20 minutes and the supernatant (ethanolic extract) was
collected and concentrated under reduced pressure at 40° C. The
third and fourth samples were blended with 100 ml. of citrate-
phosphate buffer, pH 6.4, in a Waring blender for 30 and 60 minutes
respectively. The blending speed was regulated by a variable trans-
former to keep the temperature in the range of 25. 5° to 26. 6° C. ,
78° to 80° F. and to obtain adequate areation of the mixture for
enzymatic oxidation of the oxidizable phenolics by the endogenous
enzyme in peaches. Total phenolic extraction was made on the
blends as above. The ethanolic extracts were concentrated to identical
volumes of 50 ml. These extracts were used for spectrophotometric

analysis .

 

   

 

26

In addition, 50 g. samples of ground frozen peach tissue
of four varieties (Elberta, Richhaven, Sunhaven, Redskin) were
blended with 50 ml. of citrate-phosphate buffer, pH 6.4, for 0, 15,
30, 45, 60, 75, and 90 minutes respectively. Blending was carried
out on these samples as previously described. Total phenolic deter—

mination was made on the ethanolic extracts from different samples

 

at different oxidation times. The ethanolic concentrates were washed

with petroleum ether and extracted with ethyl acetate. The ethyl

I‘r

o
acetate extracts were concentrated under reduced pressure at 40 C.
to identical volumes of 55 ml. These concentrates were used later

for paper chromatography.

Spectrophotometr ic Analysis

Beckman DU and Bausch and Lomb 505 spectrophotometers
were used in this study. All phenolic substances exhibit intense ab-
sorption in the ultraviolet region. The ultraviolet absorption of the
ethanolic extracts of the control and the third and fourth samples of
the oxidation study were determined and compared. The ultraviolet
absorption was determined on 0, 2 ml. aliquots of the extracts diluted
to 10 ml. with 95% ethanol using ethanol as a blank. Spectrophoto-
metry was also used in isolation and purification of the phenolic sub-

stances from peach extract along with paper chromatography.

 

~-. w».

 

 

 

 

27

Paper Chromatography

 

Paper chromatography was used for detection, isolation
and identification of polyphenol oxidase substrates in peach extracts.
The ethyl acetate extracts were chromatographed using the ascending
method. The chromatograms were developed using the upper phase
of n-butanol-acetic acid-water (4:1:5, V/V) for 20 hours at 20°- 22°
C. They were then dried in an air current at 20° C. for 2 hours.

For the isolation and identification of the peach phenolics,
one cm. wide streaks (about 15 m1. ethyl acetate concentrate) were
placed on Whatman No. 3 chromatographic paper, 22" x 18" and de-
veloped. The chromatograms were examined under UV light at 366
mu“ before and after exposure to ammonia vapor, the fluorescent
areas marked and their Rf values determined. Small side strips
were cut from the paper and were treated with a freshly prepared
mixture of 0. 3% FeCl3 - 0. 3% K3Fe(CN) 6 for color development.
These were then matched with the center portion of the paper, and
the bands or combination of bands were cut out. These were then
eluted with about 300 ml. 95% ethanol at 20° C. for 24 hours. The
elutes were concentrated under reduced pressure at 40° C. and
representative aliquots were examined spectrophotometerically to
determine their maximum absorbance in the ultravidlet region.

The concentrates of the different fractions were also rechromato-

graphed on Whatman No. l chromatographic 6" x 22" strips. Those

 

 

 

 

 

28

having Rf values of 0 to 0. 4 were developed with BAW for 20 hours
at 20° C. and those with Rf values of 0.4 to 0. 7 with 5% acetic acid
for five hours at 20° C. Representative chromatograms were sprayed
with 0. SN NaOH, 0. 6 N HCL in propanol, Folin-Denis reagent followed
by 10% NaZCO3, diozotized sulfanilic acid (DSA), 3% paratoluene sul-
fonic acid (PTSA) , mixture of 0. 3% FeCl3 and 0. 3% K3Fe(CN)6,
saturated sulution of 3, 5-dinitrosalsy1ic acid in 50% ethanol containing
2% NaOH (DNSA) , and 5% A1C13 for identification and comparison with
known compounds. From other representative chromatograms indi-
vidual bands were cut out and eluted with 95% ethanol for 24 hours.
The elutes were concentrated and their absorbance determined and
compared with those of known compounds.

For detection of the enzymatically oxidizable substances
in peach extracts, aliquots of non-oxidized and oxidized (blended for
2 hours) peach extracts were applied on No. 1 Whatman chromato-
graphic strips 6" x 22", developed with BAW for 20 hours and dried.
They were examined under UV light at 366 mu“ before and after ex-
posure to ammonia vapor. Some were sprayed with a mixture of
0.13% FeCl3 and 0. 3% K3Fe( CN) 6 for color development and the Rf
values of the different bands determined. Representative strips
were sprayed with partly purified peach polyphenol oxidase after a
light spray with citrate-phosphate buffer, pH 6.4, and kept in a
humid chamber for 18 hours at 20° - 22° C. and examined for color

development as indicative of enzymatic oxidation.

 

 

 

 

29

To investigate the activity of peach polyphenol oxidase
toward the different components of peach extract, 2 ml. aliquots of
ethyl acetate extract with total phenolic content of 2. 2 mg. were
applied on No. l chromatographicpaper, 6" x 22", developed with
BAW for 20 hours and dried. Successive strips of each papergram
were cut out according to Rf values. The strips were eluted with
95% ethanol for 24 hours. The elutes—were concentrated under re-
duced pressure at 40° C. to identical volumes of 10 ml. Total
phenolic determination was made on the elutes from the different
strips. The enzyme activity toward the elutes was determined
spectrophotometerically as the increase in absorbance at 420 mfl.
The reaction mixture contained 2 ml. elute, 1 ml. 0. 1 M citrate
-0. 2 M phosphate buffer, pH 6. 4 and 0. 2 m1. enzyme was added
with good mixing. The reaction was carried out for 30 minutes at
30° c.

The progress of the enzymatic oxidation of four peach
varieties by their endogenous p01yphenol oxidase was followed quanti-
tatively. Aliquots of 2 ml. of ethyl acetate extracts of the different
samples at different oxidation times (from the phenolic oxidation
study) were chromatographed on No. l chromatographic papers,

6" x 22", developed with BAW for 20 hours and dried. Representa-
tive chromatograms were examined under UV light at 366 m,“ and

sprayed with different reagents for color reactions. Three sets of

 

 

 

 

 

chromatograms representing samples were cut out into strips accord-
ing to Rf values. The strips were eluted with 95% ethanol for 24 hours
at 20° C. and the elutes concentrated under reduced pressure at 400 C.
to identical volumes of 10 ml. Total phenolic determination was

carried out on the different elutes using an aliquot of 2 ml.

was
_ _

 

 

 

 

 

 

 

 

 

RESU LTS AND DISC USSION

Maximum enzyme activity was obtained in the fraction

precipitated by 60% ammonium sulfate.

double that of the original solution.

The specific activity was

No enzyme activity was found

in the fractions precipitated below 40% or above 80% saturation

(Table l) and about 35% of the original enzyme activity was lost in

the fractionation process .

Table 1. ——Fractionation of the enzyme solution with ammonium sulfate

 

 

 

Salt Protein Enzyme Specific
conc . Volume Content Activity Activity
g. /ml. ml. mg. /m.l. units/1111. units/mg.

0. 4 50 1. 50 — -

0. 5 50 l. 10 400 440

0.6 50 0.51 820 1607

0 . 7 50 0. 43 310 720

0 . 8 50 0. 20 60 300

0. 9 50 — -

 

The pattern of protein separation and enzyme activity as

fractionated ona Sephadex G-100 column is shown (Figure 2) . Most

of the inactive protein emerged first from the column.

31

The first

1 man

 

 

 

3

Enzyme Activity AAbsorbance/Min. x 10

 

 

 

32

 

100

90

80

70

60

 

O---—-O protein content mg. /ml.
0-——-O enzyme activity

 

50
40 - 0
30 r
20 -
/ ”/°‘
10 - I
/ \\
°\‘ ’0-
\ s
f \ / ‘*~
0 l l l y‘ .‘1’ 1 *‘h ---
18 20 22 24 26 28

Figure 2. -- The chromatography of partly purified peach polyphenol oxidase

Fraction Number

on Sephadex G-100 column ( 1. 5x 70 cm.) .

30

 

2.0

Protein Content Mg. /ml.

—---a-

 

 

 

 

 

’1':
ll

 

 

33

enzyme activity appeared in fraction 24 and continued till fraction 30.
The highest enzyme activity was found in fraction 27 with a specific
activity of 8000 units/mg. This was about a tenfold purification. The
recovery of enzyme activity from the Sephadex column was 75%. No
further separation was obtained by passing fraction 27 through the
Sephadex column a second time and both protein and enzyme activity
emerged as one peak.

The enzyme extracts from the eight freestone peach
varieties showed the same pattern of protein separation and enzyme
activity in the fractions. Elberta peaches were found to have the
highest enzyme activity and Redskin the lowest level of activity,
about 56% of that of the Elberta. The other varieties ranged between
these two (Table 2) .

The enzyme extracts from all varieties showed the same
substrate specificity. The activity of the crude enzyme preparations
from all varieties toward catechol is shown (Figure 3) .

Effect of pH and Temperature
on Enzyme Activity

The rate of catechol oxidation (increase in absorbance
at 420 m J‘ ) for a given amount of enzyme was found to be markedly
dependent on the pH of the system (Figure 4). The enzyme is only
5. 5% as active at pH 4.0 as it is at pH 6. 5. It is worth noting that

while the enzyme is most stable at pH 8.0, it is most active at pH 6.4.

 

.&m.1'.1—'P" 53‘41'fr- '~ 1 '

   

 

 

 

deans 3:2an .28 no one To ma 3 323 8.338% 8.93258 2 So no .28 2
.Hocooueo $300.0 mo .HE a .cowusHOm enhance .HE ~.0 vegan—Goo c.2555 doggone.” 9.3.0.3.

.mcofifiocoo chewed pudendum ea..— uopg 355.8
nod ~00 . mo ooddQHOmnm 5 smudge .m 0350 a? 22$ cause 00 undoEm och. ”fins sandman

 

 

 

 

 

02 .0 mm 000 .0 0m minced
o2. .2 no oem .o om 55:32
one .2 E. ooo .2 2. 51,232
4 one .2 on ono .2 2. 532252
3 00m .3 0w. 00M .: «in Gourmgcdm
ovm .2 om. one .2 S 55.2332
00m .2 mm SN .3 pm co>m££oam
210.2 on one .2 on 332m
ywidwflimofi X annadwg\mofi X

unduw 00:32.53 Kaomv Hm oocmnuoawnmd sham 00:31.5 income. on ooGeQHOmnmd

ogncm mo .02 eff/50¢ magnum ogncm mo .02 >fl>fio< magnum >uowum>
SOmmom mom: acmmom «00H

 

 

.mogueem mo mofiofluw; economfip eat no >fi>$om ocEnNco 00 220352380..- .N 3nt

 

xvi—mum
j» _ .~ *1] .

 

 

 

 

A Absorbance at 420 mJt x 103

90

80

70

60

50

40

30

20

10

 

35

 

    

 

Elberta
Richhaven
Kalehaven
- Sunhaven
F airhaven
Redhaven
Halehaven
Redskin

memithH
I

 

l l

 

3 4 5 6

Time, Minute 8

Figure 3. -- Enzyme activity of the crude enzyme preparations
(0.001 M catechol, 0. 1 ml. enzyme and citrate-phosphate

buffer, pH 6.4) .

Enzyme Activity A Absorbance at 420 mtfl

 

180

160

140

120 '

100

80

60

40

36

 

 

20

L l l

 

5 6 7
pH

Figure 4. --Inf1uence of pH on peach PPO activity.

 

 

 

 

 

 

37

The maximum activity of the peach PPO in citrate-phosphate buffer
was found to be in the pH range of 6. 3 to 6. 6 with optimum activity at
pH6,4, Reyes and Luh ( 1960) reported an optimum pH range of 5. 9
to 6. 3 for Elberta PPO using a crude peach extract containing per-
oxidase and PPO.

Peach PPO showed optimum activity at 42° to 44° C.
(Figure 5). The activity at 70°C. was about 13% of that at 44° C. and
no activity was detected at higher temperatures. Ponting et_a_l. ( 1948)

reported an optimum temperature of 40° C. for PPO from apples.

Stability of the Enzyme

The enzyme was most stable at pH 8. 0 at all of the tem-
peratures studied. At room temperature, 20° C. (68° F.) there was
no decrease in enzyme activity during the first 24 hours and only a
2. 6% decrease occurred after 48 hours. Activity decreased rapidly
thereafter and there was a 93. 3% loss after 6 days storage. The
samples stored at l. 10 C. , 34° F. showed a continual loss in activity
during storage and at the end of 12 weeks the loss was 83. 3% (Figure 6).
The frozen samples stored at -17.8° C., 0° F. and ~31. 7° C., -25°F.
for 12 weeks showed a loss in activity of 20 and 16. 6% respectively.
The samples that were thawed and refrozen periodically during the

twelve weeks had a continual loss in activity, and at the end of

12 weeks, the activity losses were 66. 6 and 60% respectively (Table 3).

 

 

 

 

(St/Sum 0mm honed co ousumuomgou no 0028.30.31: .m madman

oO .ousumuomgofi
on 00 0m 0* 0m 0N 0H

 

 

 

38

 

 

 

 

0M

0*.

00

00

'uitu z/ 01 x yin 027, 112 aoueqxosqv V ‘AiiAiioV auuizug
E

Per cent Remaining Activity

 

39

 

 

 

 

 

100 ‘1'). l l l i
\Q\
90 ' ‘\\\ _.
‘ \f.
\
8x
80 — ‘ \\ —
\
i \\ \\
\
7o —‘ ‘\ Q 2
. \
\ \
\ \
\\ \
.. - \ \\ _
\‘ \
b. \
\ \\
\
50 " \ ‘\ \ -
\\ \
‘ \
\\ \
\
4° ' o—-----o -25°F \\ \E
o---—-—o 0°17 ‘\
30 o——o 34°F E
- \ 0——o 70°F
20 - -
10 - l -
0 l I l I I
o 2 4 6 8 10 12

Storage Time, Weeks

Figure 6. --Stability of crude peach PPO at different storage
temperatures, and pH 8. 0.

0.360090 0.30.0 vumpcmum n3".

 

.0mH mm? 2.53.300 08.350 0a: .«0 133300 Hmcwmzo 0.00. n.

 

 

 

 

. .0032:
00 00 m S 00 Hm mg m m unvnonh mm:
n no .2 N a meanness
_ 0 v mNH 00. 00 mm 0N mdogbucoo mm:
. oesnfi
00 00 m : 0m 0m 00 m m u 22095.20 0
. N. m5u00nm
0 00 on H 00 0.2 N0. :4 0N m0 02.20.95.300 0
4
00 .wm . m 0N N0 mm «A m m Tm
0 .0 E0 v..y..>o.0>$o< 0gncm
on 00010 .0 0
280.000 00 0 m N. 0 m 0 ud0eu00uh 0.350.80800.
E0

 

 

“0.52003 NH «0 130.3200 0gnc0 no 0w0u0um mo 30me ..t .m 0300.

 

 

41

Substrate Specificity

Monophenols with the ortho-dihydroxy configuration such
as catechol, caffeic acid, D, L-dopa, L-arterenol, pyrogallol, and
dopamine acted as substrates for the enzyme, though varying in effec-
tiveness. Other monophenols such as hydroquinone, resorcinol,
orcinol, o-, m-, and p-cresol and L-tyrosine were not oxidized.

Catechin and chlorogenic acid, polyphenolic compounds containing the

 

ortho-dihydroxy grouping in their molecules were natural substrates
for the enzyme and were present in peaches. Other polyphenols, as
quercetin, quercitrin and rutin, did not serve as substrates, although
they have the ortho-dihydroxy groupings. Apparently the side groupings
on the phenolic ring adjacent to the one carrying the ortho -dihydroxy
configuration interfered or prevented the enzyme from getting its
active sites into the right orientation for attacking the molecule.
Michaelis constants and maximum velocities for the enzyme
reactions with the substrates in decreasing reactivity rates are given
(Table 4) . These values were calculated by the method of Lineweaver
and Burk ( 1934) . A reaction curve for catechol is shown (Figure 7).
It was also noted that the enzyme became inactivated as it
catalyzed the oxidation of its substrates. All of the data reported were
obtained during the fir st two minutes before any significant inactivation

took place. Inactivation when the enzyme reacted with catechol was

 

42

noted after about five minutes. This inactivation as the reaction pro-

ceeds is referred to as reaction inactivation, Ingraham ( 1954) .

Table 4. --Michaelis constant and maximum velocities for purified
peach polyphenol oxidase. *

 

 

 

Substrate Km, M V max, M
Caffeic acid 8.0 x 10'4 2.63 x 10'1
Catechin 1.0 x 10'3 3.53 x 10'1
D, L-arternol 1.2 x 10.3 2.42 x 10—1
Chlorogenic acid 2. 2 x 10.3 5. 26 x 10-2
Pyrogallol 2.9 x 10'3 6.66 x 10"2
Catechol 9.0 x 10'3 3. 33 x 10‘1
Dopamine 8.0 x 10—3 1.96 x 10°1
D, L-dopa 2.5 x 10'2 2.00 x 10'1

 

*Calculated by the method given by Lineweaver and Burk (1934) .

Total Phenolic Compounds

Richhaven variety had the highest phenolic content, 173. 2
mg. /100 g. and Sunhaven the lowest phenolic content, 85. 4 mg. /100 g.
(Table 5) . No linear relationship was found between the total phenolic
content and the amount enzymatically oxidized. Guadagni e_t__a_l_. ( 1949)
reported the existence of a straight -line relationship between total and
enzymatically oxidizable tannins in the range of 32 to 194 mg. per 100
grams of peaches. However there was a direct relationship between

the per cent of phenolics oxidized and enzyme activity of the peach

varieties (Table 6).

 

JP

 

.Awmwd 6.360095 xusm pom 53503054 0:» mammz >9. doaooumo “32.? OanH gamma mo 95.90 Goflomou 4 u: .N. ouzmwh

<4 .cowuwuucmucoU muduumobm

 

A
ooo .m com 4 25 J com o

o~

43
o
N

o

m
AnooIQA
' I

 

ohv

om

 

 

 

 

44

 

Table 5. ---Total extractable phenolics of the different varieties of

peaches for the 1964 and 1965 seasons.

 

 

 

 

 

1964 1965
Variety Total Phenolics Total Phenolics
mg./100 g.* mg./100 g.
Richhaven 141. 9 173. 2
Kalehaven 138. 6 160. 0
Halehaven 125. 4 135. 2
Fairhaven 104.0 122.0
Elberta 99. 8 126. O
Redskin 78. 2 94. 4
Redhaven 75. 4 86. 6
Sunhaven 62.4 85.4

 

*Calculated as mg. of catechin per 100 g. peach tissue.

Table 6. --Oxidation of extractable phenolics of 100 g. of peach tissue
from different varieties, blended with 100 mg. of distilled water for

15 minutes .

 

 

 

Percent

 

Variety Number Enzyme Total Phenolics
units/100 g. Before Before Ox1dation
Oxidation Oxidation

Elberta 16, 640 126. 0 52.42 58.4
Richhaven 13, 200 173. 2 80. 36 53. 6
Kalehaven 12, 240 160.0 77.60 51. 5
Sunhaven 11, 880 85. 4 36. 70 48. 7
Fairhaven 11,660 122.2 65.23 46.7
Redhaven 10, 650 86.6 41.40 45.6
Halehaven 10, 400 135.2 90.60 44.6
Redskin 9,180 94.4 51.00 43.4

 

 

 

45

Detection of Enzyme Substrates
in Peach Extract

One dimensional paper chromatograms of the peach extract
with BAW showed the presence of 8 bands when sprayed with FeCl3 -
K3Fe(CN) 6’ (Figure 8) . Maximum blue color developed in bands 1,

2, and 7 (Table 7) . Under ultraviolet light, bands 5 and 6 emitted a

blue fluorescence which turned green when exposed to ammonia vapor.

Band 7 emitted pale blue fluorescence under UV light and ammonia

lifiv

i
1"

vapor while band 8 appeared as a dark shade. When the chromato-
grams were sprayed with partly purified peach PPO, the bands showed
brown to pale yellow color development in the following order; 1, 2, 7,
6, 5, and 8. Bands 3 and 4 occasionally showed a very pale yellow
color.

Chromatograms of the oxidized peach extract (blended with
buffer, pH 6. 4 for 2 hours to allow oxidation with endogenous enzyme)
showed less blue color development in bands 1, 2, 3, and 4 and no
color development or fluorescence in the areas corresponding to bands
5, 6, 7, and 8. No color development was observed on the chromato-
grams of the oxidized peach extract when sprayed with enzyme. This
showed that not all of the substances represented by the first 4 bands
were completely oxidized by the endogenous peach PPO while those of
the latter 4 bands were completely oxidized. These results indicated
the presence of 3 major and at least two minor compounds capable of

being oxidized by the peach PPO with the development of yellow to

 

 

46

 

 

 

‘w‘v‘ ‘f‘ . . ."
~~.. 4...; -~

 

“0...; ‘V .‘ 1,. ~ Q'd
I .0 ~ .\ 'o‘ *0. . 0. 9 o
6“.O“:O§: .:t..}: 9...... 7 o:::.;\;‘::.:‘- g o.-
..o.‘ .:.- ‘ . .
: :9. 0.... :0 .5 .c‘::.0 .. .1:
‘03.: 38“... :.,;.'.-‘ : :0‘::.:0
' $7.3} v i. "if 1 «is... #359}. 3;,“
1- '- .. v1 “1’: - .. :3..'-.-'..t);°.< ;.,. ..
\ t? oiA‘ Q; .0 9
b. . . o \
\
‘- .\Q‘ Q .
‘§?‘.r .h\:cw.‘:i.fi‘g|
L $133.32.» 3} 2 3 , 1333:" £5»
‘ fl 9 0 ’. . ’ :
afirtigfifi ‘ I. * .1. ‘0‘ .\r\.:': 1
‘.‘~‘ ~¢’ \ '0
.. ::‘~::o‘ . u 9" o ‘ O
' 735-3... *5: 3 c o ‘ 0‘.'
“-‘.v.::; “o. 0 . ' o -
b "1ft. .0: . .1 - u '. . f ‘6 0.
‘ .‘ Q" I. n . ’
‘rakt‘ . 3' 'z'.- 4 o o \ c‘
"“112? if 9"" ' a
‘82.“...0. ‘ "d to“...
. .
:— cl I.- q
0 0| '0 “s
Q ‘0'” o . | ‘ .\ x O .
R Q. 0 O f ‘ OI ‘
f b .‘o' :I':"r’u.o‘.'.‘ _ 5 - :.‘:‘ .¢::.t:' ‘ , \ #
"'.°' '0... . 0“... ‘W.‘-'
...":. .. . Q ...‘..‘\I.
' ":..‘.‘:. ,‘o' 6 O‘q’:o“‘:‘:‘¢ru
': ;¢0°:.\.o 0"o."‘t'a .0. 0 ‘0 '0. ‘\‘ 0 0
'\ ‘8 6 3o, ‘ \‘
- .0. o. :6... n":“:-‘.‘:‘ -( — .$. 0 ' J. ‘N
- ‘v 3"». .-
Q" .3 5“ ‘0 W12") $
g 7 ‘ .\ v... ‘ O I '
‘5‘ .03“? . .‘o' 7 fight
-: ~-:'{1.'~"
b q :- cl
— - .- cl
— . I .:I.OO.. q - q
I .‘D.
‘1“ " : I'M}: . 8 .«:.‘.o‘ E“.
’96:“ 3 :1.

 

 

 

 

 

 

Figure 8. -- Paper chromatograms of Elberta peach extract

a - Treated with B"eC13-K3Fe(CN)6

b - Sprayed with partly purified peach PPO

 

 

 

 

 

 

 

brown color. Siegelman ( 1954) stated that a browning reaction on the

chromatogram after spraying with PPO was not conclusive evidence of
the participation by this substance in the browning reaction, although

he gave no supporting evidence.

Table 7. --Description of the phenolic component of Elberta peach ex-
tract as separated on one dimensional paper chromatograms.

 

 

 

 

Treatment
Band

Number Rf Value FeClB-KBFe(CN) 6 Peach Enzyme
l . 11 very dark blue brown
2 . 19 very dark blue brown
3 . 28 light blue -
4 . 32 light blue -
5 . 51 dark blue yellow
6 . 58 dark blue yellow
7 . 63 very dark blue brown
8 . 93 light blue pale yellow

 

Total Phenolic Oxidation by
Endogenous Enzymes

For total phenolic extraction, boiling the 'peach tissue in
95% ethanol'or citrate -phosphate buffer, pH 6.4, were fOund equally
effective in preventing enzymatic oxidation of the phenolic compounds.
There also was no autooxidation of the phenolic compounds when the
boiled samples were blended in a Waring blender for 60 minutes

(Table 8) . The samples blended with unheated buffer for 30 and 60

 

48

minutes, respectively, showed extreme browning and 73% oxidation of

the phenolics that can be attributed to enzyme activity.

Table 8. --Effect of blending heated and unheated samples of peach
tissue on total phenolic content.

 

 

Treatment Total Phenolics
mg. [100 g. peach tissue

 

l. Extracted with 95% ethanol
non-oxidized, unheated 126. 70

2. Extracted with 95% ethanol
after being boiled in buffer
and blended for an hour 126. 10

3. Extracted with 95% ethanol
after being blended with
buffer for an hour 34. 00

 

The ultraviolet absorbance spectra of the phenolic extracts
of the samples are shown (Figure 9). The control sample showed two
absorption maxima at 280 my“ and 324-328 mull. . The spectra of the
samples, blended for 30 and 60 minutes respectively, showed no maxi-
mum absorption at 326 mull , and therefore the substances originally
present were apparently completely enzymatically oxidized. There
was also a very marked decrease in absorption at 280 mi.“ indicating
that these substances were also oxidized, but their oxidation was not
as complete as those at 326 mu“. This indicated that either they
oxidized at a slower rate or that not all of the substances were oxidized.

Since the amount of oxidation at 60 minutes was not much greater than

 

 

 

 

 

 

Iii-ii) I
' ‘I‘ll I

 

49

 

   

 

   

 

 

 

0. 5 ‘ '
0-4 P Before Oxidation Control 2
0.3 ' "
a)
o
:1
c0
,0
u
o
,8
<1
0.2 - 7
After Oxidation
0 1 - 30 min t.
60 min-
0 ' 1 J
360 320 280 240

Wavelength, Millimicrons

Figure 9. -- Ultraviolet spectra of peach phenolic extract before
and after oxidation with endogenous PPO.

 

- "A h. '— ' ' l |
.n'.- a.

 

 

that for 30 minutes, it is apparent that most of the oxidizable sub-

stances were oxidized fairly rapidly during the initial period of blend-
ing.

Further studies on the rate of phenolic oxidation by the
endogenous enzymes showed that maximum oxidation occurred during
the first 15 minutes (Table 9). It would appear that the enzyme be-
came less active or the remaining oxidizable phenolic substances had
become limiting factors. The total amount of oxidation after 90
munutes was not significantly greater than that at 60 minutes. Guadagni
fl. ( 1949) , using a similar method of oxidation, reported that the
amount of tannins oxidized in 24 hours was not appreciably greater than
the amount oxidized in one hour.

Isolation and Identification
of Peach Phenolics

Paper chromatograms of the ethyl acetate extract of
peaches developed with BAW showed the presence of 8 spots on treat-

ment with FeCl3 - K3Fe(CN) 6° Their Rf values were 0. 12,“ 0. 20,

0. 28, O. 33, 0. 51, 0. 60, O. 63 and 0. 93 respectively. These will be
referred to as spots 1 to 8 in order of increasing R.f values. The color
reactions of spots 1 and 2 with the different spray reagents used were

typical of those reported for leucoanthocyanins and those of spots 3

and 4 for flavonols, Bate-Smith ( 1948 and 1954) , Harborne (1961)

 

 

.. it www- aw mt'x' J..— . _

”'3“.

 

 

 

 

 

 

 

 

 

 

N2 Nd» i3 6.2. a .2 8
.3 22 6.: 6.3 22. 6.3 me
a. 86m v.5 i3 0.2 0.2 23 m5. m5 3
.m :13 ~28 6.3 N2 6.3 new 83 2m: 2.
5 ~.~m a .3 mam 18 was N.Nm 23 6.8. on
0.2. 25 N2. ~18 ~23. n.5, is... 25 2
o 6.3. o 25. o 6.3 o TS o
.m 03 .we .w om\ .95 .w om\ .95 .m om\ 9mg mmudaflz
8225 3:825 68:26.0 8:865 826:6 8:825 68:26.0 8:825. E 6&5.
“5605mm 130m. unmouona H.309. unoonmnm HSOH ”:50qu 5308 Gown—egg
fixated co>m£§m Goa/.9302 «team
>uowum>

 

 

.moéuco msocomopco >9 mmwuodumcw “Genomic no 353» £06.55 mo :oSmEXO ofioaezm nu .o 3an

 

 

 

 

52

and Craft ( 1961) , (Table 10) . The combined elute of the 4 spots had
a maximum absorption peak at 280 mud. The elutes of the individual
spots showed a broad absorption maxima between 275 and 283 mM ,
(Figure 10) . These absorption maxima have been reported for leu-
coanthocyanins by Hsia fl. ( 1964) . When the elutes from spots 3
and 4 were treated with concentrated sulfuric acid they developed a
yellow color that fluoresced at 366 mull . On addition of aqueous
NaOH, a dark yellow color develoPed that turned brown on standing.
Further effort to obtain identification as to the type of flavonol was
not successful.

Spots 5 and 6 gave color reactions similar to those ob-
tained for chlorogenic acid, and that of 7 was identical to those shown
by d-catechin, (Table 10). When the elutes were rechromatographed
with chlorogenic acid and d-catechin, the same Rf values were ob-
tained, using both BAW and 5% acetic acid as solvents. The spectra
of the elutes of spots 5 and 6 were identical to that of pure chlorogenic
acid (N. B. Co.) (Figure 11) and that of 7 to commercial d-catechin
(N. B. Co.) (Figure 12).

The eighth spot appeared as a dark area under ultraviolet
light. It developed a weak blue color when sprayed with FeCl - K Fe

3 3

(CN) 6' and a yellow color when sprayed with 5% AlCl3 in ethanol that

faded out on standing. The elute in 95% ethanol was pale yellow in

color with a greenish tan fluorescence under ultraviolet light.

 

 

 

Hoco>mG wvm u .. u u u u xump #506 u no .0 w
$053 053
#5300800 owN 850.3. 60: u 30:02» u ohm: 0:05 I Nm . 0 mo .0 K.
mcfiucmum
300 no 0063
3:0mouozo mm». 3029» n u .. 3022» u :00um 093 Z. . o oo . o o
mcdvcdum
300 no 000.3
05:0wou030 wNm 30:9» .. u .. 3022» n :00um 0.93 No . o Hm . o m
Hoco>md u n u .. u 30:?» 30:0.» 30:9» wimp .. mm .o v
3 Hoco>0a u u u u n 3023» 30:9» 3030.» ”.506 u mm . o m
5 5:02» 0 x55 #55
u 935500.90: . u #506 Mum: 3020.» u #55 u u u om . o N
5. G0.» 0 £35 xca
.. caucmoosfl n u View #53. 30.29» I MGR u u u N: .o H
.335 H03 m muse: m 930: om
.3862 :06: $75 8:83, 5:- 65 4mm 5062 £0.55 :2 - 64: cam 34m 88832
55556232 modem
con—55000.4 030>
mcofiomsm uofioO osfih >D mm

 

 

.m0u5H0 3.0:“ m0 00:03:0090 0:» new madame-05 >92; E05036 “35.3 w on a macaw mo 4.530005 uoHoUnu .3 03.08

 

, R
.

   

 

 

 

 

 

 

e... 7 , I
54
a
0.4 _ 1+ 2 _
3

0.3 - ' - :f

6.2 _ -
Q)
U
c:
«a
.0
H 4
3 o 3 -
.o ' "‘
4

b
0.2 " ..
0.1 r "‘
0 l l J I 4
340 320 300 280 260 240

Wavelength in Millimicrons

Figure 10. -- Ultraviolet spectra of a-the combined elute
spots, 1, 2,, 3 and 4; and b-the individual spots.

 

Absorbance

 

55

 

O
N

 

 

0 I 1 I
360 320 280 240

Wavelength, Millimicrons

 

 

Figure 11. —— Ultraviolet spectra of a-the elutes from spots
5 and 6; and b-pure chlorogenic acid.

 

 

 

 

56

 

o
.5

Ab so rbance

O
O
U

 

 

0 I J l I

340 320 300 280 260 240

Wavelength, Millimicrons

 

 

 

Figure 12. -- Ultraviolet spectra of a-the elute of spot 7; and
b—commercial catechin.

 

 

 

 

57

It became yellow in aqueous NaOH and turned brown on standing.
Addition of concentrated sulfuric acid gave an intense yellow color
that showed a white with yellowish tan fluorescence under ultraviolet
light. It had a maximum absorption at 348 mu“ (Figure 13) . The
addition of a few drops of A1C13 or NaAc to the elute did not cause
any shift in its maximum absorption. On the basis of the color re-
actions, it appeared to be a flavonol, but which flavonol could not be
determined.

The ethyl acetate peach extract was also chromatographed
using two dimensional chromatography. BAW was used as the first
solvent and 5% acetic acid as the second solvent. Examination of the
chromatograms under ultraviolet light and by spraying with different
reagents as given previously showed the presence of 10 spots (Figure
14) . These were identified the same as previously and their Rf values
recorded. The two additional spots 5a and 6a, on the basis of their
Rf values of . 51/. 76 and .62/. 72, respectively, were trans and cis
chlorogenic acids (Hsia _e_t_a_l_. 1964) . Spots 5 and 6 are evidently the
trans and cis isomers of closely related compounds such as isomeric

chlorogenic acids since their spectral properties were the same in

the ultraviolet region.

 

 

 

58

 

0.5

Ab sorbance

 

 

 

 

l l

 

I
360 320 280 240

Wavelength, Millimicrons

Figure 13. --Ultraviolet spectrum of the eulte of spot 8.

 

 

0.4

0.6

0.8

1.0,

 

BAW, 20 Hour 3

 

5 Percent Acetic Ac'd 5 Hours

 

l g I I

 

95

Q

 

 

 

W

o T 0.2 0.4 0.6

A}.

Figure 14. -- Two-dimensional paper chromatography of peach ethyl

acetate extract.

0.8

1.0

 

 

 

 

60

Enzymatic Oxidation of the
Phenolic Components

The concentration of the phenolic substances in Elberta
peach extract as eluted from one-dimensional paper chromatograms
is shown (Figure 15) . The leucoanthocyanins constituted 38. 50%,
chlorogenic acid 24. 54%, flavonol 19. 37% and catechin 13.43% of the
total phenolics. Similar results for the proportions of these phenolic
constituents were obtained for other peach varieties (Table 11) . The
elutes were incubated with purified enzyme for 30 minutes at 30° C.
and pH 6. 4, and the absorbance at 420 mu“ determined. It was found
that the greatest change in absorbance occurred with chlorogenic acid
as the substrate, followed by leucoanthocyanins, catechin and flavonols.
It was also noted that the flavonol with Rf O. 93, although small in
quantity, resulted in a relatively large change in absorbance at 420 mill
(Figure 16).

Table 11. -- Concentration of the identified phenolic components in
four peach varieties.

 

 

Phenolic Components

 

 

Variety % Leucoantho- % Chlorogenic % Flavonols % Catechin
cyanins Acids

Richhaven 35.6 25.9 18.9 14.8

Elberta 38.5 24.6 19.4 13.4

Redskin 38.8 22.8 18.8 14.2

Sunhaven 39.4 24.1 19.3 12.0

 

.Fi

I.

 

 

 

100

80
H
O
:1
.S’
2‘

O 60
E
U)
.3
'3
C.
0
.C.‘
0..
E

,6 40
H
DD
0
H
.2
2

20

o

61

 

 

 

 

 

 

 

 

 

 

 

 

0.2 0.4 0.6
Rf

0.8 1.0

Figure 15. -- Phenolic components of Elberta peach extract as
separated on one ~dimentional paper chromatograms. l and 2
leucoanthocyanins, 3 and 4 flavonols, 5 and 6 chlorogenic acid,

7 catechin and 8 flavonol.

 

 

62

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

200-
6

160'
U)
0
‘5
.S
2
(«:1 120-
\
M
2
>4
8 73"
V‘ 7
4.)
Cd 80"
0 l
U
c:
cc
,0
I...
8
.o 2
<1

40"

3 5
4
0__ I I I
0 0 2 0.4 0.6 0.8 1.0

Figure 16. -- Peach polyphenol oxidase activity toward the
different components of Elberta peach extract.

 

}T
.u
an.

 

 

 

/ ' /
.n’ ‘ p -
.il ‘

fi—‘_‘__._ . -1 .

   

 

63

When the chromatograms were sprayed with partly purified
enzyme, the development of brown to pale yellow color did not follow
the same pattern as the change in absorbance at 420 me . Leucoan-
thocyanins developed maximum brown color followed by catechin,
chlorogenic acid and flavonols (Figure 8) .

The progress of oxidation of the phenolic components by
endogenous enzyme in Elberta, Richhaven, Sunhaven and Redskin ‘

peaches is shown (Tables 12 to 15) . It was found that 73. 16% of the

= ‘Wmiwfl

leucoanthocyanins in Elberta peaches was oxidized in 45 minutes with
no further oxidation up to 90 minutes. Oxidation of flavonols also
stopped after 45 minutes with 58. 34% oxidized. Chlorogenic acid and
catechin were completely oxidized in 45 minutes and the flavonol

(R 0. 93) was completely oxidized in 15 minutes. The results ob-

f
tained for the varieties Richhaven, Sunhaven and Redskin were similar
to those for Elberta for the leucoanthocyanin and flavonols. Chloro-
genic acid was not completely oxidized in 90 minutes. The remaining
chlorogenic'racid was oxidi'zedon addition of more enzyme. Catechin
reqired 75 minutes for completeoxidation.

These results indicated that only a fraction of leucoantho-
cyanins and flavonols were enzymatically oxidizable. Chlorogenic
acid and catechin can be completely oxidized by the enzyme when the
latter is present in high concentration (as in Elberta peaches) . Their

rates of oxidation were higher than those of leucoanthocyanins and

flavonol s .

 

 

 

 

64

Table 12. -- Progress of oxidation of the different phenolic components

of Elberta peaches by endogenous enzyme.

 

 

Identity

Oxidation Time, Minutes
R ‘0 15 30 45 60 75 90

 

Total. Phenolic s Remaining after Oxidat ion, mic g.

 

 

 

 

, 0, 1 92 45 35 22 22 22 22
Leuc°anth°cYamnS o. 2 57 26 20 18 18 18 18
F1 1 O. 3 34 20 16 13 13 13 13

aV°n° 0.4 26 16 14 12 12 12 12

. . 0. 5 20 6 - - - - -
Chlorogenic Ac1ds O. 6 75 40 15 _ _ _ _
Catechin 0. 7 52 22 10 - - - -
0. 8 6 6 ‘ 6 6 6 6 6

Unknown 0. 9 10 4 _ _ - _ _
Flavonol 1. 0 15 5 - - - - -
Table 13. -- Progress of oxidation of the phenolic components of Rich-

haven peaches by endogenous enzyme.
Oxidation Time, Minutes
Identity Rf ‘ 0 15 30 45 60 75 90

 

TotallPhenolics Remaining after Oxidation micg.

 

Leucoanthocyanins

Flavonol

Chlorogenic Acids

Catechin

Unknown

Flavonol

0.1 112 72 46 38 36 36 36
0.2 80 50 34 28 26 24 24
0.3 44 28 20 16 16 18 18
0.4 38 22 18 18 16 16 16
0.5 32 12 - - - - -
0.6 108 53 36 24 20 l6 14
0.7 80 42 30 16 8 - -
0.8 10 10 12 13 12 12 14
0.9 16 6 - - - - -
1.0 20 10 - - - - -

 

 

‘Ir-

 

 

 

 

 

65

Table 14. -- Progress of oxidation of the phenolic components of Sun-
haven peaches by endogenous enzyme.

 

 

Oxidation Time, Minute 8

 

 

Identfiy' o 15 3o_ 45 6o 75 _ ,90
Total Phenolics Remaining after Oxidation..micg.

'L ucoanthoc anhi 0.1 64 38 3o 26 24 22 22

e y 0.2 44 20 16 14 14 14 14

F1 1 0.3 25 16 14 12 12 12 12

aV°n° 0.4 18 12 8 8 8 8 8

. . 0.5 14 5 - - - - -
Chl°r°gem° Auds o. 6 52 3o 24 20 15 12

Catechin 0.7 33 18 12 10 10 6 -

O. 8 6 8 6 9 6 10 6

Unknown 0 . 9 8 _ _ _ _ _ _

Flavonol 1. 0 10 5 - - - - -

 

Table 15. -- Progress of oxidation of the phenolic components of Red-
skin peaches with endogenous enzyme.

 

 

Identity

Oxidation Time , Minute 5 5
0 15 30 45 60 75 ‘90

 

Total Phenolics Remaining after Oxidation, micg.

 

Leucoanthocyanins

Flavonols

Chlorogenic Acids
Catechin
Unknown

Flavonol

00000 OO 00
OOGQO‘U‘I I-bw NH

O"
O

74 58 45 34 28 26 26
52 38 32 28 24 20 20

29 l8 l6 14 14 14 14
20 16 12 10 10 10 10

'14 8 - - - - -
6o 38 3o 24 19 14 12
46 32 26 18 12 6 -

8 8 9 8 10 8 8
10 4 - - - - -
12 4 — - - - -

 

 

 

 

 

 

 

   

 

CONC LU SIONS

Polyphenol oxidase activity differs considerably among the
peach varieties investigated. Elberta showed the highest level of en-
zyme activity and Redskin the lowest. The enzymes of all eight varie-

ties had the same substrate specificity. The enzyme solution was not

 

very stable at room temperature, 22. 20 C., 720 F. , losing 93. 3% of

its activity in 6 days. The enzyme was more stable at low temperatures
losing only 20 and 16.6% activity in 12 weeks at -l7.8o C. , 0° F., and
-31. 70 C. , -250 F. , respectively. The enzyme was most active at pH
6.4 while most stable at pH 8. 0.

The phenolic constituents of peach extract in order of
prominence were leucoanthocyanins, 35. 6 to 39.4%, chlorogenic acid,
22. 8 to 25. 9%, catechin, 12. 0 to 14.8% and flavonols, 18.8 to 19. 4%.
Catechin exhibited the highest reactivity with the enzyme followed by
chlorogenic acid, leucoanthocyanins and flavonols. However, their
contributions to discoloration in enZymatic browning of peaches did
not follow the same pattern as reactivity to the enzyme. Leucoantho-
cyanins contributed most of the browning followed by catechin, chloro-
genic acid and flavonols. The degree of discoloration of frozen peaches

during storage or on thawing is primarily attributed to the original

66

 

67

activity of the enzyme in the varieties that have from moderate to high
total phenolic content. In such cases the enzyme could cause discolora-
tion to a profound extent before the substrate became a limiting factor,
which seems to be the case in all eight varieties investigated in this
study. However, in some varieties distinguished for low total phenolic
content, the substrate could be the limiting factor for browning since
there is not much for the enzyme to do without the substrate. Elberta
peaches showed a higher rate of browning (phenolic oxidation by peach
PPO) than the other varieties. This was due to the higher enzyme
concentration in Elberta peach tissue as compared with these varieties.

Spectrophotometric and chromatographic methods were
used to identify and to determine the concentration of the different
phenolic substances in peach extract. More work is still needed to
establish the identity of the flavonols in peaches since this work identi-
fied them only as a group through their color reactions with spray

reagents.

 

Q», '1.

 

 

 

 

 

REFERENCES

Arnon, D. I. (1948) "Localization of Polyphenol-oxidase in the
Chloroplasts of Beta Vulgaris. " Nature. 162: 34-342.

Arnon, D. I. (1949) "Copper Enzymes in Isolated Chloroplasts.
Polyphenol -oxidase in Beta Vulgaris. " Plant Psysiol.
24: 1-150

 

Arther, J. C. Jr. and T. A. McLemore. (1956) ”Properties of
Polyphenolases Causing Discoloration of Sweet Potatoes
During Processing." Ligr. Food Chem. 4: 553-555.

Arther, J. C. Jr. and T. A. McLemore. (1959) "Interactions
Between Copper Ions and Sweet Potato Polyphenolase
Oxidized Substrates." J. Air. Food Chem. 7: 714-716.

Asimov, I. and C. R. Dawson. (1950) "On the Reaction Inactivation
of Tyrosinase During the Aerobic Oxidation of Catechol. "
J. Am. Chem. Soc. 72: 820-828.

Bailey, M. E., E. A. Fieger and A. F. Novak. (1959) "Phenol
Oxidase in Shrimp and Crab. " Food Research. 25: 565-
572.

 

Bailey, J. L. (1960) ”Purification of Latent Phenolase from Broad-
bean (Vicia Faba) Leaves." Biochem. J. 79: 514-516.

 

Balls, K. A. ( 1947) "Enzyme Action and Food Quality. " Food Tech-
nology. 1: 245-251.

 

Barton, G. M., R. S. Evans and J. A. F. Gardner. (1952) "Paper
Chromatography of Phenolic Substances. " Nature. 170:
249-250.

Baruah, P. and T. Swain. ( 1953) "The Effect of L-ascorbic Acid on
the in Vitro Activity of Polyphenoloxidase from Potato. "
Biochem. J. 55: 392-399.

 

68

 

69

Basset, H. P. and F. Tompson. (1911) "The Preparation and
Properties of an Oxidase Occurring in Fruits. " J. Am.
Chem. Soc. 33: 416-423.

 

Bate-Smith, E. C. ( 1954b) "Flavonoid Compounds in Foods. "
Advances in Food Research. 5: 262-300.

 

Bate-Smith, E. C. (1948) "Paper Chromatography of Anthocyanins
and Related Substances in Plant Extracts. " Nature. 161:
835-838.

Bate-Smith, E. C. (1954a) "Astringency in Foods." Food. 23:
124-135. *

 

Bate-Smith, E. C. (1957) "Oxidation of Plant Phenolics." Nature.
179: 1283-1284.

Bauernfeind, J. C. and G. F. Sieners. (1945) "Adding Ascorbic
Acid to Peaches Before Freezing. " Food Inds. 17: 79-80.

Bauernfeind, J. C. and G. F. Sieners. (1945) "Retardation of Dis-
coloration in Frozen Sliced Peaches by L-ascorbic Acid. "
Quick Frozen Foods. 7: 46, '60.

 

Behm, R. C. and J. M. Nelson. (1944) "The Aerobic Oxidation of
Phenol by Means of Tyrosinase. " J. Amer. Chem. Soc.
66: 711-716.

 

Blake, M. A. and O. W. Davidson. (1941) "Some Results of Acidity
and Catechol Tannin Studies of Peach Fruits. " Proc. Am.
Soc. Hort. Sci. 39: 201-204.

 

 

Blake, M. A. ( 1942) "Additional Studies of the Acidity and Tannin
Content of Mature Peach Fruits. ” Proc. Am. Soc. Hort.
§_c_i. 40: 153-156. 7

 

Boardonan, N. K. and S. M. Partridge. (1955) "Separation of Neutral
Proteins on Ion-exchange Resins." Biochem. J. 59: 543-
552.

 

Boscan, L., W. D. Powrie and O. Fennema. (1962) "Darkening of
Red Beets, Beta Vulgaris." Food Research. 27: 574-582.

 

Boswell, J. G. and G. C. Whiting. (1938) "A Study of the Polyphenol
Oxidase System in Potato Tubers." Ann. Botany. 2: 847-
863.

 

 

 

 

 

 

7O

Bouchilloux, Simone, P. McMahill and H. S. Mason. (1963) "The
Multiple Forms of Mushroom Tyrosinase. Purification

and Molecular Properties of the Enzymes." J. Biological
Chem. 238: 1699-1707.

Brown, F. C. and D. N. Ward. (1958) "Studies on Mammalian Tyro-
sinase. Chromatography on Cellulose Ion Exchange Agents. "
J. Biological Chem. 233: 77-80.

 

Bunzell, H. H. ( 1912a) "The Measurement of the Oxidase Content of as
Plant Juices." U. S. Dept. Bur. Plant Ind. Bull. 238: :3
1-40.

Bunzell, H. H. ( 1912b) "The Measurement of the Oxidase Content of
Plant Juices." J. Am. Chem. Soc. 34: 303-316.

 

 

Bunzell, H. H. (1916a) "The Mode of Action of the Oxidases. " ._1_
Biol. Chem. 24: 91-102.

 

Bunzell, H. H. ( 1916b) "The Relative Oxidase Activity of Different
Organs of the Same Plant." J. Biol. Chem. 24: 103-110.

 

Bunzell, H. H. ( 1916c) "The Relationship Existing between the Oxi-
dase Activity of Plant Juices and their Hydrogen Ion Con-

centration with a Note on the Cause of Oxidase Activity in
Plant Tissues." J. Biol. Chem. 28: 315-333.

 

Burn, E. R. (1963) "Methods of Tannin Analysis for Forage Crop
Evaluation." Georgia Agr. Exp. Stat. Technical Bulletin.
N. S. 32.

Caushing, M. L. (1948) "The Oxidation of Catechol -type Substrates
by Tyrosinase." J. Am. Chem. Soc. 70: 1184-1187.

 

Cherman, L., B. Luh and E. Hinreiner. (1953) "Flavor Evaluation
of Canned Cling Peaches." Food Technology. 7: 480.

 

Clayton, R. A. (1959) "Properties of Tobacco Polyphenol Oxidase. "
Arch. Biochem. Biopth. 81: 404-417.

 

Collins, P. R., L. B. Warner and L. Paige. (1963) "The Tyrosinase
Activity of Strobilomyces Strobilaceus. " Mycologia. 55:
764-744.

Corse, J. (1953) "A New Isomer of Chlorogenic Acid from Peaches. "
Nature. 172: 771-772.

 

 

 

 

 

 

71

Craft, C. C. (1961) "Polyphenolic Compounds in Elberta Peaches

Cruess,

Cruess,

Cruess,

Cruess,

Cruess,

Cruess,

Cruess,

Dawson,

Dawson,

DuBois,

DuBois,

during Storage and Ripening." Am. Soc. for Hort. Sci.
78: 119-131.

 

W. V. and W. Y. Fong. (1926) "Observations on the Oxi-

dizing Systems of Fruits. " Fruit Products J. 6: 13-15.

 

W. V. and W. Y. Fong. (1929a) "The Effect of Sulphur

Dioxide on the Oxidase of Fruits." Fruit Products J.
8: 21. '

 

W. V. and W. Y. Fong. (1929b) "The Effect of pH Value

and Hydrogen Peroxide Concentration on Fruit Oxidase
Activity. " Plant Physiol. 4: 363-366.

 

W. V., P. J. Quin and E. M. Mark. (1932) "Observation

on the Browning of Canning Peaches. " Fruit Products J.
12: 38 ~40.

 

W. V. and C. L. Alsberg. (1934) "The Bitter Glucoside of

the Olive." J. Am. Chem. Soc. 56: 2115-2117.

 

W. V. (1948) Commercial Fruit and Vegetable Products.

 

McGraw-Hill Book Co., New York. pp. 870-875.

W. V. and J. Sugihara. (1948) "The Oxidase of Olive. "

Arch. Biochem. 16: 39-45.

 

C. R. and J. M. Nelson. (1938) "On the Mechanism of

Enzymatic Oxidation of Catechol. I. --The Influence of
Catechol on the Stability of O-benzoquinone in Aqueous
Solutions." J. Am. Chem. Soc. 60: 245-249.

 

C. R. and B. J. Ludwig. (1938) "On the Mechanism of

the Catechol-tyrosinase Reaction. II. --The Hydrogen
Peroxide Question." J. Am. Chem. Soc. 60: 1617-1621.

 

C. W. and D. L. Colvin. (1946) "Loss of Added Vitamin C

in the Storage of Frozen Peaches. " Fruit Products J.
25: 101-103.

 

C. W. ( 1949) "Ascorbic Acid and Color in Food Products. "

Food Technol. 3: 119-121.

 

Eiger, Irena A. and C. R. Dawson. (1949) "Sweet Potato Phenolase--

Preparation, Properties, and Determination of Protein
Content." Arch. Biochem. 21: 194-209.

 

 

 

 

 

 

 

72

El Bayomi, M. A. and Earl Frieden. (1957) "A Spectrophotometric
Method for the Determination of Catecholase Activity of
Tyrosinase and Some of its Applications. " J. Am. Chem.

Soc. 79: 4854-4858.

 

El Tabey, A. M. and Cruess, W. V. (1949) "The Oxidase of Apricot."
Plant Physiology. 24: 307-316.

 

Evans, R. H., W. H. Parr and W. C. Evans. (1949) "Partition
Chromatography of Phenolic Substances. " Nature. 164:
674-675.

Fahraeus, G. and H. Ljunggren. (1961) "Substrate Specificity of a
Purified Fungal Laccase." Biochim. Biophys. Acta. 42:
22-32.

Fong, W. Y. and W. V. Cruess. (1929) "Comparison of Several
Indicators for Fruit Oxidase." Am. J. Botarg. 16: 799-
802.

Frieden, Earl and M. Ottesen. ( 1959) "A Simplified Method for the
Purification of Mushroom Polyphenol Oxidase, " Biochim.
Biophys. Acta. 34: 248-251.

 

Flodin, P. , B. Gelotte and J. Porath. (1960) "A Method for Concen-
trating Solutes of High Molecular Weight. " Nature. 188:
493-494.

Folin, O. and W. Denis. (1912) "On Phosphotungstic-phosphomolybdic
Compounds as Color Reagents." J. Biol. Chem. 12: 239-
251.

 

Folin, O. and W. Denis. (1915) "A Colorimetric Method for the
Determination of Phenols (and Phenol Derivatives) in
Urine." J. Biol. Chem. 22: 305-308.

 

Fox, A. S. and J. B. Burnett, (1958) "Kinetics of Tyrosine Oxidation
by Crude Tyrosinase from Neurospora Crassa. " Proc. Soc.
Exp. Biol. Med. 98: 110-114.

 

Goodwin, T. W. and R. A. Morton. (1946) "The Spectrophotometric
Determination of Tyrosine and Pryptophan in Proteins. "
Biochem. J. 40: 628-632.

 

Graubard, M. (1939) "A Comparative Study of Some Oxidases and
Peroxidases. " Enzymologia. 5: 332-345.

 

 

 

 

 

 

 

 

73

Graubard, M. and J. M. Nelson. (1935) "Tyrosinase Action on
Mono- and Di-hydric Substrates." J. Biol. Chem. 111:
757-770.

 

Gregg, Donald C. and J. M. Nelson. (1940) "The Action of Tyro-
sinase on Hydroquinone." J. Am. Chem. Soc. 62: 2510-
2512.

 

Grice, M. R., H. D. Brown and R. C. Burrell. (1952) "Varietal
Characteristics Influence Browning of Frozen Peaches. "

Food Eng. 24: 131-139.

Griffiths, L. A. ( 1959) "Detection and Identification of the Poly-
phenol-oxidase Substrate of the Banana. " Nature. 184:
58-59.

 

Guadagni, D. C., D. G. Sober and J. S. Wilber. (1949) "Enzymatic
Oxidation of Phenolic Compounds in Frozen Peaches. "
Food Tech. 3: 359-364.

 

Harborne, J. B. ( 1961) "Chromatography of Phenols. " Ch. 24 in
Chromatography ed. by Erich Heftmann. Reinheld Pub.
Co. New York. pp. 607-622.

 

Hathway, D. E. and J. W. T. Deakins. (1957) "Enzymic Oxidation
of Catechin to a Polymer Structurally Related to Some
Phlobatannins." Biochem. J. 67: 239-244.

 

Hess, E. H. (1958) "The Polyphenolase of Tobacco and its Partici-
pation in Amino Acid Metabolism. I. --Manometric Studies. "
Arch. Biochem. Biophys. 74: 198-208.

 

Hillis, W. E. and T. Swain. (1959) "The Phenolic Constituents of
Prunus Domestica. II. --The Analysis of Tissues of the
Victoria Plum Tree." J. Sci. Food Agric. 10: 135-144.

 

Hillis, W. E. and T. Swain. (1959) "Phenolic Constituents of Prunus
Domestica. III. --1dentification of the Major Constituents

in the Tissues of Victoria Tree. " J. Sci. Food ALric.
10: 533-537.

 

Hillis, W. E. and G. Urbach. (1958) "Leucoanthocyanins as the
Possible Precursors of Tannins." Nature. 182: 657-688.

 

 

 

 

   

74

Hjerten, H. S. and R. Mosbach. (1962) "Molecular-sieve Chroma-
tography of Proteins on Columns of Cross-linked Polyacry-
lamide. " Anal. Biochem. 3: 109-118.

 

Horwitz, N. H. and S. C. Shen. (1952) "Neurospora Tyrosinase."
J. Biol. Chem. 197: 513-520.

 

Hsia, C. L., L. L. Claypool, J. L. Abernathy and Paul Esau. (1964)
"Leucoanthocyan Material from Immature Peaches." 1.
Food Science. 29: 723-729.

 

Hussein, A. A. and W. V. Cruess. (1960) "Properties of the Oxi-
dizing Enzymes of Certain Vinifera Grapes. " Food Research.
25: 637-648. '

 

Ingraham, L. L. and B. Makower. (1953) "Chronometric Method of
Determining Polyphenol -oxidase Activity. Potintiometric
Device for Automatic Determination of End Point." Anal.
Chem. 27: 916-918.

 

Ingraham, L. L. (1955) "Reaction-inactivation of Polyphenol-oxidase:
Catechol and Oxygen Dependence. " J. Am. Chem. Soc.
77: 2875-2876.

 

Ingraham, L. L. (1954) "Reaction-inactivation of Polyphenol-oxidase:
Temperature Dependence." J. Am. Chem Soc. 76: 3777-
3780.

 

Ingraham, L. L. (1956) "Determination of Polyphenol Oxidase Acti-
vity by Rotating Platinum Electrode. " Anal. Chem. 28:
1177.

 

Ingraham, L. L. ( 1956) "Effect of Ascorbic Acid on Polyphenol-
oxidase." J. Am. Chem. Soc. 78: 5095-5097.

 

Jackson, H. (1939) "The Oxidation of Catechol and 1:2:4 Trihydroxy-
benzene by Polyphenol-oxidase. " Biochem. J. 33: 1452-
1480.

 

Jimenez, M. A. (1947) "A Study of the Oxidizing Enzymes of Guava. "
Food Research. 12: 300-310.

 

Johnson, G., E. M. Foreman and M. M. Mayer. (1950) "The Ultra-
voilet Absorption of Polyphenolic Substances from Various
Fruits." Food Tech. 4: 237-241.

 

 

 

 

75

Johnson, G. and D. G. Guadagni. (1949) "Treatment of Fruits to
Prevent Browning." U. S. Patent 2, 475, 838.

Johnson, G., M. M. Mayer and D. K. Johnson. (1951) "Isolation
and Characterization of Peach Tannins. " Food Research.
16: 169—180.

 

Joslyn, M. A. (1934) "The Present Status of Methods of Improving
Quality of Frozen Fruit and Fruit Products. " Fruit Pro-
ducts J. 13: 142-145, 153-155. I

 

Joslyn, M. A. and L. A. Hohl. (1943) "The Commercial Freezing
of Fruit Products." Calif. Agr. Expt. Sta. Bull. 703,
pp. 1-108.

Joslyn, M. A. and J. D. Ponting. (1951) "Enzyme-catalyzed Oxida-
tive Browning of Fruit Products." Advances in Food Re-
search. 3: 1-44.

Joslyn, M. A. and L. Goldstein. (1964) "Astringency Principles:
Changes in Phenolic Content in Persimmons during
Ripening and Processing." J. Agr. Food Chem. 12:
511-520.

Joslyn, M. A. and J. L. Goldstein. (1964) "Astringency of Fruits
and Fruit Products in Relation to Phenolic Content. "
Advances in Food Research. 13: 179-217.

Karkhanis, Y. and E. Frieden. ( 1961) "On the Induction Period and
a Protein Inhibitor of Mushroom Tyrosinase. " J. Biol.
Chem. 236: Preliminary Communication.

 

Karkhanis, Y. and E. Frieden (1961) "Tyrosinase Inhibition by its
Apoenzyme and the Transformation of a Protein Inhibitor
into Tyrosinase." Biochem. Biophys. Res. Commun. 4:

303-308.

Kastle, J. H. and H. Loevenhart. ( 1901) "On the Nature of Certain
of the Oxidizing Ferments." Am. Chem. J. 26: 566.

Kastle, J. H. (1910) "The Oxidases and Other Oxygen-catalysts
Concerned in Biological Oxidation." Hyg. Lab. Washington

Bull. 59: 1-164.

Keilin, D. and T. Mann. (1938) "Polyphenol Oxidase: Purification,
Nature and Properties." Proc. Roy. Soc. London. B125:

187-2040

 

 

 

 

76

 

"The Action of Tyrosinase on Monophenols."

 

 

Kendal, L. P. (1949)
Biochem. J. 44: 442-454.
Kertesz, Z. I. ( 1933) "The Oxidase System of a Non-browning
Yellow Peach." New York Agr. Expt. Sta. Tech. Bull.
219, pp. 1-14.
Kertesz, Z. I. (1934) "The Browning of Yellow Peaches. " Fruit
Products J. 13: 304-306.
Kertesz, Denis and Romano Zito. (1957) "Polyphenoloxidase (Tyro-
sinase): Purification and Molecular Properties. " Nature.
179: 1017.
Kertesz, Denis. (1957) "State of Copper in Polyphenoloxidase (Tyro-
sinase) ." Nature. 180: 506.

Kertesz, D. and O. Azzopardi. (1962) "L'oxydation Indirecte des
Substances par la Polyphenoloxidase; L'oxydation de l' Hydro-
oquinone et des Monohydroxyphenols. " Biochim. Biophys.
Acta. 64: 149-152.

Kertesz, Denis and Romano Zito. (1962) "Kinetic Studies of the Poly-
phenoloxidase Action, Kinetics in the Presence of Reducing
Agents." Biochim. Biophys. Acta. 64: 153-167.
Kirk, L. P. ( 1947) "The Chemical Determination of Proteins. "
Advances in Chem. M. L. Anson and John T. Edsall. 2:
139-161.
( 1963) "Correlation of Structure and Function
142: 1533-1540.

Science.

Koshland, D. E. Jr.
in Enzyme Action. "
"The Role of Reducing Agents in the Action
76: 87-96.

Krueger, R. C. (1959)
of Tyrosinase." Arch. Biochem. Biophys.
Lamb, J. and H. B. Sreerangachar. (1940a) "Studies on the Fer-
mentation of Ceylon Tea. 1. --The Nature of the Enzyme
System." Biochem. J. 34: 1472-1484.
Lamb, J. and H. B. Sreerangachar. (1940b) ”Studies on the Fer-
mentation of Ceylon Tea. 11. —-Oxidizing Enzymes." Bio-

chem. J. 34: 1485-1492.
( 1895) "Sur 1'oxydation du tannin de la pomme a cidre. ”
120: 370-372.

Lindet, M.
Compt. rend.

 

 

 

.’ /
\ ./
' \
, \.
, ,7 ,
, ° /
. 1.|~ _ . _‘ . I

N7.fl."~fl"" ,

 

77

Lineweaver, H. and D. Burk. (1934) "The Determination of Enzyme
Dissociation Constants." J. Am. Chem. Soc. 56: 658-666.

 

Ludwig, J. and J. M. Nelson. (1939) "Inactivation of Tyrosinase in
the Oxidation of Catechol." J. Am. Chem. Soc. 61: 2601-
2606.

 

Mallette, M. F., and C. R. Dawson. (1947) "Measurement of the

Cresolase Activity of Tyrosinase. " J. Am. Chem Soc.
69: 466-467. I

 

Mallette, M. F., S. Lewis, S. R. Ames, J. M. Nelson and C. R.
Dawson. ( 1948) "The Preparation of Mushroom Tyro-
sinase." Arch. Biochem. 16: 283-289.

 

Mallette, M. F. and C. R. Dawson. (1949) "On the Nature of Highly
Purified Mushroom Tyrosinase Preparations. " Arch.
Biochem. 23: 29-48.

Mason, H. S. (1949) "The Chemistry of Melanin. VI. --Mechanism
of the Oxidation of Catechol by Tyrosinase. " J. Biol.
Chem. 181: 803-812.

Mason, H. S., W. L. Fowlks and E. Peterson. (1955) "Oxygen
Transfer and Electron Transport by the Phenolase Com-
plex." J. Am. Chem. Soc. 77: 2914-2915.

 

Mason, H. S. (1956) "Structures and Functions of the Phenolase
Complex." Nature. 177: 79-81.

Mason, H. S., E. Spencer and I. Yanazaki. (1961) "Identification
by Electron Spin Resonance Spectroscopy of the Primary
Product of Tyrosinase Caralyzed Catechol Oxidation."
Biochem. Biophys. Res. Commun. 4: 236-238.

McElroy, W. D. and Bentley Glass. (1950) Copper Metabolism;
A Symposium on Animal, Plant, and Soil Relationships.
The John Hopkins Press, Baltimore. p. 443.

Miller, W. H. and C. R. Dawson. (1941) "A New Method for the
Measurement of Tyrosinase Catecholase Activity. " J.
Am. Chem. Soc. 63: 3375-3382.

 

Miller, W. H. and C. R. Dawson. (1942) ”Factors Influencing the
Cresolase Activity of Tyrosinase. The Effect of Gelatin
and p-cresol Concentration. " J. Am. Chem. Soc. 64:
2344-2348. '

 

 

 

 

 

 

78

Miller, W. H., M. F. Mallette, L. J. Roth and C. R. Dawson.
(1944) "A New Method for the Measurement of Tyrosinase
Catecholase Activity. 11. --Catecholase Activity Based on
the Initial Reaction Velocity. " J. Am. Chem. Soc. 66:
514-519.

 

Mondy, N. 1., B. P. Klein and I. 1. Smith. (1959) "The Effect of
Maturity and Storage on Phenolic Content, Enzymatic
Activity and Discoloration of Potatoes. " Food Research.

24: 693-705.

 

Nakamura, T. (1958) "Purification and Physico-chemical Properties
of Laccase with Oxygen." Biochim. Biophys. Acta. 42:
499-5050

 

Nakamura, T. ( 1960)» "On the Mechanism of the Reaction of the Re-
duced Laccase with Oxygen." Biochim. Biophys. Acta.
42: 499-505.

 

Nelson, J. M. and C. R. Dawson. (1944) "Tyrosinase." Advances
in Enzymology. Interscience Pub. Inc. New York. 4:
99-152.

 

Onslow, M. W. (1920) "Oxidizing Enzymes. III.--The Oxidizing
Enzymes of Some Common Fruits. " Biochem. J. 14:
541-547.

 

Onslow, M. W. and M. E. Robinson. (1928) "Oxidizing Enzymes.
X. --The Relationship of Oxygenase and Tyrosinase. "
Biochem. J. 22: 1327-1331.

 

Onslow, M. W. (1931) The Principles of Plant Biochemistry. Cam-
bridge University Press, Cambridge, England. pp, 123-
1720

Osaki, S. (1963) "The Mechanism of Tyrosine Oxidation by Mush-
room Tyrosinase." Arch. Biochem. Biophys. 100:
378-384.

 

Palmer, J. K. (1963) "Banana Polyphenoloxidase; Preparation and
Properties." Plant Physiology. 38: 508-513.

 

Parkinson, G. G. Jr. and J. M. Nelson. (1940) "On the Nature of
the Enzyme Tyrosinase." J. Am. Chem. Soc. 62: 1793-
1797.

 

 

 

 

 

 

 

79

Patil, S., H. J. Evans and P. McMahill. (1963) "Electrophoretic
Separation of the Phenolases from Potato Tubers. "
Nature. 200: 1322—1323.

Patil, S. S. and M. Zucker. (1965) "Potato Phenolases. Purifica-
tion and Properties." J. Biol. Chem. 240: 3938-3943.

Peterson, A. E. and A. H. Sober. (1956) "Chromatography of Pro-
teins. I. --Cellulose Ion-exchange Adsorbents.” J. Am.
Chem. Soc. 78: 751-755.

Pomerantz, S. H. (1963) "Separation, Purification and Properties
of Two Tyrosinases from Hamster Melanoma. " J. Biol.
Chem. 238: 2351-2357.

 

Ponting, J. D. (1944) "Catechol Test for Frozen Fruits. ” Quick
Frozen Foods. 7: 31, 46.

Ponting, J. D. and M. A. Joslyn. (1948) "Ascorbic Acid Oxidase
and Browning in Apple Tissue Extract. " Arch. Biochem.
19: 47-63.

Ponting, J. D. (1954) "Reversible Inactivation of Polyphenol Oxidase. "
J. Am. Chem. Soc. 76: 662-663.

Porath, J. (1960) "Gel Filtration of Proteins, Peptides and Amino
Acids. " Biochim. Biophys. Acta. 39: 193-207.

 

Porath, J. and P. Flodin. ( 1959) "Gel Filtration: A Method for De-
salting and Group Separation. " Nature. 183: 1657—1659.

Pridham, J. B. (1963) Enzyme Chemistry of Phenolic Compounds.
Proceedings of the Plant Phenolics Group Symposium,
Liverpool, April, 1962. Pergamon Press Ltd, Oxford,
England.

Pridham, J. B. (1964) Methods in Polyphenol Chemistry. Proceed-
ings of the Plant Phenolics Group Symposium. Oxford,
April 1963. Pergmon Press Ltd., Oxford, England.

Reyes, Philip and B. S. Luh. (1960) "Characteristics of Browning
Enzymes in Fay Elberta Freestone Peaches. " Food Tech-
nology. 14: 570-575.

 

 

 

80

Roberts, E. A. H. and D. J. Wood. (1950) "The Fermentation Pro-
cess in Tea Manufacture. II. --Oxidation of Substrates by
Tea Oxidase. " Biochem. J. 47: 175-186.

 

Robinson, E. S. and J. M. Nelson. (1944) "The Tyrosine-tyrosinase
Reaction and Aerobic Plant Respiration. " Arch. Biochem.
4: 111-117.

 

Rosenblatt, M., and J. V. Peluso. (1941) "Determination of Tannins
by Photocolorimeter." J. Ass'n Off. Agr. Chem. 24:
170-180.

 

Rothchild, M. L. and R. Macvicar. (1949) "Studies on Copper-
containing Oxidases in Higher Plants." Federation Proc.
8: 245-246.

 

Roux, D. G. (1957) "Some Recent Advances in the Identification of
Leucoanthocyanins and the Chemistry of Condensed Tannins. "
Nature. 180: 973-975.

Roux, D. G. and S. R. Evelyn. (1958) "Condensed Tannins. 1. A
Study of Complex Leucoanthocyanins Present in Condensed
Tannins." Biochem. J. 69: 530-538.

 

Rudkin, G. O. and J. M. Nelson. (1947) "Chlorogenic Acid and
Respiration of Sweet Potatoes." J. Am. Chem Soc. 69:
1470-1475.

 

Russell, A. (1935) "The Natural Tannins." Chem. Revs. 17: 155-
186.

Samisch, R. ( 1935) "The Measurement of Phenolase Activity." J.
Biol. Chem. 110: 643-654.

 

Samisch, R. and W. V. Cruess. (1934) "Enzymic Darkening in
Apricots." Proc. Am. Soc. Hort. Sci. 31: Suppl. 28-31.

 

Samisch, R. ( 1937) "Contribution to the Knowledge of Plant Pheno-
lases. " Plant Physiol. 12: 499-508.

 

Sando, C. E. ( 1924) "The Isolation and Identification of Quercetin
from Apple Peels." J. Agr. Research. 28: 1243-1245.

 

Sater, L. E., M. E. Kirkpatrick, K. E. Stein, H. B. Murray and
D. G. Skinner. "The Effect of Various Pretreatments of
Frozen Peaches on Quality. " Food Freezing. 2: 128-131,
144, 162.

 

 

 

 

 

 

 

 

 

81

Scharf, Walter and C. R. Dawson. (1958) "The Effect of Ascorbic
Acid on the Inactivation of Tyrosine. " J. Am. Chem.
Soc. 80: 4627-4631.

 

Schwimmer, S. (1953) "Enzyme Systems of the White Potato. ” J.
Agr. Food Chem. 1: 1063-1069.

 

Siegelman, H. W. ( 1955) "Detection and Identification of Polypheno-
loxidase Substrates in Apple and Pear Skins." Arch. Bio-
chem. Biophys. 56: 97-102.

 

 

 

Smith, F. G. and E. Stotz. (1949) "A Colorimetric Method for the
Determination of Phenol Oxidase in Plant Material. " J.
Biol. Chem. 179: 865-880.

 

 

Smith, Jack L. and R. C. Kruger. (1962) "Separation and Purifica-
tion of the Phenolases of the Common Mushroom. " J.
BioLChem. 237: 1121-1128.

 

Sober, H. A. and E. A. Peterson. (1954) "Chromatography of Pro-
teins on Cellulose Ion-exchangers. " J. Am. Chem. Soc.
76: 1711-1712. ‘

 

Sumner, J. B. and G. F. Sommers. (1943) Chemistry and Methods
of Enzymes. Academic Press, New York.

 

 

Sussman, A. S. ( 1961) "A Comparison of the Properties of Two
Forms of Tyrosinase from Neurospora Crassa. " Arch.
Biochem. Biophys. 98: 407-415.

 

Swain, T. and W. E. Hillis. (1959) "The Phenolic Constituents of
Prunus Domestica. 1. --The Quantitative Analysis of
Phenolic Constituents." J. Sci. Food Agric. 10: 63-68.

 

Tauber, H. ( 1949) The Chemistry and Technology of Enzymes.
John Wiley and Sons, New York, pp. 453-456.

 

Wada, E. and M. lhida. (1957) "The Enzymic Oxidation of Chloro-
genic and Caffeic Acids in the Presence of Nornicotine. "
Arch. Biochem. Biophys. 71: 393-402.

 

Wagreich, H. and J. M. Nelson. (1936) "On the Oxidation Product
of Catechol When Oxidized by Means of Tyrosinase. "
J. Biol. Chem. 115: 459-465.

 

 

 

 

 

 

82

Wagreich, H. and J. M. Nelson. (1938) "On the Mechanism of the
Catechol-tyrosinase Reaction." J. Am. Chem. Soc. 60:
1545-1548.

Walter, E. M. and J. M. Nelson. (1945) "Further Studies on Tyro-
sinase in Aerobic Plant Respiration. " Arch. Biochem.

6: 131-138.

Warburg, O. and W. Christian. (1942) Biochem. Z. pp. 310,384.

 

Wendler, S. H. and R. B. Gage. (1949) "Paper Chromatography of
Flavonoid Pigments." Science. 109: 287-289.

Wright, C. L. and H. S. Mason. (1946) "The Oxidation of Catechol
by Tyrosinase." J. Biol. Chem. 165: 45-53.

 

 

Yasunobu, K. T. (1959) "Mode of Action of Tyrosinase. " in Pigment
Cell Biolm ed. by M. Gordon. Academic Press, New

York.