AN ABSTRACT OF THE THESIS OF
Kimberly Warner Wissemann for the degree of Doctor of Philosophy in Food Science and Technology presented on November 11, 1982
Title: Purification and Properties of d'Anjou Pear (Pyrus communis, L.) Polyphenol Oxidase
Abstract approved: ^ . ~~ DK Morris W. M^ntgomer^
Polyphenol oxidase (PPO) from d'Anjou pears was purified and some molecular properties were studied. The extraction of PPO was accomplished in the presence of the phenolic binder AG 2X-8 and Triton X-100. Chlorophyll pigment was removed by chromatography resulting in a clear, colorless enzyme extract. Three fractions of pear PPO were purified by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, followed by chromatography on DEAE-cellulose and hydroxyl- apatite (HA). The best resolution was achieved when the columns were developed at room temperature. Reproducibility of the entire scheme was excellent with chromatography on the hydrophobic resin as a key to the procedure's success. The three fractions of pear PPO were homogeneous when stained for protein with the silver stain after polyacrylamide gel electrophoresis and corresponded to relative mobilities of 0.41 (HA 1), 0.43 (HA 2), and 0.73 (DEAE 3). However, other minor protein bands were noticed on lithium dodecyl sulfate, LDS, electrophoresis. Dimethylsulfoxide, DMSO, was found to significantly increase the PPO activity over the control. An 80% increase in enzyme activity was observed when 5% DMSO was added to the enzyme fraction. Chlorogenic
acid reacted with one of the purified fractions (HA 2) at pH 7 to yield 5 additional PPO bands upon electrophoresis. However, no electrophoretic changes were observed with another fraction (DEAE 3) or when 2,3-dihydroxybenzaldehyde was reacted with the two PPO
fractions.
The molecular weights of the three multiple forms of pear PPO were similar and were 45,300 ± 5% daltons by Sephacryl S-300 gel
filtration and 59,600 ± 3% daltons by LDS electrophoresis in 10% and
gradient polyacrylamide slab gels. The isoelectric points of the
three PPO fractions were: HA 1, pi 4.3 and 4.5; HA 2, pi 4.5 and 4.7;
and DEAE 3, pi 6.9. The three fractions differed in amino acid
composition with HA 1 having a higher proportion of hydrophobic amino
acids and less charged residues than either HA 2 or DEAE 3. All three
forms of PPO contained a high proportion of the acidic amino acids
and/or their amides, and glycine. Purification and Properties of d'Anjou Pear (Pyrus communis, L.) Polyphenol Oxidase
by
Kimberly Warner Wissemann
A THESIS
submitted to
Oregon State University
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Completed November 11, 1982
Commencement June 1983 APPROVED:
Professor of Food Science in e'Karge oWnajor
Head of Department of FOJW Science and Technology
Dean of Graduate SchedY
Date thesis is presented November 11, 1982
Typed by Jill Nowac for Kimberly Harner Wissemann This thesis is dedicated with love, to my husband, Michael
and to my parents, Charles and Jacqueline Warner ACKNOWLEDGEMENTS
I wish to express my gratitude to my advisor, Dr. Morris
Montgomery, for the encouragement and wisdom that he provided.
Appreciation is also extended to the members of my committee, and the faculty and staff of the Department of Food Science for their understanding and help in providing educational advice.
I would also like to thank my fellow graduate students who provided me with many fond memories of OSU. A special thanks is extended to Mark Einerson, Luis Sayavedra, and Pedro Wesche-Ebeling, for their valuable advice, assistance, and friendship. TABLE OF CONTENTS
Page
INTRODUCTION 1
LITERATURE REVIEW 3
Reactions of Polyphenol Oxidase 3 Assay of Polyphenol Oxidase 7 Physiological Role of Polyphenol Oxidase 7 Intracellular Enzyme Location 9 Enzyme Extraction 11 Enzyme Purification 14 Enzyme Multiplicity 17 Amino Acid Composition and Copper Content of Polyphenol Oxidase 21 Enzyme Molecular Weight and Quaternary Structure 25 Fruit Polyphenol Oxidase 28
MATERIALS AND METHODS 38
Pears 38 Polyphenol Oxidase. Assay 38 Protein Determination 39 Enzyme Extraction Procedure 40 Cleaning of anion exchange resin 40 Extraction procedure 41 Pigment extraction 41 Purification Procedure 43 Bio-gel P-6 column chromatography 43 Phenyl Sepharose CL-48 column chromatography 44 DEAE-Cellulose column chromatography 45 Hydroxylapatite column chromatography 46 Effect of DMSO on Enzyme Activity 47 Molecular Weight Determination by Gel Filtration 47 Eiectrophoresis 49 Polyacrylamide slab gel eiectrophoresis 49 Detection of PPO isozymes 50 Protein stain 50 Molecular weight determination by LDS eiectrophoresis 52 Isoelectric Focusing 54 Amino Acid Analysis 55 Protein-Phenolic Interactions 57
RESULTS 58 Purification of Polyphenol Oxidase 58 Eiectrophoresis 72 Enzyme Stability 80 Effect of DMSO 80 Page
RESULTS (Continued)
Molecular Weight Determination 85 Isoelectric Focusing 92 Ami no Acid Composition 96 Protein-Phenolic Interactions 98 DISCUSSION 103
Enzyme Extraction 103 Purification of Polyphenol Oxidase 106 Purity and Stability of Pear Polyphenol Oxidase Ill Effect of DMSO 113 Molecular Weight Determination 114 Isoelectric Focusing 120 Amino Acid Composition 121 Protein-Phenolic Interactions 125
CONCLUSION 132
BIBLIOGRAPHY 135 LIST OF FIGURES
Figure Page 1. Purification outline 42
2. Absorption scan from 700 to 300nm of an ethyl ether extraction of the crude enzyme preparation 60
3. Effect of temperature on the elution of pear PPO during column chromatography 62
4. Phenyl Sepharose CL-4B column chromatography of pear PPO 65
5. DEAE-Cellulose column chromatography of pear PPO 67
6. Hydroxylapatite column chromatography of pear PPO 70
7. Polyacrylamide electrophoresis on 1% slab gels of pear PPO during purification 74
8. Polyacrylamide electrophoresis on 7% slab gels of selected fractions of pear PPO 77
9. Effect of Triton X-100 in enzyme extraction procedure of pear PPO 79
10. Stability of a purified d'Anjou pear PPO fraction 82
11. Effect of DMSO on the enzyme activity of d'Anjou pear PPO... 84
12. Molecular weight of d'Anjou pear PPO by Sephacryl S-300 Superfine chromatography 87
13. LDS 10% polyacrylamide slab gel electrophoresis of pear PPO stained for protein 89
14. LDS gradient polyacrylamide slab gel electrophoresis of pear PPO stained for protein 91
15. Molecular weight of d'Anjou pear PPO by LDS gradient polyacrylamide electrophoresis 94 16. Isoelectric focusing of d'Anjou pear PPO 95
17. 7% Polyacrylamide slab gel electrophoresis on PPO-phenolic interactions 101
18. Interaction of polyphenol with protein-bound lysine 131 LIST OF TABLES
Table Page 1. Comparison of the amino acid composition of PPO from various sources 22 2. Purification of d'Anjou pear PPO 71
3. Amino acid composition of d'Anjou pear PPO 97
4. Protein-phenolic interactions with d'Anjou pear PPO 99
5. Comparison of pear PPO amino acid analysis to PPO from other higher plants 124 PURIFICATION AND PROPERTIES OF D'ANJOU PEAR (Pyrus communis, L.) POLYPHENOL OXIDASE
INTRODUCTION
Polyphenol oxidase (PPO) is a problem to the Northwest fruit and vegetable processors because of the enzymic browning that occurs when the fruit is damaged during harvesting, handling, and processing. PPO catalyzes the oxidation of phenolic compounds to quinones which are further oxidized and polymerized to the brown pigment melanin.
Undesirable changes in flavor, odor, and nutritive value usually accompany the browning reaction, and further decrease the quality and consumer acceptance of the product.
PPO is believed to be ubiquitous in the plant kingdom (Whitaker,
1972), but its activity is particularly high in those fruits and vegetables containing significant levels of phenolic compounds. In part, because of the economic losses which can be caused in the fruit and vegetable industries, PPO has been extensively studied in this past century. As a result there is a considerable amount of data on its catalytic properties, multiple forms, latency and activation, location, physiological role, and activity during maturation and storage of fruits and vegetables. However, in spite of the abundant amount of literature many questions remain unanswered and, in fact, the more that is learned about this enzyme the more baffling it becomes.
Recently, the isolation and purification of PPO from plant tissues has been greatly facilitated through the use of effective phenolic binding agents to remove interfering phenols. With a pure 2 enzyme system it would be possible to study PPO in more detail, including its molecular structure and catalytic site. This information could help to explain the mode of action of PPO and how to control its undesirable effects. However, the number of PPO preparations that have been purified to homogeneity from higher plants is very limited.
The purpose of this dissertation was to purify d'Anjou pear PPO and use this purified enzyme preparation to study some molecular properties; including molecular weight, multiplicity, isoelectric point, and amino acid composition. PPO from d'Anjou pears was chosen because this fruit is economically important to the Pacific Northwest.
There has been at least a 50% increase in the production of this pear cultivar over the last decade due to its good storage and eating qualities (Lombard ert al_., 1980). Also, recent work by Smith (1980) has led to an improved extraction procedure of PPO from d'Anjou pears making this fruit a good model for further research. LITERATURE REVIEW
Reactions of Polyphenol Oxidase
Polyphenol oxidase (PRO) is a copper containing enzyme found widely in plants and animals. It was among the first enzymes to be studied which catalyzed a reaction with oxygen (Bourquelot and
Bertrand, 1895; Bertrand, 1895; Bertrand, 1896). As early as 1896
Bertrand labeled this enzyme as an oxidase and suggested that it was a catalytic metalloprotein (Malmstom, 1982; Owen, 1982). Since that time, PPO has been referred to by a variety of names including: mon- ophenol monooxygenase, tyrosinase, phenolase, catecholase, catechol oxidase, monophenol oxidase, and cresolase depending on the particular substrate being studied and the preference of the author. In 1975 the
International Union of Biochemists classified this enzyme as monophenol, dihydroxyphenylalanine: oxygen oxidoreductase; EC
1.14.18.1 (Enzyme Nomenclature, 1975).
One of the most interesting aspects of PPO is its bifunctional ability to catalyze two quite different reactions. These are the hydroxylation of monophenols to yield o-diphenols and the oxidation of 4 o-diphenols to the corresponding o-quinones (Whitaker, 1972). The hydroxylation of monophenols requires molecular oxygen plus an electron donor, AH^, as illustrated in the following reaction sequence:
OH OH 0H Q + o2 + AH2 - (fS + A + H20 " R
This is frequently referred to as cresolase activity since one of the most common and simplest substrates is jD-cresol (R-CH3). The second reaction catalyzed by PRO is the oxidation of o-diphenols as shown below with catechol as a substrate:
0H 8 n
The observed stoichiometry with catechol is 2 moles of diphenol dehydrogenated per mole of 0^ consumed, with the (L undergoing a
net 4 electron reduction to water (Walsh, 1979).
In spite of much effort, the reaction mechanisms for both activities remains unclear. It is known that, for the cresolase oxidation reaction, there is a requirement (both in initiation and during steady-state catalysis) for a diphenol molecule (Hamilton,
1974; Mason, 1957a). Mason (i957a; 1956) has suggested that the
£-diphenol added to the reaction is oxidized and acts as a continuous
source of electrons for the hydroxylation of the monophenols, and thus 5 acts as a cosubstrate in this reaction (Mason, 1965). Vanneste and
Zuberbuhler (1974) have proposed a reaction mechanism which includes both of the apparently different activities of PPO. This mechanism is consistent with the following evidence: 1) only o-diphenols are oxidized in the catecholase reaction (jD-diphenols are unreactive), 2) no evidence has been found for free radical intermediates, 3) a binuclear copper active site is involved, 4) there are two-electron oxidations of the o-diphenol showing that the semiquinone is not an intermediate, and 5) the two enzyme cycles overlap explaining the need for an o-diphenol to initiate and sustain monophenol hydroxylation. A series of recent reports by Lerch and associates (Winkler et al.,
1981; Ruegg and Lerch, 1981; Himmelwright et a_l_., 1980; Kuiper et al.,
1980) on the use of derivatives to investigate the active site of tyrosinase, has also led to a better understanding of the role of copper in the reaction mechanism of PPO.
The catecholase reaction alone has been investigated and the work indicates an ordered Bi Bi mechanism, with oxygen binding to the enzyme first, followed by the binding of the substrate (Ingraham,
1957; Hamilton, 1969; Mason, 1957b; Whitaker, 1972).
Recently Koiner and Pawelek (1982) have described a third reaction that is catalyzed by mammalian tyrosinase in the biosynthesis 6 of melanin. This reaction is the conversion of 5,6-dihydroxyindole to melanochrome as shown below: ™co -xu and requires dihydroxyphenylalanine (DOPA) as a cofactor and is inhibited by tyrosine. The level of tyrosine, DOPA, and
5,6-dihydroxyindole appears to act in the regulation of melanin biosynthesis.
Food scientists are primarily concerned with the catecholase reaction of PPO since this reaction leads to the discoloration of food products. The PPO enzymes of higher plants oxidize a great variety of monophenolic and o-diphenolic compounds, but the vast majority of phenolics in fruits and vegetables are the dihydroxyphenols (Mathew and Papria, 1971). The reason for the concern over the oxidation of these various phenol ics by PPO is the production of the resultant quinones. Quinones are extremely reactive compounds which can undergo a number of rapid secondary reactions (Mason, 1955). These reactions include complexing with amino acids and proteins, coupling oxidations with other compounds, and condensation and polymerization reactions leading to the formation of brown pigments (Mathew and Papria, 1971;
Sizer, 1953). Enzyme inactivation during reaction has long been associated with PPO (Miller and Dawson, 1941) as the quinones produced will combine with and inactivate the enzyme; aptly termed suicide inactivation (Abeles and Maycock, 1976; Robb, 1981). Assay of Polyphenol Oxidase
The above interactions make finding an accurate enzyme assay for
PPO from product formation or substrate depletion measurements somewhat difficult (Mayer and Harel, 1979). Several methods have been reported including manometric, polarographic, chronometric, spectrophotometric, and colorimetric techniques (Arnon, 1949; Miller and Dawson, 1941; El-Bayoumi and Frieden, 1957; Ponting and Joslyn,
1948; Harel et aj_., 1964). Many variations of these methods exist depending on the phenolic substrate and the source of the enzyme.
More recently methods have been described using tritiated tyrosine
(Kahn and Pomerantz, 1980), quinone trapping or coupling agents to measure product formation (Pifferi and Baldassari, 1973; Esterbauer et a!., 1977), and a fluorometric assay (Warren and Routley, 1970). The most convenient of these assays is to follow the initial rate of browning spectrophotometrically at 420 or 410 nm. However, most reports indicate that measuring initial oxygen uptake by the polarographic method is convenient and more accurate than other assay methods (Mayer et a_l_., 1966; Mayer and Harel, 1979).
Physiological Role of Polyphenol Oxidase
It is generally accepted that PPOs are responsible for many of the enzymatic browning reactions occurring throughout the phylogenetic scale. They function in the biosynthesis of the skin pigment melanin in vertebrates (Korner and Paweiek, 1982), in sclerotization of insect cuticles (Nellaiappan and Ramalingam, 1980), and in the biosynthesis 8 of brown polyphenolic polymers in plants and fungi (Vanneste and
Zuberbuhler, 1974). However, in spite of its widespread distribution and the efforts of many researchers, the specific function(s) of plant
PPO remain unresolved. Several functions that have been suggested are the synthesis of o^-diphenols; regulation of plant growth; involvement in electron transport, aerobic respiration, and photosynthesis; effect on the water permeability of seed coats; role in wound healing and in disease resistance (Mayer and Hare!, 1979). Recently, a link with boron requirements has also been proposed (Lewis, 1980; Shkolnik and
Krupnikova, 1981) and with the C,g fatty acid, linolenate (Golbeck and Cammarata, 1981).
The theories on wound healing and disease resistance have evolved from the observation that following injury, quinones are produced, plant tissues rapidly brown, and lignification may occur. The initial reactions are catalyzed by PPO. The protective actions of phenolic oxidation products are associated with the toxicity of the quinones and the increase in insoluble polymers and condensation products that limit further invasion of the pathogen by forming a physical barrier
(Butt, 1977; Van Sumere et aj_., 1975; Butt and Lamb, 1981). An increase or activation of latent PPO activity is frequently observed in response to pathogenic invasion (Tokimoto, 1980; Pangelova-
Shturkova, 1980; Friend, 1977; Martyn et aj[. * 1979). The quinones inhibit the cell wall degrading enzymes of the invading organisms, namely polygalacturonase, or they may inhibit the spore germination of some fungi (Friend, 1977; Friend, 1981). It has also been claimed that the normal browning reaction of apples can be suppressed by the 9 infecting fungus Penicillium expansum by inhibiting the host PPO
(Walker, 1970; Walker, 1969). This implies that the pathogen itself can produce compounds which act against the fruit's own defense mechanism. But, in spite of the numerous reports on host-pathogen relations the specific role of PPO in the protection mechanisms remains unclear (Mayer and Harel, 1979).
In a recent article Mayer and Harel (1981) remind us that the function of fruit is primarily to assist in the distribution of seeds.
Therefore, the role of PPO in fruits might be to protect the seeds prior to their being mature. This could be accomplished through the tanning of proteins to lower their digestibility, and/or the oxidation of phenolics in host-pathogen interactions. Once the seeds are ready to be dispersed, the fruit ripens and decays; no longer requiring a high level of PPO activity. In conjunction with this idea, several fruits that have been studied during maturation reveal high levels of
PPO activity early in the developmental stages, followed by a decline before and after harvest (Wissemann and Lee, 1980a; Flurkey and Jen,
1978; Mayer and Harel, 1981).
Intracellular Enzyme Location
It is important to consider the subcellular location of an enzyme before discussing methods of its extraction and purification. There are many different methods of isolation and some are more applicable in cases of membrane bound and membrane associated enzymes versus those found in aqueous portions of the cell. A knowledge of the cellular environment also helps to predict interactions that may 10 interfere with the extraction methods. This is particularly true in plant systems where tanning reactions, taking place after the disruption of tissues rich in phenols, may cause binding of an otherwise soluble enzyme to a particulate fraction (Mayer and Hare!,
1979; Loomis et aj_., 1979; Craft, 1966). Many workers have stressed the importance of the prevention of protein-phenolic interactions during the extraction of plant enzymes (Coggon et aj_., 1973; Anderson,
1968; Loomis, 1969, 1974; Rhodes, 1977; Sanderson, 1965).
In a classic experiment, Sanderson (1964) showed that PRO from tea leaves remains soluble if the isolation is carried out in the presence of insoluble polyamide, which absorbs endogenous phenols, and thus prevents the appearance of the enzyme in the particulate fraction. These results suggested that polyphenols inactivate enzymes by causing their denaturation and precipitation. However, many enzymes that were initially classified as soluble, have been shown to be bound within delicate lipoprotein vesicles and associated with cell organelles (Tolbert, 1971; Rhodes, 1977). In fact, Kato et al_. (1976) has reported that PRO is still associated with particulate fractions even after the use of phenol adsorbents.
It is therefore not surprising to find that plant PPOs have been located in a variety of cell fractions, both tightly bound to chloroplast (Parish, 1972) or mitochondrial (Walker and Hulme, 1965) membranes and in soluble cell fractions (Craft, 1966; Hare! et al.,
1964; Coombs et aj_. > 1974). The majority of the work, however, seems to indicate that this enzyme is mainly associated or present in chloroplast membranes (Parish, 1972; Henry et al., 1981; Arnon, 1949; 11 Sato, 1967; Hare! and Mayer, 1971; Meyer and Biehl, 1982). Density gradient centrifugation experiments with marker enzymes have also shown PPO to be localized in the grana lamellae containing fractions
(Ben-Shalom et al_., 1977b; Kintsurashvili et a/L, 1980; Henry et al.,
1981; Czaninski and Catesson, 1972, 1974; Martyn et al_., 1979; Vaughn et al., 1981).
Recently Vaughn and Duke (1981) reported using both cytochemical and biochemical fractionation methods to locate PPO in sorghum. Their results indicated that PPO was associated with chlorophyll proteins and was present in mesophyll plastids but absent from bundle sheath and guard cell plastids. These authors were even advocating the use of PPO as a "marker enzyme" for mesophyll chloroplasts in sorghum.
Enzyme Extraction
The isolation of PPO has typically been a difficult task. The main problem lies in the interference of endogenous phenolic compounds during the extraction. The enzyme and phenolic substrates are spatially separated in the intact tissue (Bendall and Gregory, 1963), but as soon as the material is disintegrated in order to extract the enzyme, they come in contact and give rise to quinones and other condensation products. As indicated earlier, these products react with the enzyme to inactivate or modify the protein molecule
(Sanderson, 1965; Loomis and Battaile, 1966; Anderson, 1968). In addition to the covalent attachment of quinones through sulphydryl and amino groups, the unoxidized phenols can also interact with proteins by hydrogen, hydrophobic, and ionic bonding (Loomis, 1974). 12
Several methods of extraction for use with plant enzymes include
compounds which are phenolic scavengers or inhibit phenolic oxidation.
Anderson (1968) suggested the use of copper chelating agents and
reducing agents such as thiols, ascorbate, dithionite, and metabisulphite to control the protein-phenolic reactions. However, all of these compounds act by either interfering with or inhibiting
the action of PPO which is not desirable in the isolation of this enzyme.
The use of organic solvents, such as acetone in the preparation
of an acetone powder, has frequently been used as a first step in the
isolation of PPO (Ponting and Joslyn, 1948; Patil and Zucker, 1965;
Dizik and Knapp, 1970; Ben-Shalom et al_., 1977a; Wissemann and Lee,
1981). Acetone lowers the dielectric constant of the aqueous solution which results in a decrease in the solubility of the proteins, thus
causing them to precipitate (Wharton and McCarty, 1972). In addition,
the acetone is capable of hydrogen bonding to the phenolic compounds
to solubilize them into the filtrate (Loomis, 1969). However, acetone
does not extract all of the phenolic compounds, allowing considerable
browning of the extract to still occur (Patil and Zucker, 1965;
Wissemann, 1980; Smith, 1980).
Another aid in the separation of PPO from phenols, which has been
strongly advocated by Loomis (1966, 1969, 1974, 1979), is the use of
phenol-complexing agents. These include polycaprolactam powder
(Sanderson, 1964), polyethylene glycol (Badran and Jones, 1965;
Benjamin and Montgomery, 1973), insoluble polyvinylpyrrolidone (PVPP)
(Loomis and Battaile, 1966; Andersen and Sowers, 1968), Amberlite XAD 13 resins (Loomis, 1974; Gray, 1978; Loomis et al_., 1979), and anion exchange resins (Lam and Shaw, 1970; Gray, 1978). These adsorbents remove phenolic compounds through the formation of hydrogen, hydrophobic, and ionic bonds (Loomis and Battaile, 1966; Loomis et al_., 1979; Fields and Tyson, 1973; Olsson et al_., 1976).
There has been some debate over the effectiveness of these phenolic scavengers since it has been speculated that the enzyme may also bind to the resin (Walker and Hulme, 1965; Jones et aK, 1965;
Lam and Shaw, 1970). However, since all plant materials contain different types and quantities of phenolic compounds there is no single compound or combination of compounds that is universally applicable (Rhodes, 1977). Therefore, each plant system must be examined individually to optimize the enzyme extraction and minimize protein-phenolic interactions. Smith (1980) and Wesche-Ebeling (1980) have developed such extraction procedures for d'Anjou pear and strawberry, respectively, based on a knowledge of the phenolics present in the fruit and various trials with different levels and combinations of PVPP, XAD, and anion exchange resins. Unfortunately, even under the best conditions most protein preparations made from plant tissues probably contain some attached phenolic molecules which may well modify the behavior of the protein even when enzymatic activity is not impaired (Rhodes, 1977; Fields and Tyson, 1973).
Another factor in the extraction of PRO is the association of PPO with membranous material. The strength of this bond seems to vary depending on the tissue and stage of development of the plant (Mayer and Hare!, 1981). The natural ripening of the fruit appears to render 14 the enzyme more soluble than is observed in the green fruit
(Ben-Shalom et a_l_., 1977b; Kidron et al_., 1978). When necessary, harsh homogenization, detergents, butanol, and proteolysis have been used to release the PPO from particulate matter (Mayer and Harel,
1979; Mayer, 1964). The use of Triton X-100 has been the most common detergent used for this purpose (Harel et aK, 1965; Walker and Hulme,
1966; Harel and Mayer, 1971; Parish, 1972; Wissemann and Lee, 1980a;
Galeazzi et aT_., 1981). The popularity of this detergent is due to the fact that it has a low critical mi cellar concentration which causes it to be less disruptive and have a minimal effect on protein conformation (Maddy and Dunn, 1976). One of its drawbacks however, is that it tends to release the chlorophyll also present in the fruit to yield a green enzyme extract (Walker and Hulme, 1965; Parish, 1972).
The use of any detergent may also result in modification of the enzyme structure and properties (Rhodes, 1977; Mayer and Harel, 1979; Maddy and Dunn, 1976).
Enzyme Purification
The isolation of PPO from plant tissues has been greatly facilitated by the advent of effective phenolic binding agents. Once the phenolic materials have been removed and the enzyme is solubilized it is then necessary to separate the PPO from the many other proteins which may be present. Frequently these proteins have similar properties and this problem is further complicated by the fact that
PPO is generally present in very low concentrations in plant cells
(Mathew and Parpia, 1971). 15 Polyphenol oxidase has been partially purified from many plants, fungi, and fruits. However, there are very few reports on its purification to homogeneity and amino acid composition from plant tissues. The majority of the information has been obtained from mushrooms (Smith and Krueger, 1962; Bouchilloux et_ al_., 1963; Jolley et al_., 1969a; Kertesz and Zito, 1965) and Neurospora (Fling et al.,
1963; Katon and Galcun, 1975; Lerch, 1976; Lerch 1978; Robb and
Gutteridge, 1981). The only reports from plants that indicate that the enzyme has been purified to apparent homogeneity are from potato
(Patil and Zucker, 1965; Balasingham and Ferdinand, 1970), Bartlett pear (Rivas and Whitaker, 1973), spinach beet (Vaughan et al_., 1975),. grapes (Kidron et aj_., 1977), eggplant (Sharma and Ali, 1980), peaches
(Flurkey, 1979) spinach (Golbeck and Cammarata, 1981), and yam tubers
(Anosike and Ayaebene, 1981). But only one of these researchers reported that the initial extract was free from tanning and further found the enzyme to be homogeneous by SDS electrophoresis and sedimentation velocity studies (Vaughan et &]_., 1975). In the purification of any protein whose purity is determined by electroporesis the detection of the presence of contaminating proteins is only as good as the protein stain that is used. All of the above reports used either the Coomassie blue or the less sensitive amido black protein stain. Recently a highly sensitive silver stain for visualizing proteins in polyacrylamide gels has been developed
(Switzer et aj_., 1979; Wray et aK, 1981). The silver stain is 100 times more sensitive than the conventional Coomassie blue stain
(Switzer et al., 1979) and able to detect proteins at the nanogram 16 level (Merril et al_., 1981). This stain allows detection of proteins at concentrations which previously could be revealed only by autoradiographic techniques (Switzer jet al_., 1979). This allows for a stricter criterion of enzyme purity than was possible before.
The most common techniques used in the purification of PPO involve ammonium sulfate precipitation, dialysis, and chromatography on various adsorption, ion-exchange, and gel filtration columns. The most popular being DEAE-cellulose and hydroxylapatite columns.
Affinity chromatography has been used to purify mushroom tyrosinase
(Gutteridge and Robb, 1973; O'Neill et al_., 1973), but there were problems using phenol derivatives since they are oxidized by the enzyme (O'Neill et al_., 1973).
Recently the use of hydrophobic interaction chromatography has been shown to be very useful in the purification of PPO (Flurkey and
Jen, 19078; Jen and Flurkey, 1979; Flurkey and Jen, 1980a; Wissemann and Lee, 1980b). This is a relatively new technique in the separation of proteins (Hofstee, 1973; Rosengren et al_., 1975; Yon, 1978;
Hjerten, 1981b; Carson and Konigsberg, 1981). This procedure allows for the separation of proteins based on the different size and shape of accessible hydrophobic regions on their surface (Shaltiel, 1974;
Shaltiel and Halperin, 1979; Morris, 1976). These hydrophobic pockets interact with immobilized hydrophobic groups supported on insoluble, hydrophilic matrices, such as beaded agarose (Yon, 1978). The optimum resolution of individual proteins is then achieved by gradually changing the nature of the eluting solvent, its ionic strength, salt concentration, pH, polarity, or temperature (Hjerten, 1973, 1981a). 17
The work with hydrophobic chromatography and PPO involved the uncharged media, Phenyl-Sepharose CL-4B. This resin has the general structure:
Sepharose ^ - O - CHj - CH - CH2 -O- (i A
(Hjerten et al., 1974),
Sepharose CL-4B is a cross-linked agarose gel manufactured by
Pharmacia, Uppsala, Sweden. The phenyl groups are attached to the monosaccharide units of the matrix via uncharged, stable ether linkages at a concentration of about 0.2 moles of ligand per mole of galactose (Pharmacia, 1976).
Enzyme Multiplicity
Isozymes is the name given to multiple forms of an enzyme occurring in a single species. They are similar but chemically distinct protein molecules. The International Union of Biochemists have suggested a classification of multiple forms, based on those whose synthesis is controlled by distinct stretches of DNA versus those derived by post-synthetic modifications of a single gene product
(Dixon et a^., 1979). The first includes both genetically independent proteins that are coded for by separate genes and genetic variants
(alleles) of the same gene. There are differences, either great or small, in the primary sequence that effect the charge and mobility of these proteins on electrophoretic gels or in ion-exchange 18 chromatography. The second group of isozymes are different due to nongenetic phenomena, resulting from post-translational modifications
(Kaplan, 1968). These modifications may involve an extra peptide non-covalently attached, partial proteolysis, carbohydrate attachment, deamidization and decarboxylation, aggregation of a single polypeptide chain, and conformationally different forms (Dixon et aj.., 1979; Kaplan, 1968; Market, 1968).
The role of isozymes in living systems is not known with certainty. But it appears that enzymes with different kinetic parameters are needed to fulfill different physiological functions.
Thus, substrate concentrations may vary between different tissues; between different organelles; and at different developmental stages of an organism (Metzler, 1977).
Multiple forms of PPO were first reported by Mallette and Dawson
(1949) for mushroom tyrosinase. They noted a number of similar tyrosinases with different physical and enzymatic properties that they attributed to chemical changes or the fragmentation of one native form during the purification process. Smith and Kruegar (1962) and
Bouchilloux et_ a]_. (1963) disagreed and claimed that the multiplicity of their enzyme extracts was not a result of the method of preparation but the consequence of the actual occurrence of various enzyme species in the mushroom. Since then multiple forms of PPO have been isolated from a wide variety of sources including: peaches (Wong et a!.,
1971), apples (Harel et al_., 1965; Harel and Mayer, 1968), bananas
(Montgomery and Sgarbieri, 1975; Galeazzi e_t al_., 1981), avocados
(Dizik and Knapp, 1970; Kahn, 1976), grapes (Harel and Mayer, 1971; 19 Wolfe, 1976; Wissemann and Lee, 1981), pears (Rivas and Whitaker,
1973; Halim and Montgomery, 1978), and many other plant tissues.
A discussion on the multiplicity of PPO, or any other enzyme, would not be complete however, without presenting the possibility that these multiple forms are not true isozymes but instead artifacts of the purification procedure. The early work of Bendall and Gregory
(1963) showed that tea leaf phenolase will tan itself if supplied with an o-diphenolic substrate, and that the behavior of the enzyme on
DEAE-cellulose columns could be modified considerably by tanning of the protein molecule. Smith (1980) further demonstrated that with an optimized extraction procedure, using phenolic adsorbents, she could decrease the number of isozyme bands on electrophoresis gels from eleven to three. Apparently, she was removing the endogenous phenolics and therefore not allowing them to combine with the enzyme and alter the charge and mobility of PPO during electrophoresis.
Other alterations in protein structure due to isolation techniques involve the possibility of fragmentation, proteolysis, activation of latent forms, and the release of membrane bound forms
(Mayer and Hare!, 1979). Flurkey and Jen (1980c) found several apparent isozyme forms in crude preparations of peach fruit, which were eliminated when protease inhibitors were used in every step of the purification. They reported that some of the forms were probably the result of proteolytic action by peach proteases while others were the result of the association of PPO with carbohydrate materials.
Kidron et al_. (1977) also speculated that partial degradation may have occurred in their previous (Harel et al_., 1973) isolation of grape 20
PPO. However, many of the enzyme forms that they isolated had the same molecular weights but different charge distributions. In a very thorough study on several isozymes of PPO from spinach leaves Lieberei et a_l_. (1981) found no difference in the primary protein structure by immunological methods. The different forms were attributed to differential binding of membrane components.
Another problem in the isolation of PPO that can lead to multiple bands on electrophoresis gels is apparent latency and activation. The activation of latent or existing PPO forms has been shown to occur after exposure to acid, alkali, detergents, limited proteolysis, acetone, urea, or C,g fatty acids (Mayer and Friend, 1960; Yamaguchi et al., 1970; Robb et al_., 1964; Mayer, 1964; Ohnishi et aK, 1970;
Lerner et al_., 1972; Golbeck and Cammarata, 1981). Spinach chloroplast PPO is also spontaneously activated by its release from thylakoid membranes during homogenization and storage (Meyer and
Beihl, 1982). It has been speculated that this activation phenomena is either due to the removal of an inhibitor (Sato, 1977; Keilin and
Mann, 1938) or involves conformational changes (Lerner and Mayer,
1975; Swain et a]_., 1966). Changes in enzyme multiplicity as a result of aging or storage have also been reported (Zaprometov et aj_., 1979;
Meyer and Biehl, 1980, 1981; Adamson and Abigor, 1980). The importance of the appearance or disappearance of these various enzyme forms due to changes in the purification procedure are not well understood. The arguments of whether these are true isozymes or artifacts only make it more evident that great care should be taken in the interpretation of these multiple forms. 21
In spite of the above precautions there is good evidence for differences in the primary structure between the isozymes of PPO isolated from mushroom (Jolley et al_., 1969a) and Neurospora (Horwitz et aj_., 1961). Horowitz et^ aj_. (1961) also illustrated that the four isozymes of Neurospora were the result of four different alleles originating from one gene locus. Also, the interconversion of subunits to yield isozymes of different polymeric molecular weights has been reported for mushroom (Jolley et a_L, 1969b)and apple (Hare! and Mayer, 1968) PPO.
Amino Acid Composition and Copper Content of Polyphenol Oxidase
There are very few reports on the amino acid composition of PPO from higher plants due to the difficulty in isolating the enzyme in sufficient purity and quantities. There is, however, a remarkable resemblence in the amino acid compositions of polyphenol oxidases that have been isolated from a diverse variety of sources. Table 1 illustrates the similarity between Neurospora (Lerch, 1978; Fling et al., 1963), mushroom (Jolley e_t &]_., 1969a), potato (Balasingam and Ferdinand, 1970), grape (Kidron jet a]_., 1977) and spinach beet
(Vaughan et a]_., 1975) PPO. The differences in the reported molecular weight of the enzyme subunit will affect the values slightly.
The similarity in the basic amino acids (lysine, arginine, and histidine) is particularly noticeable, as well as the content of aspartic acid, glycine, alanine, threonine, and isoleucine. The content of the hydrophobic residues (leucine, isoleucine, proline, valine, and phenylalam'ne) is consistently about 30% of the total. In Table 1. Comparison of the amino acid composition of PPO from various sources.
Species: Neurospora Neurospora0' Mushroom Potato6 Grapes Spinach beet^ Amino Acid
Cysteic acid 1 0 5 5 1 7 Aspartic acid + asparagine 44 31 35 39 41 Methionine 3 2 5 6 0 7 Threonine 22 16 20 17 18 19 Serine 42 32 15 22 24 24 Glutamic acid + glutamine 34 22 30 31 28 18 Proline 31 23 18 17 26 28 Glycine 25 19 23 25 22 26 Alanine 33 24 17 24 25 23 Valine 23 17 16 21 18 26 Isoleucine 13 9 15 17 17 14 Leucine 33 24 21 28 21 30 Tyrosine 20 12 11 10/11 18 9 Phenylalanine 24 17 15 14 12 17 Lysine 17 12 13 20 18 18 Histidine 10 6 8 6 6 8 Arginine 20 14 11 11 10 Tryptophan 12 8 8 ^ - 2
Based on MW of 46,000 32,000 32,400 36,000 40,000 40,000
Residues per molecule fBalasingham and Ferdinand (1970) Lerch (19'78) TKidron et aK (1977) "jFling et aK (1963) ^Vaughan et aK (1975) aJolley et al. (1969a) not determined
l\3 23 an attempt to explain the association-dissociation phenomena seen in
PPO several authors have quoted Van Holde's (1966) findings that globular proteins may be classed as single chain or multiple chain according to whether their hydrophobic content is less than or more than 30%, respectively. Proteins with about 30% hydrophobic residues are on the dividing line and are likely to exhibit an interconversion of forms (Jolley et a]_., 1969a; Balasingam and Ferdinand, 1970;
Vanneste and Zuberbuhler, 1974; Mayer and Hare!, 1979).
There is a relatively high content of proline, glycine, and asparagine residues in Neurospora (Lerch, 1978), grape (Kidron et a!.,
1977) and spinach beet (Vaughan et al_., 1975) PPO which are known to occur preferentially in 3-turn structures. Analysis of the primary structure by Lerch (1978) revealed the presence of 45 B-turns. The amino acid sequence of a single polypeptide chain included 407 amino acids, an acetylated serine for the amino terminus, and a phenylalanine residue on the carboxyl terminal. He described the secondary structure of Neurospora tyrosinase to be composed of 31%
B-turn, 34% a-helix, 15% 3-sheet, and 20% coil structure; and suggested a globular conformation for PPO.
The values reported for the sulfur containing amino acids vary;
Robb et al_. (1965) found no methionine or cysteine in their analysis and very low to negligible levels were found in Neurospora (Fling et al., 1963) and grapes (Kidron et al_., 1977). Lerch (1978), however, did find an unusual thioether linkage between a single cysteinyl residue (#96) and histidyl residue 94. He postulated that this 24 structure could be involved in the binding of the active site copper or play a role in enzyme catalysis.
Reports on the binding of non-amino acid constituents to the protein molecule include the presence of carbohydrate and RNA
(Balasingham and Ferdinand, 1970; Bull and Carter, 1973; Stelzig et ai., 1972). PRO was the first enzyme in which the copper content was related to activity (Kubowitz, 1937). If the copper is removed by prolonged dialysis, aging, diethyldithiocarbamate, or cyanide, the activity is lost. This can be restored by adding copper back (Owen, 1982; Kertesz and Zito, 1965). For several years the number of copper atoms and its valency in PRO were debated. Since the metal cannot be detected by electron para-magnetic resonance (EPR) (Bouchilloux et aj_., 1963) and the values for the copper content suggested one atom per polypeptide
(Kertesz and Zito, 1965; Robb et al_., 1965; Fling et al_., 1963; Kidron et aj_., 1977), it was assumed that the enzyme contained one copper atom in the Cu state.
However, the copper content can be complicated by the apparent loss of copper from the enzyme during purification (Mayer and Hare!,
1979). Also the work by Makino jet _al_. (1974), Duckworth and Coleman
(1970), Ruegg and Kerch (1981), Lerch (1976), Dienum et aj_. (1976), and Kuiper et ajL (1980) strongly supports the presence of two cupric atoms per functional unit so close together (3-6A0) that they become antiferromagnetically coupled. Furthermore, Moore and Vigee (1982) recently synthesized binuclear copper (II) complexes and found that they were able to catalyze the air oxidation of several o-diphenols in 25 aqueous solution. Himmelwright et a_l_. (1980) and Winkler et al.
(1981) have proposed a structural model for the binuclear copper active site shown below bridged by Op and bound to nitrogens on the enzyme molecule. R is an unidentified endogenous protein bridge
(Eickman et aT_., 1979).
N ^ © 0= • N Cu(2) Cu(2)
Using two distinct photo-oxidation techniques histidine has been suggested to be present in the active site and involved in copper binding of Neurospora tyrosinase (Gutteridge et al_., 1977; Pfiffner and Lerch, 1981). Structural studies on plant PPOs are now required to evaluate to what extent these results obtained for fungi are representative of higher plants.
Enzyme Molecular Weight and Quaternary Structure
PPO enzymes isolated from different sources and by different researchers seem to differ markedly in their molecular weights (MW).
PPO can undergo association-dissociations which change the observed MW
(Jolley et ak, 1969b, Bouchilloux et al_., 1963; Hare! and Mayer,
1968; Zaprometov et al., 1979). Heat, high ionic strength, dilution, sodium dodecyl sulfate (SDS), urea, EDTA, and proteolytic enzymes are all effective in increasing the amount of lower MW species
(dissociation) (Jolley et al., 1969b; Harel et al., 1973). 26
Bouchilloux et a]_. (1963) found under conditions of neutral pH, low temperature, low ionic strength, and increased protein concentration that a tetrameric state prevailed.
Dizik and Knapp (1970) reported four PPO fractions from avocados with MW values of 14, 28, 56, and 112 thousand. Harel and Mayer
(1968) found PPO with MW's of 30, 60, and 120 thousand in apple fruits. In both cases the results fell into multiples indicating a monomer, dimer, and higher polymeric forms of the enzyme. Nakamura et al_. (1966) isolated a dimeric mushroom PPO with a MW of 61,000.
Several authors have reported the presence of only a single form with a MW of about 120,000 (Kertesz and Zito, 1965; Sharma and Ali, 1981;
Anosike and Ayaebene, 1981).
Initially the minimal MW observed was 30,000 and the enzyme was supposed to consist of four identical subum'ts each containing one copper atom (Duckworth and Coleman, 1970; Fling jet aj_., 1963;
Bouchilloux et^ aj_., 1963; Mayer, 1966). However, now that there is strong evidence that the copper in PPO occurs in pairs a reinvestigation of the MW has shown it to be greater than previously thought. A value of 40 to 46 thousand has been reported for
Neurospora (Lerch, 1976; Robb and Gutteridge, 1981), spinach beet
(Vaughan £t aK, 1975), and spinach (Golbeck and Cammarata, 1981;
Lieberei et al_., 1981) PPO. Lerch (1978) calculated the MW of the
Neurospora enzyme to be 46,000 from the determined amino acid sequence. Kidron et a_h (1977) reported that the MW of grape PPO was 80,000 with indication from its copper content that it was the monomeric form of the enzyme and not a dimer. However, in a recent 27 review Mayer and Harel (1979) indicate that this probably is the dimeric form.
Investigations on the quaternary structure of PPO have been reported for the enzyme isolated from mushroom (Strothkamp et al.,
1976) and Neurospora (Robb and Gutteridge, 1981). PPO from mushrooms was found to contain two types of polypeptide chains by SDS electrophoresis; one was heavy (MW 43,000) and one was light (MW
13,400). They proposed that the enzyme was a tetramer composed of
L2H2 with a MW of 120,000. Robb (1979) further found the occurrence of at least two types of heavy subunits, H and H , so that a range of tetrameric structures is possible. The work of Robb and Gutteridge (1981), however, has found that the Neurospora enzyme contains only one subunit, which is similar in size (MW 46,000) to the larger subunit from tyrosinase isolated from mushrooms. They claim that the Neurospora enzyme is distributed among a number of forms, from the monomer to the tetramer, all of which are fully active. Robb and Gutteridge (1981) also confirmed the results of Strothkamp et al.
(1976) for mushroom PPO. Thus, the presence of two distinct molecular structures for PPO in fungi have been found.
The subunit structure of spinach PPO was found to be 42,500 by lithium dodecyl sulfate gel electrophoresis (Golbeck and Cammarata,
1981). A higher MW tetramer (158,000) was also found by these workers in freshly isolated preparations by gel filtration. Vaughan et al.
(1975) working with spinach beet found only a single enzyme species of
MW 40,000 by SDS electrophoresis. There was no evidence for a tetrameric quarternary structure for spinach beet PPO. 28
Fruit Polyphenol Oxidase
There are many parameters that characterize a particular enzyme, such as amino acid composition, molecular weight, cofactors, activators, multiple forms, location, and physiological function, all of which have already been outlined for PPO. But the rate of an enzyme catalyzed reaction is also influenced by pH, temperature, substrate, and the presence of inhibitors, which have not been discussed above. There are several good recent reviews on PPO in plants that compile the data that has been reported (Mayer and Harel,
1979; Wesche-Ebeling, 1980; Mayer and Harel, 1981; Vamos-Vigyazo,
1981). For ease of discussion only the information that is related to fruit, and particularly to pear, PPO will be mentioned here.
The literature provides information on a number of different pH optima for various plant PPOs, but generally the range is from pH 5.0 to 7.0. Above pH 7.0 autooxidation of the substrate makes it difficult to measure enzymatic activity (Scott, 1975; Gregory and
Bendall, 1966; Loomis, 1974). Enzyme activity has not been demonstrated below pH 3.0 but two different preparations from grapes retained over 50% of their maximum activity at pH 3.5 (Cash et al.,
1976; Wissemann and Lee, 1981), the normal pH of grape juice.
The optimum pH of PPO activity has also been reported to vary with the source of the enzyme, substrate, buffer, and purity of the enzyme (Benjamin and Montgomery, 1973; Reyes and Luh, 1960; Gregory and Bendall, 1966; Vamos-Vigyazo, 1981). Enzyme preparations obtained from the same fruit at various stages of maturity have shown different 29 pH optima (Jen and Kahler, 1974). Isozymes may have distinctly different pH maxima as reported by Wong et al_. (1971) for four purified isozymes from peach PPO.
The presence of two pH optima or a pH activity peak and a noticeable shoulder at another pH has been reported for grapes (Harel and Mayer, 1971), apples (Harel et aJL, 1965; Stelzig et aK, 1972), and peaches (Jen and Kahler, 1974). Examples of a few of the reported fruit PPO pH maxima are: cherry PPO - pH 7.0 (Benjamin and
Montgomery, 1973), date PPO - pH 4.5-6.5 (Hasegawa and Maier, 1980), apple PPO - pH 5.0 (Walker and Hulme, 1966), banana PPO - pH 7.0
(Palmer, 1963), grape PPO - pH 5.5 (Wissemann and Lee, 1981), and peach PPO - pH 6.2 (Lerch and Phithakpol, 1972). The values for pear fruit include PPO isolated from Bartlett pears - pH 4.0 (Rivas and
Whitaker, 1973) and pH 6.2 (Tate et ah, 1964) and from d'Anjou pears
- pH 7.0 (Halim and Montgomery, 1978) and pH 5.1 (Smith, 1980).
The temperature for optimum activity has been studied less frequently for PPO than the pH optimum. But the factors affecting the optimum temperature seem to be essentially the same as those previously mentioned for the pH optimum. Typical values for the optimal temperature for enzyme activity include: 250C - 30oC for grape PPO (Cash et ah, 1976), 250C for apricots (Vamos-Vigyazo, 1981), 370C for peaches (Jen and Kahler, 1974) and 30oC for pears
(Tate et ah , 1964).
PPO is not considered to be an extremely heat-stable enzyme.
Blanching operations in the food industry are frequently used to inactivate PPO to prevent the undesirable browning caused by this 30 enzyme. Short exposures to temperatures of 70 to 90oC are, in most cases, sufficient for partial or total irreversible, destruction of its catalytic function (Vamos-Vigyazo, 1981). Halim and Montgomery
(1978) found that the heat inactivation of d'Anjou pear PPO followed first order kinetics and the half-life was 11.7 min. at 70oC, 6.25 min. at 750C, 2.25 min. at 80oC, and 1.1 min. at 850C. Heat stability experiments have shown differences between isozyme forms (Wong et al.,
1971); and factors such as pH and substrate concentration also have an effect on the thermotolerance of PPO. The presence of thermal stabilizing or destabilizing compounds in the vicinity of the enzyme molecule can also contribute to the observed effect of heat on activity. When the enzyme is present in tissues or in a fruit pulp the heat penetration can be influenced by the size of various particles and be different than when the enzyme is in a clear juice
(Scott, 1975). Svensson and Erickson (1974) reported that lipoxygenase was more thermal stable in pear press juice than when isolated and tested in a buffer solution.
The enzyme activity of PPO may also be affected by exposure to temperatures below zero, but generally there is a slow decrease in activity at lower temperatures. Jen and Kahler (1974) found that at
30C the activity of peach PPO did not drop below 50% of the maximum value. Freezing fruit tissues will slow down the action of PPO, but in most cases, on thawing, the fruits will turn brown; indicating that the enzyme has not been inactivated (Scott, 1975; Vamos-Vigyazo,
1981). 31 Another parameter that has not been adequately discussed in this review is the substrates of PPO. Fruits contain a wide variety of phenolic compounds. However, only a relatively small part of these serve as substrates for PPO. Moreover, it appears that individual
PPOs isolated from different sources show a preference for certain phenolic substrates. Frequently, this preferred substrate is the most abundant phenolic in the plant tissue (Corse, 1964; Mayer and Harel,
1979). While in other instances the main substrate does not even occur in the plant that the enzyme has been isolated from (Harel et al_., 1964; Jen and Kahler, 1974). Coggon et al_. (1973) also found that, tea PPO becomes more specific when the enzyme extract is purified.
The most important natural substrates of PPO in fruits are flavonoids and cinnamic acid esters. In particular these include the o-diphenols catechin, epicatechin, chlorogenic acid, and caffeic acid, with chlorogenic acid as the most widespread (Corse, 1964;
Vamos-Vigyazo, 1981). Although free simple phenols are not commonly found in fruit tissue, catechol (the simplest o^dihydroxyphenol) tyrosine and £-cresol (monophenols) are frequently used as model substrates in enzymatic oxidation studies (Mathew and Parpia, 1971).
The site of substitution of dihydroxy phenols also is a factor in the affinity of PPO for that substrate. Substituents in the 3 position (i.e. 3-methyl catechol) cause a decrease in the reactivity of the substrate, probably owing to steric hindrance (Vamos-Vigyazo,
1981). The presence of an electron-donating group in position 4 (i.e.
4-methyl catechol, chlorogenic acid) tends to increase the affinity of 32 the enzyme for the substrate (Harel et al_., 1964). Substrates with smaller substituent groups on the ring (position 4) of diphenols were oxidized by banana PPO at a faster rate than those with larger groups
(Montgomery and Sgarbieri, 1975). However, purified peach PPO (Wong et aj_.; Luh and Phithakpol, 1972) and strawberry PPO (Wesche-Ebeling,
1980) were most active with d-catechin, indicating that it is difficult to make predictions on the reactivity of PPO toward substrates based on the size of attached groups. From the work of
Hasegawa and Maier (1980), Lanzarini et aj_. (1972), and Mayer et al.
(1964) it is apparent that the presence of an electron-abstracting group in the para position of the dihydroxyphenol acts to greatly reduce or even inhibit PPO activity. This includes compounds such as
3,4-dihydroxybenzoic acid, 2,3-naphthalene diol, and 4-nitro catechol.
The substrate specificity of most PPOs obtained from fruits is primarily towards £-diphenols (Wong et aj_., 1971; Reyes and Luh, 1960;
Benjamin and Montgomery, 1973; Jen and Kahler, 1974; Hasegawa and
Maier, 1980; Wissemann and Lee, 1981). None of the above researchers found any activity when monophenols were used as substrates. Harel and Mayer (1971) working with grapes, Harel £t al_. (1965) working with apples, and Montgomery and Sgarbieri (1975) working with bananas did find some monophenolase activity, but it was very low. It appears that the primary reaction catalyzed by fruit PPO is the oxidation of o-dihydroxy phenols.
The phenolic constituents in assorted pear varieties have been characterized and the compounds identified include catechin, epicatechin, chlorogenic acid, caffeic acid, £-coumaryl-quinic acids, 33 quercetin, arbutin, leucoanthocyanidins, and various other flavonol glycosides (Cartwright et^ al_., 1955; Hulme, 1958; Sioud and Luh, 1966;
Durkee et aK, 1968). Of the above compounds the predominant polyphenols in pear are chlorogenic acids, catechins, and leucoanthocyanidins, which constitute about 90% of the total phenolics
(Sioud and Luh, 1966; Ranadive and Haard, 1971).
The PPO in pear has been shown to be specific for o-dihydroxyphenols (Rivas and Whitaker, 1973; Tate ejt al_., 1962; Halim and Montgomery, 1978). Chlorogenic acid, catechin, catechol, and
4-methyl catechol are all rapidly oxidized, although the last two compounds do not normally occur in pears (Rivas and Whitaker, 1973).
It has been suggested that chlorogenic acid and catechin are the major natural substrates for pear PPO (Siegelman, 1955; Hulme, 1958; Walker,
1964, Weurman and Swain, 1953; Ranadive and Haard, 1971).
Vamos-Vigyazo and Nadudvari-Markus (1982) recently studied the initial rate of browning of five pear cultivars and found the catechins to be more important than chlorogenic acid in the enzymatic browning of pears. While Smith (1980) showed that the browning of an enzyme extract of d'Anjou pear PPO could be prevented by using anion exchange resins in the extraction step, which have a high affinity for chlorogenic acid (Gray, 1978). Ranadive and Haard (1971) found the browning tendency of four different pear varieites to correlate well with the content of both chlorogenic acid and catechins.
Lack of information on the exact catalytic mechanism of PPO makes it difficult to understand the mode of inhibition and binding of various PPO inhibitors. Many factors can influence the degree of 34 inhibition such as pH, temperature, buffer, ionic strength, concentration of enzyme, substrate concentration, and concentration of inhibitor (Whitaker, 1972). Ionizing groups on both the enzyme and inhibitor may be responsible for the pH effects (Webb, 1966).
Different PPO isozymes may also show differential sensitivity to inhibitors (Wong et aj_., 1971; Montgomery and Sgarbieri, 1975).
There are several types of PPO inhibitors. These include reducing agents, quinone scavengers, reagents which interact with the copper prosthetic group, compounds which affect the substrate binding site, and natural PPO inhibitors. Reducing agents and quinone scavengers do not act directly on the enzyme molecule but on the reaction products to inhibit the formation of the secondary brown pigments. Therefore, they are considered to be part of the more general group called browning inhibitors instead of PPO inhibitors
(Vamos-Vigyazo, 1981). In many cases, it is not easy to distinguish between the two types of inhibitors since some act on both the enzyme and the substrate or product, such as sulphur dioxide (Embs and
Markakis, 1965) and some thiol compounds (Pierpoint, 1966).
Reducing agents, including ascorbic acid, act by reducing the formed quinones back to the £-diphenols. These compounds are consumed in the process of inhibition, through their own oxidation, and thus provide only temporary protection against browning, unless they are present in high concentrations (Vamos-Vigyazo, 1981). Quinone couplers include cysteine, sulphur dioxide, glutathione, dithiothreitol, and benzenesulphinic acid. These compounds complex 35 with the quinones and remove them from further participation in secondary browning reactions (Mathew and Parpia, 1971).
Among the chemicals used to treat non-enzymatic and enzymatic browning, sulphur dioxide (SCL) and sulphite salts are very important. SCL is used extensively in the fruit processing industry
(Mathew and Parpia, 1971). Several reviews have indicated that SCL acts by reducing quinones back to o-diphenols (Mathew. and Parpia,
1971; Mayer and Harel, 1979; Vamos-Vigyazo, 1981). However, using radioactive sulphite and paper chromatography, Embs and Markakis
(1965) were able to show that the sulphite was consumed, but not oxidized to sulphate, during the reaction (Haisman, 1972). The appearance of a new UV absorption peak was also reported. Their results indicated that SCL was not acting as a reducing agent, but was forming a colorless quinone-sulphite complex. Embs and Markakis
(1965) also noticed that when PPO was preincubated with SCL its enzymatic activity was gradually lost; suggesting that SCL is also acting directly on the enzyme. Unfortunately, the actual mode of inhibition by sulphur dioxide is not thoroughly understood. Recent work in our laboratory has shown that SCL does indeed inhibits the enzyme molecule in an irreversible manner (Sayavedra, 1982).
Compounds which complex with the enzyme's prosthetic group are copper chelators and include ethylenediaminetetra acetic acid (EDTA), cyanide, diethyldithiocarbamate (DIECA), thiourea, azide, and phenylthiourea. Their inhibitory ability depends upon the stability of the complex formed between the copper and the chelator versus the stability of the complex between the copper and the enzyme (Whitaker, 36 1972). EDTA showed different degrees of inhibition of PPO. EDTA strongly inhibited the PPO from bananas (Palmer, 1964) and grapes
(Cash et al_., 1976). Luh and Phithakpol (1972) and Wong et aJL (1971) reported low inhibition of peach PPO by EDTA. PPO isolated from pears was shown to be very sensitive to DIECA (Tate et ajk, 1964; Halim and Montgomery, 1978).
Another group of PPO inhibitors are compounds which affect the substrate binding site of PPO. Some common competitive inhibitors include benzoic acid (Webb, 1966; Duckworth and Coleman, 1970; Rivas and Whitaker, 1973), 2,3-naphthalene diol (Mayer et aj_., 1964); resorcinol (Knapp, 1965), and 4-chlorophenol (Robb et aj_., 1966).
£-Coumaric acid was named as a simple, linear noncompetitive inhibitor of pear PPO (Rivas and Whitaker, 1973) and phenylhydrazine as an irreversible inhibitor of catechol oxidase from grapes (Lerner et al.,
1974). The soluble polymer polyvinylpyrrolidone (PVP) has also been cited as a competitive inhibitor of PPO from higher plants (Walker and
McCallion, 1980). Several natural inhibitors, some phenolic compounds and others which are undefined at present, have also been found which inhibit the action of PPO (Macrae and Duggleby, 1968; Walker, 1970;
Bull and Carter, 1973; Walker and Wilson, 1975).
The specifics on the work that has been reported for pear fruit
PPO (Walker, 1964; Tate et al_., 1964; Rivas and Whitaker, 1973; Halim and Montgomery, 1978; Smith, 1980; Vamos-Vigyazo and Nadudvari-Markus,
1982) has been reviewed. The above studies were complete in their presentation of the characterization of pear PPO in terms of pH optima, temperature optima and stability, substrate specificity and 37
affinity, as well as behavior toward several inhibitors. Smith (1980)
also proposed a more efficient method for enzyme extraction using
phenolic adsorbents, that prevented browning of the enzyme extract,
and should help in isolating PPO from the interfering phenols. It is with this basis that the following dissertation was undertaken, with
the purpose of isolating a pure enzyme preparation and characterizing
d'Anjou pear PPO with respect to molecular weight, subunit structure,
and amino acid composition. 38 MATERIALS AND METHODS
Pears
d'Anjou pears (Pyrus communis L.) used in this study were grown in the orchards at the Mid-Columbia Experiment Station, Hood River,
Oregon during the 1981 season, picked in September, and placed in -1°C storage for 7 months. Upon removal from storage the pears were green, hard, and in good condition. They were quartered, cored, frozen in liquid nitrogen, sealed under vacuum in cryovac bags, and stored at
-40oC until used.
Polyphenoloxidase Assay
Polarographic measurements of enzyme activity were performed using the Yellow Springs Instruments (YSI) model 53 Biological Oxygen monitor equipped with a Clark electrode, following the recommendations given in the manufacturer's instructions and by Cooper (1977). The reaction chamber was maintained at 250C by a Lauda K2/R (Brinkmann
Instruments) constant temperature circulating water bath.
Standardization of the instrument to 100% was with 3 ml of air- saturated distilled water. Oxygen content of air-saturated water at
250C is 258 nmoles oxygen/ml (Cooper, 1977).
To determine PPO activity 2.3 ml of 0.1 M citrate - 0.2 M sodium phosphate buffer, pH 5.0, and 0.2 ml of enzyme extract were added to the reaction chamber and allowed to equilibrate to 250C for 4 min. To initiate the reaction 0.5 ml of 0.1 M 4-methyl catechol in 0.01 M citrate-0.02 M sodium phosphate buffer, pH 3.6, was added. A Linear 39 recorder, Model 585, was used to monitor the oxygen consumption over time with the linear portion of the curve used to calculate the initial rate of oxygen consumption. Enzyme activity was expressed as nmoles of oxygen/min/ml of enzyme extract under the conditions described above. When it was necessary to dilute the enzyme extract
0.1 M citrate - 0.2 M sodium phosphate, pH 5.0, buffer was used.
Between enzyme assays it was necessary to rinse the reaction cell with
IN hydrochloric acid followed by several washings of distilled water to prevent the adhesion of PPO onto the surface of the reaction vessel.
Protein Determination
Protein content was determined by the dye binding method of
Bradford (1976) using the Bio-Rad dye reagent (Bio-Rad, 1977).
Standard procedure for 20-140 pg protein was used on the crude enzyme extracts. A microassay method for 1-20 yg protein was used with the purified extracts. Protein solutions of bovine serum albumin (Sigma) were made daily in water or the appropriate buffer and used to prepare the standard curves. Absorbance at 595 nm was determined in a Perkin
Elmer spectrophotometer, model 550, against a reference cuvette containing buffer plus the dye reagent. Protein content was expressed as yg/ml of enzyme extract.
Samples containing 1% or more of Triton X-100 cannot be assayed by the Bradford method due to severe interference (Bio-Rad, 1977); therefore, it was necessary to remove the Triton X-100 from the crude extracts before protein determination. This was accomplished by the 40 procedure of Holloway (1973) using Bio-Beads SM-2 (Bio-Rad
Laboratories. Moist copolymer beads (3.0 g) were added to a 5 ml sample of the crude extracts containing 2% Triton X-100. The samples were stirred at 40C for 120 min and filtered through glass wool to remove the beads. A spectrophotometric scan from 400-200 nm, before and after the batch procedure, revealed that the 275 nm peak for
Triton X-100 had been removed. No loss in enzyme activity was observed.
Enzyme Extraction Procedure
Cleaning of anion exchange resin
AG 2-X8, (200-400 mesh, Bio-Rad Laboratories), was prepared for use following the method of Cooper (1977) and Smith (1980). The resin was suspended in distilled water several times to remove the fines.
After rinsing twice with 95% ethanol, twice with 2.0 N hydrochloric acid, and boiled in 2.0 N hydrochloric acid for 5 min, the resin was rinsed three times with 2.0 N acetic acid, three times with 1.0 M sodium acetate, and once with 0.1 M acetic acid. This was followed by washing with distilled water until the pH of the effluent was the same as the water being added. The resin was stored hydrated at 40C.
The day before it was going to be used the resin was poured into a Buchner funnel lined with Whatman No.l filter paper, washed twice under vacuum with distilled water and left to drain to a constant weight. This resin contained 63% water, which had to be considered when weighing the resin. The AG 2-X8 was equilibrated overnight in the extraction buffer, before use. 41
Extraction procedure
The PPO was initially extracted from the pears by a modification of the procedure developed by Smith (1980) and Wesche-Ebeling (1980).
Frozen pear quarters which were chilled in liquid nitrogen until brittle, broken into smaller pieces and ground under liquid nitrogen in a stainless steel Waring Blendor until a fine powder resulted. The pear nitrogen powder was poured into a Dewar flask and stored in liquid nitrogen until used. The extraction buffer consisted of 0.1 M citrate- 0.2 M sodium phosphate, pH 5.6, containing 2% Triton X-100 v/v and 5 mM phenylmethylsulfonyl fluoride (PMSF). Either PMSF or trasylol was added throughout the entire isolation and purification procedure on the recommendation of Flurkey and Jen (1980c). The extraction procedure is outlined in the first section of Figure 1 on the overall purification scheme. Pear nitrogen powder (12.5g) was combined with 33.8 g AG 2-X8 equilibrated in 100 ml of the extraction buffer. The solution was stirred in the cold room (40C) for 60 min, filtered through glass wool, and centrifuged at 7,700 xG for 15 min at
20C in a Sorvall RC-5 centrifuge. The supernatant was collected and
0.33 ml of trasylol (Aprotinin, Sigma) per 100 ml of supernatant was added.
Pigment extraction
The crude enzyme extract, after centrifugation, had a slight green tint, presumably from the skin of the green pears. To investigate the source of this color an enzyme extract was prepared 42 Pear Nitrogen Powder AG 2-X8 Phenolic Adsorbent 2% Triton X-100 Protease Inhibitors: PMSF, Aprotinin
Stir, filter, centrifuge
Bio-gel P-6 Column
Adjust eluent to 1 M ammonium sulfate, pH 6.5, filter
Phenyl Sepharose CL-4B Elute with decreasing concentrations of equilibration buffer
I PS 1 PS 2 PS 3 most active fractions dialyze and concentrate I DEAE-Cellulose Chromatography Elute with increasing concentration of phosphate
r T ] DEAE 1 DEAE 2 DEAE 3 I I HA column HA column Elute with increasing concentration of phosphate, KC1, and (NH4)2S04 I I HA 1 HA 2
Figure 1. Purification outline 43 with 5 g of pear nitrogen powder, 13.5 g of AG 2-X8, and 40 ml of extraction buffer in the same manner as previously described. After centrifugation, 30 ml of supernatant was extracted with 30 ml of ethyl ether in a separatory funnel and allowed to settle. Two layers formed, with the color in the ether layer. The ether extract was collected and concentrated to 1 ml under nitrogen.
An absorption scan was performed in the range from 700 nm to 300 nm with a Perkin Elmer recording spectrophotometer, at a scan speed of
120 nm/min and a chart speed of 6 cm/min. The ether extract was read against a reference of ethyl ether.
Purification Procedure
d'Anjou pear polyphenoloxidase was extracted and purified according to the procedure outlined in Figure 1. A major portion of the purification was designed after the work by Flurkey (1979),
Flurkey and Jen (1980), Flurkey and Jen (1978), Wissemann (1980), and
Wissemann and Lee (1980b). Glass distilled water was used throughout the following purification procedure.
Bio-gel P-6 column chromatography
A Bio-gel P-6 (Bio-Rad Laboratories) desalting column with a 20 ml bed volume was prepared and equilibrated with 1 mM citrate - 2 mM
sodium phosphate, pH 5.0, buffer. The combined supernatants of two
100 ml crude enzyme extracts were passed through the column and collected in the void volume. 44
Phenyl Sepharose CL-4B column chromatography
Preswollen Phenyl Sepharose CL-4B resin (Pharmacia Fine
Chemicals) was degassed and equilibrated in 4 volumes of deaerated equilibration buffer (EB, 1 M ammonium sulfate, 1 M potassium chloride, and 0.05 M sodium phosphate, at pH 6.5, and stored overnight in a refrigerator. A 30 cm x 1.0 cm SR 10/50 column (Pharmacia) was packed at room temperature and rinsed with several bed volumes of EB.
To the enzyme extract collected from the Bio-gel P-6 column 0.33 ml of
Aprotinin was added to 100 ml of extract. This solution was made to 1
M ammonium sulfate by adding solid crystals of the salt, adjusted to pH 6.5 with 10 N sodium hydroxide, and filtered through Whatman No. 1 filter paper.
The filtrate was applied onto the hydrophobic column and the proteins bound to the resin were eluted by decreasing the EB concentration in a stepwise manner: (A) 100 ml EB, (B) 60 ml 0.8 EB
(48 ml EB + 12 ml water), (C) 60 ml 0.6 EB (36 ml EB + 24 ml water),
(D) 60 ml 0.4 EB (24 ml EB + 36 ml water), (E) 60 ml 0.2 EB (12 ml EB
+ 48 ml water), (F) 60 ml 0.05 EB (3 ml EB + 57 ml water), (G) 60 ml
50% ethylene glycol, and (H) water. The column was developed at room temperature. Flow rate was regulated to 60 ml/hr with a Laboratory
Data Control, model 711-31, pump (Milton Roy Co.) and 6 ml fractions were collected by an Instrumentations Specialties Co. (ISC0) model
400, Volumeter and ISC0 model 270 and 272 fraction collector. Each fraction was assayed for PP0 activity and protein content was determined by measuring absorbancy at 280 nm against a reference of water. 45 The most active fractions were combined and dialyzed overnight against 2 changes of 5 mM sodium phosphate, pH 7.0, buffer at 40C.
The dialysis membrane was Spectrapor 1 with a molecular weight cutoff of 6,000-8,000. The tubing was rinsed and soaked in water for 4 hr before use. After dialysis the samples in the dialysis bags were concentrated to 20 ml with Aquacide II A (Calbiochem).
The Phenyl Sepahrose resin was regenerated according to the manufacturer's recommendations (Pharmacia, 1976). To avoid passing organic solvents through the pump, the resin was removed from the column and placed in a filter lined with Whatman No. 1 filter paper.
The gel was then washed with one bed volume of water, 95% ethanol, n-butanol (twice), 95% ethanol, and finally with water until the n-butanol odor was removed.
DEAE-Cellulose column chromatography
DEAE-cellulose 32 in microgranular form was purchased from
Whatman Ltd., England. The resin was prepared according to manufacturer's specifications for precycling, degassing, equilibration, and removal of fines and was equilibrated by repeated suspensions in 10 mM and, finally, 5 mM sodium phosphate buffers at pH
7.0. A 27 ml bed volume (1.0 cm x 35 cm SR 10/50) column was packed at room temperature and rinsed with several washings of 5 mM sodium phosphate, pH 7.0, buffer. Aprotinin (0.33 ml/100 ml of extract) was added to the dialyzed and concentrated sample from the hydrophobic column. This solution was applied onto the ion exchange column and washed with 40 ml of 5mM 46 sodium phosphate, pH 7.0, buffer. Proteins were eluted by a 200 ml linear gradient (5 to 100 mM sodium phosphate, pH 7.0); followed by a
100 ml linear gradient from 100 to 200 mM sodium phosphate. Fifty milliliters of 400 mM sodium phosphate, pH 7.0, buffer was used to wash out any remaining proteins on the column. A flow rate of 60 ml/hr was used to develop the column at room temperature and 6 ml fractions were collected. Each fraction was assayed for PPO activity and 280 nm absorbing material.
Three peaks of PPO activity were eluted from the ion exchange column and labeled DEAE 1, 2, and 3. The fractions from each peak were pooled and the three samples were stored at 40C until used.
Hydroxylapatite column chromatography
Bio-gel HTP hydroxylapatite (HA, Bio-Rad Laboratories) was prepared by swirling 1 part dry powder with 6 parts of starting buffer
(1 mM sodium phosphate, pH 7.0). After settling for 10 min the fines were removed by decanting to the settled bed. Six parts of starting buffer were added and the suspension was left in the refrigerator overnight. After a final decanting the material was resuspended for column pouring.
A 30 cm x 1 cm SR 10/50 column was packed by gravity at room temperature and equilibrated with 1 mM sodium phosphate, pH 7.0, buffer. Samples DEAE 1 and DEAE 2 were each run on the HA column separately. After sample application the column was developed with:
(A) 40 ml of starting buffer (1 mM sodium phosphate, pH 7.0), (B) 100 ml of a linear gradient from 1 mM sodium phosphate to 50 mM sodium 47 phosphate, 1 M potassium chloride, pH 7.0, (C) 100 ml of a linear gradient from 50 mM sodium phosphate, 1 M potassium chloride to 50 mM sodium phosphate, 1 M potassium chloride, 1 M ammonium sulfate, pH
7.0, (D) 50 ml 400 mM sodium phosphate, pH 7.0.
The column was run at room temperature at a flow rate of 50 ml/hr and 6 ml fractions were collected. Each fraction was assayed for PP0 activity and 280 nm absorbing material. Fractions containing enzyme activity were combined and stored at 40C until further use.
Effect of DMSO on Enzyme Activity
The effect of dimethyl sulfoxide, DMSO, on enzyme activity was investigated using the most active peak from the Phenyl Sepharose column (PS 1). Five ml solutions were prepared containing 1 ml of the enzyme fraction and from 1 to 80% DMSO made to volume with 0.1 M citrate - 0.2 M sodium phosphate buffer, pH 5.0. A 0.2 ml aliquot of this solution was assayed for enzyme activity in the usual manner.
The control contained 1 ml of enzyme extract plus 4 ml of the citrate-phosphate buffer.
Molecular Weight Determination by Gel Filtration
Sephacryl S-300 Superfine (Pharmacia Fine Chemicals) with a wet bead diameter range of 40-105 microns was purchased pre-swollen and prepared according to the manufacturer's instructions. Important aspects on the properties and use of Sephacryl columns were also obtained from the papers by Johansson and Lundgren (1979) and Hoff and
Easterday (1978). 48
The resin was packed at 2 ml/min and equilibrated in a Pharmacia
K 26/100 column to a height of 72 cm giving a total bed volume (Vt) of
385 err. The elution buffer consisted of 0.1 M sodium phosphate, 0.1
M sodium chloride, and 0.01% Merthiolate at pH 7.0. Blue dextran 2000
(2 mg/ml) was used to establish the void volume (V ), which equalled
139 ml. Proteins of known molecular weight were purchased from Sigma
Chemical Company to calibrate the column. They consisted of: thyroglobulin (669,000), glucose oxidase (186,000), bovine serum albumin (67,000), ovalbumin (43,000), chymotrypsinogen (25,000), and ribonuclease (13,700). Two to 3 mg/ml were applied to the column.
Tyrosine (MW 181) was used to estimate the elution time of a molecule that was totally included in the gel. Flow rate was carefully regulated to 0.90 ml/min with a pump. The absorbance at 280 nm was monitored with a Beckman DB spectrophometer equipped with a flow through quartz cuvette and attached to a Perkin Elmer Model 500 recorder. The chart speed was set at 10 cm/hr. Elution volumes (V ) were determined for all of the standards.
Purified fractions DEAE 3, DEAE 2, and HA 1, were applied to the column in 2 ml volumes with a Pharmacia 25 ml applicator and developed in a manner identical to the standards. Elution volume of PP0 was determined by assaying 3 ml fractions for enzyme activity. It was necessary to assay the fractions immediately after elution as the sodium chloride appeared to have a slight inhibitory effect on pear
PP0. The inclusion of 0.1 M sodium chloride was necessary, however, to ensure that there was no adsorption of proteins onto the gel matrix 49 (Hoff and Easterday, 1978). Average enzyme activity recovered was 68% of the total applied.
The molecular weight of PPO was determined by calculating the partition coefficient, K , for each of the standard proteins as av described by Andrews (1970):
K e 0 Kav =
o o
A plot of K vs. log MW was used as the standard curve and the molecular weight of PPO fractions was calculated from K values. 3 av
Electrophoresis
Polyacrylamide slab gel electrophoresis
Polyacrylamide gel electrophoresis was performed under the discontinuous nondenaturing conditions described by Davis (1964) and
Ornstein (1964), in a Bio-Rad Model Protean 16 cm (Bio-Rad
Laboratories) slab gel unit. The gel slabs were 0.15 cm thick and 14 cm wide and consisted of 12 cm of a 7.0% polyacrylamide separation gel and 2 cm of a 4.5% large pore stacking gel. Fifteen sample wells were formed with a well forming comb used during the casting of the stacking gel. All solutions used were filtered through a 1.2 v Millipore filter. To 1 ml of enzyme extract was added 0.25 ml of a
40% sucrose solution containing a very small amount of bromophenol blue. One hundred micro!iters of this solution was injected per slot in the slab gel. 5n
The electrode buffer, (0.025 M Tris - 0.16 M glycine) was adjusted to pH 8.3 with IN hydrochloric acid and used to fill the upper and lower reservoirs. Slabs were cooled throughout the run by circulating cold water from a Lauda K2/R waterbath, through the cooling channels of the electrophoretic cell. A Buchler 3-1500 constant power supply (Buchler Instrument, Inc.) was used to maintain
15 mA per slab and run until the tracking dye migrated to within 1 to
2 cm from the bottom (about 5 to 6 hr).
Detection of PPO isozymes
The procedure to detect PPO activity in the electrophoresis gel slabs was similar to that described by Smith (1980) and Benjamin and
Montgomery (1973). The slabs were immersed in 15 mM catechol in 0.1 M citrate - 0.2 M sodium phosphate buffer, pH 5.0, containing 0.05% j)-phenylenediamine for about 1 hr. To stabilize the color and decrease the non-specific background color, the slabs were rinsed in 1 mM ascorbic acid for 5 min, soaked in water overnight and stored in
30% ethyl alcohol.
Protein stain
Slab gels were stained for protein by a modification of the silver stain procedure of Wray et a_l_. (1981) developed by Einerson
(1982). Distilled, deionized water was used to prepare all of the solutions. The slab gels were immersed in 10% TCA for 30 min, rinsed
in water for 20 min, and placed in 50% methanol, 0.05% formaldehyde overnight (ca. 12 hr). The gels were then allowed to swell in water 51 for 30 min, with one change of the water after 15 min, and transferred to a solution containing 50% methanol and 0.05% formaldehyde for at least 2 hr.
The gel was placed in water for 10 min before the staining solution was added. To prepare the silver staining solution 4 g silver nitrate was dissolved in 20 ml water and 105 ml 0.36% sodium hydroxide plus 7 ml concentrated ammonium hydroxide were mixed. The silver nitrate solution was added dropwise to the solution of bases with rapid mixing and the total was made up to 500 ml with water.
This staining solution had to be used within 5 min of preparation.
The gel was stained for 20 min with gentle agitation on a mechanical shaker. After staining, the gel was rinsed with water and then agitated in water for 5 min. The water was removed and the gel developed by the addition of freshly prepared developer solution that consisted of 2.5 ml 1% citric acid and 0.25 ml formaldehyde in a total volume of 500 ml. Development was usually complete in 15 to 30 min, after which the gel was returned to 50% methanol.
To remove unwanted background the gels were destained by immersion in Kodak Rapid Fix (film strength) containing 5% methanol.
When the desired amount of background had been removed, the Rapid Fix was removed with a 5 min water wash, a 20 min wash in Kodak Hypo
Cleaning Agent (film strength), another 5 min water wash, and finally the gel was returned to 50% methanol. 52 Molecular weight determination by LDS electrophoresis
The molecular weight of PPO was analyzed using lithium dodecyl sulfate (LDS) in both 10% acrylamide slabs and in 7.5 to 15% linear acrylamide gradient slabs. The method of Laemmli (1970) was used to prepare the 10% acrylamide slab gels with the exception that sodium dodecyl sulfate (SDS), which is insoluble below 10oC (Cooper, 1977), was replaced with LDS (BDH Chemicals). A 12 cm x 0.15 cm x 14 cm 10% separation gel was used with a 2 cm x 0.15 cm x 14 cm 4.5% stacking gel. Both the separation and stacking gels contained 0.1% LDS. The upper reservoir buffer (pH 8.3) consisted of 0.021 M Tris-HCl, 0.16 M glycine, and 0.1% LDS. The lower reservoir buffer was the same except there was no LDS added.
Bio-Rad low molecular weight standards (Bio-Rad Laboratories) were used as molecular weight markers. They consisted of phosphorylase B (92,500), bovine serum albumin (66,200), ovalbumin
(45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor
(21,500), and lysozyme (14,400). Five microliters of the standards were diluted with 295 yl of a solution conaining 0.0625 M Tris-HCl (pH
6.8), 2% LDS, 10% glycerol, 0.001% bromophenol blue, and 5%
3-mercaptoethanol.
This was incubated at 100oC for 3 min before 20 yl was applied in a sample well on the slab gel. To the purified enzyme extracts 2%
LDS, 10% glycerol, and 5% 3-mercaptoethanol was added. The samples were then placed in boiling water for 3 min and applied in 100 yl aliquots onto the gel slabs. 53 Linear acrylamide gradient slab gels, 12 cm x 0.15 cm x 14 cm, consisting of 7.5 to 15% acrylamide (Einerson, 1982) were also used to determine the molecular weight of pear PPO. A 5 to 17.5% sucrose gradient was used to stabilize the acrylamide. The gels were poured with a Buchler gradient former and a Buchler peristalic pump. The light acrylamide solution consisted of 5% sucrose, 0.375 M Tris-HCl, pH 8.8, 7.5% acrylamide, 0.2% bisacrylamide, 0.1% ammonium persulfate,
0.07% TEMED, and water. The heavy acrylamide solution contained 17.5% acrylamide, 0.4% bisacrylamide, 0.1% ammonium persulfate, 0.07% TEMED, and water. The gradients were poured at a flow rate of 8 ml/min in the cold room and overlayed with water saturated butanol. After polymerization the top of the gel was rinsed with water and a 2 cm x
0.15 cm x 14 cm 4.5% stacking gel (0.125 M Tris - HC1, pH 6.8, 4.5% acrylamide, 0.12% bisacrylamide, 0.1% ammonium persulfate, 0.05%
TEMED) was formed.
The upper and lower reservoir buffers, the molecular weight standards, and the samples were prepared as previously described for a
10% acrylamide homogeneous LDS gel. Gels were electrophoresed at 12 mA per gel constant amperage, at 40C until the bromophenol blue tracking dye was 2 cm from the bottom of the gel (about 17 to 19 hr).
The silver stain procedure, as previously described, was used to visualize the protein bands. Measurements of relative mobility were determined by measuring the distances traveled by each protein divided by the dye front. The log MW vs. relative mobility were plotted and the molecular weights of the unknown samples were calculated from the slope and y intercept. 54
Isoelectric Focusing
Gel tube isoelectric focusing was performed under nondenaturing conditions by a modification of the procedures used by Vlasuk and Walz
(1980), Wrigley (1968), and Hoyle (1977). Important aspects on the design and use of the isoelectric focusing technique were also obtained from the papers by An der Lan and Chrambach (1981), Gaal et aT_. (1980), and Catsimpoolas (1979). Gel tubes 140 mm x 3 mm were precoated with Sigmacote (Sigma
Chemical Company), dried and a 10 mm parafilm plug was inserted in the bottom of the tube. The gels were filled to 10 mm from the top with
5% acrylamide, 0.27% bis-acrylamide, containing 1.5% pH 3 to 10 ampholytes (Bio-Rad Laboratories), 0.5% pH 3 to 5 ampholytes, 0.07%
TEMED and 0.025% ammonium persulfate. After 1 hr of polymerization the water formed on top was removed and replaced with 20 Ml of ampholyte buffer (1.5% pH 3 to 10 ampholytes, 0.5% pH 3 to 5 ampholytes, and 10% glycerol), overlayed with water, and left for 2 hr. The parafilm plug was removed from the bottom of the tube, the well filled with ampholyte buffer, and the end of the tube covered with dialysis tubing secured with a small rubber band, taking care to exclude air bubbles.
The gel tubes were placed in a Bio-Rad Model 155 gel electrophoresis cell, and the ampholyte buffer replaced with a fresh
20 pi of ampholyte buffer. The upper reservoir, anode, was filled with 50 mM phosphoric acid and the bottom chamber (cathode) with degassed (for 30 min) 40 mM sodium hydroxide. The gels were 55 prefocused at 200 volts for 15 min, 300 volts for 30 min, and 400 volts for 30 min. The upper reservoir was emptied and the ampholyte buffer was removed from the surface of the gels.
Purified enzyme extracts were prepared for electrofocusing by the addition of 20% glycerol, 1.5% pH 3 to 10 ampholytes, and 0.5% pH 3 to
5 ampholytes. One hundred micro!iters of this solution were placed on top of each gel and overlayed with 10 yl of sample overlay solution
(10% glycerol, 0.75% pH 3 to 10 ampholytes, and 0.25% pH 3 to 5 ampholytes). The upper buffer chamber was refilled, the gels were focused at 200 volts for 15 hr followed by 500 volts for 2 hr at constant voltage. After isoelectric focusing the gels were stained for PP0 activity as previously described.
The pH gradient established in the isoelectric focusing gels was measured by cutting a representative gel into 5 mm segments, 2 ml of deionized, degassed water added, and the pH measured after several hours. Alternatively, the pH of the gel was measured directly with a
Ml-410 Micro-Combination Glass Contact pH Probe (Microelectrodes,
Inc., Londonberry, NH).
Ami no Acid Analysis
Amino acid analysis were determined by Becker and Francis in the
Dept. of Biochemistry at Oregon State University. Fractions HA 1, HA
2, and DEAE 3 were dialyzed against two changes of distilled, deionized water for 24 hr at 0oC, lypholized, and stored at -40oC until used. Each sample contained 4 yg of protein. Before analysis 56 the samples were hydrolyzed in 6 N hydrochloric acid at 110oC for 22 hr in evacuated sealed tubes.
The fluorimetric procedure of Roth and Hampai (1973) was employed to determine the amino acids. A Durrum DC-4A anion exchange resin
(Durrum Chemical Corp.) was used in a 0.32 X 30 cm column and the amino acids were eluted in a stepwise manner with the Durrum
Fempto-Buffer System I (Durrum Chemical Corp.) which employs four sodium citrate buffers. The system utilizes an £-phthalaldehyde solution to give an intense blue fluorescence with all a-amino acids.
Fluorescence was detected and measured by a Gil son fluorometer. A spectrophysics Autolab System IV B integration system was used for the recording of the data. An advantage of fluorimetric detection is that the signal is a linear function of the concentration, which permits easy evaluation of peaks after linear recording (Roth and Hampai,
1973). The total run time was 96 min.
Tryptophan was destroyed during the hydrochloric acid hydrolysis of the protein and was not determined by an alternative method. The hydrochloric acid also reacts with asparagine and glutamine to release free ammonia and the corresponding acid. Thus, the values for asparagine and aspartic acid, as well as glutamine and glutamic acid are reported as the acids. Finally, proline does not react with o-phthalaldehyde sothe proline content of the samples is unknown. 57
Protein-Phenolic Interactions
Two separate reaction conditions were chosen to test the association of the pure enzyme system with phenolics, patterned after the work of Sato (1976) and Smith (1980). The phenolics used were
2,3-dihydroxybenzaldehyde (DHBA) and chlorogenic acid. In the first set the pH of the reaction was 7.0 and the enzyme fractions DEAE 3 and
DEAE 2 were used. Five mM of DHBA and 0.5 mM DHBA were mixed with the fraction and allowed to react for 20 hr at 40C. Three mM of chlorogenic acid was mixed with another sample of DEAE 2 and 3 and allowed to react at 40C for 20 hr and at 250C for 30 min. After the specified time period the samples were electrophoresised in a 7% acrylamide slab gel by the Davis-Ornstein system and stained for PPO activity and protein as previously described.
The second set of reactions were allowed to proceed at pH 5.6.
Separate aliquots of enzyme fractions DEAE 2 and DEAE 3 were mixed with 3 mM chlorogenic acid and 5 mM DHBA at 40C for 20 hr. The samples were then electrophoresised as described above. 58
RESULTS
Purification of Polyphenol Oxidase
The majority of this work was concerned with the purification of d'Anjou pear PPO. An outline of the procedure that was developed is illustrated in Figure 1. The phenolic adsorbent AG 2-X8 (1.0 g dry weight/g pear tissue) was used in the extraction procedure to remove the endogenous pear phenolics. Initial work on the purification procedure was conducted without the use of 2% Triton X-100 during extraction. Without Triton X-100 the supernatant of the crude extract was colorless and clear. The detergent was added because 30% or more enzyme activity was extracted from the pears. However, Triton X-100 also extracted the green pigment from pear skins. To determine the nature of this faint green tint an ether extract of the crude PPO sample was prepared and concentrated under nitrogen. An absorption scan from 700 to 300 nm of this extract is shown in Figure 2. Peaks can be seen at 660, 470, and 430 nm. The extract was not scanned below 300nm due to the large absorbance at 275nm from the Triton
X-100.
The crude enzyme extract was passed through a Bio-gel P-6 desalting column and the enzyme was collected in the void volume.
Column yields of 85-100% were obtained with a slight increase in the
purification.
The remainder of the column chromatography steps were initially
run in a cold room at 40C with unsuccessful results. The effect of
temperature on the elution of PPO from three columns is shown in 59
Figure 2. Absorbance scan from 700 to 300 nm of an ethyl ether extraction of the crude enzyme preparation. '00 600 500 400 300 WAVELENGTH (nm) Figure 2
ON O 61
Figure 3. Effect of temperature on the elution of pear PPO during column chromatography.
Phenyl Sepharose Elutants: A. Equilibration Buffer (EB) B. 0.8 EB C. 0.6 EB D. 0.4 EB E. 0.2 EB F. 0.05 EB G. 50% ethylene glycol
Hydroxylapatite Elutants: A. Linear gradient ( ) from ImM sodium phosphate to 50mM sodium phosphate, 1M potassium chloride.
DEAE Column Elutants: A. Linear gradient ( )from 5mM to lOOmM sodium phosphate.
B. Linear gradient from lOOmM to 200mM sodium phosphate. 62
Room Temperature 40C
12,500 Phenyl Sephorose
7500- no/ eluted E r | 2500 - A B C Dj Vk 6 A B C D E F G J I r, T.r, T ) i 40 80 120 40 80 120 c 1 r ^ 10,000 Hydroxylopatite o CO I 6000 c
i i i 1 ' ■ ■ 30 45 15 30 45
DEAE Column
2001-
1000-
■ 10 30 50 70 0 10 20 30 FRACTION NUMBER Figure 3 63
Figure 3. Several runs were tried with the Phenyl Sepharose column at
40C using different pHs, gradients, and elution buffers but the enzyme was bound too tightly to the hydrophobic media and was never eluted.
The results from the hydroxylapatite (HA) and DEAE columns at 40C were the reverse. As can be seen in Figure 3, the PPO came off in the void volume of the HA column and was not well separated on the ion-exchange column. Only after the columns were run at room temperature were sharp peaks and good resolution obtained. Also, with the Phenyl
Sepharose column without Triton X-100 in the initial extract, the elution of the PPO was erratic and difficult to predict.
The elution profile from the hydrophobic column^ run at room temperature and with Triton X-100, is illustrated in Figure 4. The proteinaceous material was monitored at 280 nm and the majority of the
PPO activity corresponded to one protein peak. This first peak was labeled PS 1. The two smaller peaks of activity, following the major peak, were labeled PS 2 and PS 3, respectively. PPO in PS 1 was released from the column following the addition of elutant D, which was a 0.4 concentration of the equilibration buffer. The remainder of the 280 nm absorbing material was eluted off the column in large amounts before and after the PPO. There was a 24-fold increase in the specific activity in PS 1 over the crude extract. Total column yield was over 100% recovery of the total activity units applied to the column.
Chromatography of PS 1 on DEAE-cellulose columns at pH 7.0 resulted in the separation of three major chromatographic PPO forms
(Figure 5). These isozymes were labeled DEAE 1, 2, and 3 and eluted 64
Figure 4. Phenyl Sepharose CL-4B column chromatography of pear PPO.
Elutants: A. Equilibration Buffer (EB) B. 0.8 EB C. 0.6 EB D. 0.4 EB E. 0.2 EB F. 0.05 EB G. 50% ethylene glycol H. water
PPO Peaks: PS 1 fractions No. 66-72 PS 2 fractions No. 76-79 PS 3 fractions No. 80-82
The equilibration buffer consisted of 0.05 M sodium phosphate, 1 M potassium chloride, 1 M ammonium sulfate, pH 6.5. KOOOp -l0.7
JU 12,000- 0.6 I l l « F 10,000 0.5 E c O Cvi 00 O C\J 8000 0.4 UJ o u ■z. < ^ 6000 A 0.3 ccCD o en > \ CD 4000 - / 0.2 < O < O Q. 2000 0.1 CL c
0 J l_ 0 0 20 40 60 80 100 120 FRACTION NUMBER Figure 4 66
Figure 5. DEAE-Cellulose column chromatography of pear PPO.
Elutants: A. 5mM sodium phosphate, pH 7.0 B. Linear gradient from 5mM to lOOmM sodium phosphate C. Linear gradient from lOOmM to 200mM sodium phosphate
PPO Peaks: DEAE 1 fractions No. 23-26 DEAE 2 fractions No. 27-29 DEAE 3 fractions No. 43-46 PPO ACTIVITY (nmoles 02/mn/rc\\) C ro OJ 4^ m Cfi o o o o O 0 o O - 8 § 8 8 o O u ^ 1' 1 i i 1 —1
•M ^ ^ O
*.'*"'*■- ^ • ^ ^ • **-N O • J • =— 70 < 'T* ""-^■ > *«-. J ' "^^ O w s* ^. ''--•"" 0 ,—' H f *.>. •" Q ( r \ Z? \ t
5° ^^ ^ • • CD ^"'^v • / • • XC o"» «
-->
(D _ *■ ^^ ■••«-o O - ^-' *'
->l O 1 1 1 t 1 | t-^ o o o o o P o "o o o (D o ro OJ ^ Ul en ABSORBANCE 280nm
i i i i 1 i < 8 o o8 o DWHQD UATF rr»N irrMTRATinM
19 68 at 50, 62, and 118 mM phosphate concentration, respectively. The total column yield was 95%. The resolution of the three forms was good, with DEAE 3 appearing homogeneous by slab gel electrophoresis, as will be discussed later. The other two PPO forms (DEAE 1 and 2) were each applied onto a HA column and the elution profiles are illustrated in Figure 6. PPO from DEAE 1 was resolved from the other
280 absorbing material and eluted in one protein peak at 36 mM phosphate concentration (pH 7.0) and 0.72 M potassium chloride (Figure
6a). The recovery of enzyme activity from this column was approximately 90% with 5 fold purification over the DEAE column. When
PPO activity of DEAE 2 was subjected to HA chromatography (Figure 6b) elution was at a higher phosphate and salt concentration (50 mM phosphate, 1 M potassium chloride, 0.08 M ammonium sulfate) than DEAE ++ 1, indicating that PPO of DEAE 2 had a greater affinity for the Ca groups in the HA resin.
Table 2 is a summary of the purification of d'Anjou pear PPO.
An overall purification fold of 148 was achieved for HA 1 and 103 fold for HA 2. DEAE 3 was only purified 7 fold which was due to the high
(13.2 yg/ml) protein level for that fraction. The overall yield for
HA 1 was 28% and 7% for HA 2, with respect to the crude extract.
The highest fold increase in specific activity was due to the
Phenyl Sepharose column, suggesting that the use of this resin was important to the successful purification of d'Anjou pear PPO. The reproducibility of the entire procedure was excellent. Similar elution profiles were obtained for all of the chromatographic columns from trials with several pear nitrogen powders. 69
Figure 6. Hydroxylapatite column chromatography of pear PPO.
Elutants: A. ImM sodium phosphate, pH 7.0. B. Linear gradient from 1 mM sodium phosphate to 50mM sodium phosphate, 1M potassium chloride. C. Linear gradient from 50 mM sodium phosphate, 1M potassium chloride to 50mM sodium phosphate, 1M potassium chloride, 1M ammonium sulfate. D. 400mM sodium phosphate
6a - sample was DEAE 1: PPO Peak HA 1 fractions No. 23-25
6b - sample was DEAE 2: PPO Peak HA 2 fraction No. 22
'*' - represents the linear gradients described above 70
D 0.04 a. 14,000 0.03 J 10,000 H H ii II 0.02 i 'i 6000- I I c .", 'E 0.01 E A /• » c 2000h / ? O ♦ 00 O ^ N i i •. C\J in Q> O O E z: c b. D - 0.04 § > • 1400 _ o • • CO > 00 c ..■•" - 0.03 < O < 1000 ,1 o •• QL •• -0.02 Q- • • • • 600 B A /\ / - 0.01 2/ ^C-"' 200 -v r t ! /. il \. 1 1 10 20 30 40 50 FRACTION NUMBER Figure 6 Table 2. Purification of d'Anjou pear PPO.
Specific Volume PPO Activity Total Protein Activity Yield Purification Sample (ml) (nmoles02/min/ml) Units (yg/ml) (Units/ug) {%) (Fold)
Crude supernatant 182 1410 257,000 135 10 100 1 Bio-gel P-6 eluent 180 1220 224,000 69 18 87 2
(NH4)2S04 filtrate 176 1180 212,000 75 16 83 2
Phenyl Sepharose:
-most active fractions 24 6140 151,000 26 236 59 24 -after dialysis + concentration 20 8650 181,000 30 288 69 29
DEAE - Cellulose:
Peak 1 24 3090 92,700 10 297 36 30 Peak 2 18 900 20,200 8 118 8 12 Peak 3 24 880 26,400 13 68 10 7
Hydroxylapatite: Peak 1 18 3120 73,100 2 1480 28 148 Peak 2 6 1030 18,500 1 1030 7 103 72
Electrophoresis
After the isolation of PPO the purity of the preparations and the multiplicity of the enzyme were examined by 1% polyacrylamide slab gel electrophoresis. The results of the various runs are illustrated in Figure 7a for the enzyme stain and Figure 7b for the protein stain.
The samples 1 through 6 represent the progression in purification.
Streaking with the Bio-gel P-6 sample on the protein stained gel
(Figure 7b) was due to the presence of Triton X-100 in the extract.
On the enzyme stained gel (Figure 7a) this sample revealed three PPO bands with high activity and two bands with minor activity. After the
Phenyl Sepharose column only the three most active PPO bands were present on the enzyme stained gel and these were separated on the
DEAE-cellulose and hydroxylapatite columns.
The silver stained gel (Figure 7b) revealed the presence of several other proteins in the eluate of the PS column, including two very prominent bands with low relative mobilities. The third sample well represents the most active PPO peak from the DEAE column (DEAE 1) and, although the two large bands at the top of the gel had been removed, two minor protein bands that were not PPO remained. These were eliminated after the HA column, leaving a pure, active PPO band at a relative mobility of 0.41 (sample 6). DEAE 3, relative mobility
0.73, (sample 5) was also pure, although it had about 3 to 4 times less enzyme activity than HA 1. The third PPO fraction, relative mobility 0.43, (DEAE 2) is free from other protein contaminants but 73
Figure 7. Polyacrylamide electrophoresis on 1% slab gels of pear PPO during purification.
7A - Gel stained for polyphenol oxidase activity.
7B - Gel stained for protein.
Samples: 1. Bio-gel P-6 eluent 2. PS 1 3. DEAE 1 4. DEAE 2 5. DEAE 3 6. HA 1
(Two wells were used for each sample) 74
2 3 4 5
B
'2345
Figure 7 75 has a trace of the DEAE 1 form present that was not removed even after an HA column.
The results in Figure 8 are presented to show the multiplicity of some of the other fractions, particularly from the Phenyl Sepharose column. Sample 1 on the protein stain (Figure 8a) is PS 1, as shown previously, but samples 2 and 3 were PPO peaks PS 2 and 3, respectively. Several oth.er protein bands were present that were not found in the PS 1 PPO peak. Figure 8b is an enzyme stained gel showing the multiple of PPO forms in the PS peaks 2 and 3 (samples 3 and 4). These bands are faint, but are definitely present. These two enzyme peaks from the Phenyl Sepharose column were not investigated further. Another fraction illustrated on this gel is the ammonium sulfate filtrate, sample 1. This fraction also contained several enzyme forms, but they were all eliminated after the subsequent column chromatography.
Figure 9 is a photograph of several PS and DEAE fractions that were tested when the column procedures were being developed. Among them are two samples that were run to evaluate the use of Triton X-100 in the initial enzyme extraction procedure. Sample C is PS 1 when
Triton was used and sample F is PS 1 when the extraction mixture did not contain Triton. The photograph illustrates that there were no differences in pear PPO multiplicity due to the addition of Triton
X-100. The same three bands, corresponding to HA 1, HA 2, and DEAE 3, were observed in both samples. 76
Figure 8. Pol acry!amide electrophoresis on 7% slab gels of selected fractions of pear PPO.
8A - Gel stained for protein.
Samples: 1. PS 1 2. PS 2 3. PS 3 4. HA 1 5. HA 1
8B - Gel stained for polyphenol oxidase activity.
Samples: 1. Ammonium sulfate filtrate 2. PS 1 3. PS 2 4. PS 3 5. DEAE 2 6. DEAE 3 7. DEAE 1 77
12 3 4 5
B -rm-*^
• -^,,
• 2 3 4 5 6 7
Figure 8 78
Figure 9. Effect of Triton X-100 in enzyme extraction procedure of pear PPO. Results shown on 7% polyacrylamide slab gel electrophoresis. Sample: C. PS 1 after PPO had been extracted with Triton X-100. F. PS 1 after PPO had been extracted without Triton X-100. A,B,D, and E are PS and DEAE fractions that were tested when the column procedures were being developed. 79
.
+
-r -^'^w i « without Triton X- 100rt Triton X-100
Figure 9 80
Enzyme Stabiliby
Stability of the purified PPO extract (HA 1) was monitored for
120 days after purification and the results are shown in Figure 10.
The enzyme extract was very stable when stored at 40C in the elution solution from the HA column (36 mM phosphate, 0.72 M potassium chloride, pH 7.0). Within the first 15 days 25% of the initial activity of the extract was lost, but remained at that level for 120 days when mold growth was noted. This stability made it possible to store the extract at refrigeration temperatures and continually use the purified extract for 2 months after purification with no detrimental effects. Initially the enzyme extracts were frozen and stored at -40oC, however, 50% of the original activity was lost upon thawing.
Effect of DMSO
The effect of dimethylsulfoxide, DMSO, on enzyme activity was studied using the most active peak eluted from the Phenyl Sepharose column (PS 1). DMSO was initially investigated as a possible component of the elution buffer for the hydrophobia column and Figure
11 details the results from the study. DMSO in the enzyme mixture significantly increased the PPO activity over the control, with the maximum level obtained at 5% DMSO. With 50% DMSO in the solution activity was increased 25%. At 80% DMSO, however, enzyme activity decreased 70%. 81
Figure 10. Stability of a purified d'Anjou pear PPO fraction, stored at 4QC in 0.7M potassium chloride, 30mM phosphate buffer, pH 7.0. 82
60- cr o 40- o
20- O DC U CL 0 20 40 60 80 100 120 DAYS AFTER PURIFICATION AT 40C Figure 10 83
Figure 11. Effect of DMSO on the enzyme activity of d'Anjou pear PPO. Activity measurements were performed with PS 1. 100% activity represents the enzyme activity in the absence of DMSO. 84
I80r- IM „.,Nl
160-
140-
P ,20
< feioo I 5 10 25 50 ^Oi: PERCENT DMSO y 8o[ IT a. 60
40
20L-
Figure II 85
Molecular Weight Determination
The molecular weight of each of the three forms of d'Anjou pear
PPO was investigated using Sephacryl S-300 gel chromatography and LDS electrophoresis in 10% and gradient polyacrylamide slab gels. A tabulation of the results from the Sephacryl S-300 column are presented in Figure 12, a plot of log molecular weight versus Kav.
Each of the three PPO isozymes (HA 1, DEAE 2, and DEAE 3) were subjected to gel filtration both separately and combined. The results consistently revealed a single PPO peak at 45,300 daltons ± 5%. When all three forms were combined there was a slight shoulder on the tailing edge of the Sephacryl PPO peak. But when each fraction was collected and electrophoresed there was no difference in the mobility of the three fractions. The resolution of six protein standards on this column was excellent with a correlation coefficient of r2=0.985.
The three purified forms of PPO were also studied with LDS electrophoresis to determine if they differed from each other on the basis of charge or molecular weight and also to further investigate their purity. Figure 13 is a photograph of a LDS 10% polyacrylamide slab gel stained for protein. Several minor protein bands were present in all of the fractions. Sample 2 is a DEAE 2 fraction before further purification. The other samples representing duplicates of HA
1 (samples 1 and 4) and DEAE 3 (samples 3 and 5) show one major band at a relative mobility of 0.50. This protein band corresponded to a molecular weight of 60,000 daltons. 86
Figure 12. Molecular weight of d'Anjou pear PPO by Sephacryl S-300 Superfine chromatography.
• are molecular weight standards
X is polyphenol oxidase 87
0.60r^ Ribonuclease r2= 0.985
0.50- ^ Chymotrypsinogen
^\Ovolbumin
0.40- 45,300 \ Bovine serum albumin K av 0.30 Glucose oxidase 0.20
0.10 Thyroglobulin
4.0 4.4 4.8 5.2 5.6 6.0 LOG MOLECULAR WEIGHT Figure 12 88
Figure 13. LDS 10% polyacrylamide slab gel electrophoresis of pear PPO stained for protein.
Samples: 1. HA 1 2. DEAE 2 3. DEAE 3 4. HA 1 5. DEAE 3 STD. Molecular Weight Standards. 89
2 STD. 3 STD. 5
Figure 13 90
Figure 14. LDS gradient polyacrylamide slab gel electrophoresis of pear PPO stained for protein.
Samples: 1. DEAE 3 2. HA 2 3. HA 1 STD. Molecular Weight Standards 91
+
Figure 14 92
A representative LDS 7.5-15% acrylamide gradient gel is shown in
Figure 14. The molecular weight standards are flanked by the samples
DEAE 3, HA 2, and HA 1 from left to right. The presence of minor contaminating protein bands is not as noticeable in the gradient gels as they were in the LDS 10% gels. The molecular weight of each of the three isozyme forms appears to be the same from the results obtained with the 10% acrylamide LDS electrophoresis. A plot of relative mobility versus log molecular weight (Figure 15) revealed a molecular weight estimate of 59,600 daltons ± 3%. One other protein band at a relative mobility of 0.65 continually appeared in the DEAE 3 enzyme fraction. The molecular weight of that band corresponded to a value of 39,000 daltons.
Isoelectric Focusing
To further assess the differences between the three PPO isozymes isoelectric focusing in polyacrylamide gels was performed. Several difficulties were encountered with this procedure due to the low amount of protein available in the extracts. The silver stain for protein is extremely sensitive but it does not work well in tube gels and not enough protein was present to use the Coomassie Blue or Amido
Black protein stains. Therefore, it was necessary to depend solely on enzyme activity to stain the isoelectric focused gels. The color of the enzyme stained bands would fade, so it was necessary to record the results immediately after staining the gels.
The results of the isoelectric focusing experiment are illustrated in Figure 16. A linear pH gradient was created in the 93
Figure 15. Molecular weight of d'Anjou pear PPO by LDS gradient polyacrylamide electrophoresis.
• are molecular weight standards
X is polyphenol oxidase 94
5.0r- Phosphorylase B
X Bovine serum albumin O 4.8h UJ 59,600 \ Ovalbumin §4.6 _J =) %Carbonic anhydrase U LM 4.4 '^Soybean Trypsin 2 inhibitor O 4?- r = 0.990 Lysozyme
4.0. 0.2 0.4 0.6 0.8 .0 RELATIVE MOBILITY Figure 15 95
3.6
4.3 4.3 4.5 4.5 4.7 5.2
6.2
6.9 7.3
8.0
pH HA I HA 2 DEAE3 gradient
Figure 16 Isoelectric Focusing of d'Anjou Pear PPO 96 tube gels from pH 3.6 to 8.0. Two prominent bands were obtained for
PPO fraction HA 1, at pi 4.3 and 4.5. HA 2 separated into two faint bands at pi 4.5 and 4.7. The third fraction, DEAE 3, revealed one band on the isoelectric focused gel, at pi 6.9. Thus, from three single bands on regular electrophoresis gels four bands appeared during electrofocusing; three with acidic pi and one with a neutral
Pi.
Ami no Acid Composition
The ami no acid composition was determined for each of the three purified pear PPO fractions; HA 1, HA 2, and DEAE 3. Due to the low concentration of protein in the extracts the amino acids were analyzed by the fluorimetric method which has good precision at the level of
0.5 nmole amino acid (Roth and Hampai, 1973). The results are tabulated in Table 3 and are given as a fraction of the total amount of amino acids present in each sample. The percent of amino acids in each fraction that were hydrophobic, acidic, and basic was also calculated and is shown in Table 3. The hydrophobic amino acids
(alanine, valine, isoleucine, leucine, and phenylalanine) represented
35% of the total in HA 1 compared to only 20% in HA 2 and DEAE 3. HA
1 also had less total charged amino acids with 36% versus 54 and 41% for HA 2 and DEAE 3, respectively. The values for the acidic amino acids (aspartic acid and glutamic acid) also include the amides of these two amino acids due to the hydrolysis of the amino group during acid hydrolysis. Thus, unfortunately, it is not known what percentage of these "acidic" amino acids are actually due to the acid forms. 97 Table 3. Amino acid composition of d'Anjou pear PPO
Fraction of total amino acids present Amino Acid WT RFT —mrr
Cysteic acid .01 .03 .01 Asparagine & Aspartic acid .11 .24 .17 Threonine .06 .03 .04 Serine .06 .07 .12 Glutamine & Glutamic acid .13 .13 .18 Glycine .14 .15 .21 Alanine .12 .09 .08 Valine .07 .04 .03 Methionine None None None Isoleucine .04 .02 .02 Leucine .08 .04 .04 Tyrosine .03 .01 .02 Phenylalanine i .03 .01 .02 Histidine .03 .04 .03 Lysine .05 .04 .02 Arginine .04 .06 .02 Unknown None .04 .02
Percent of total amino acids present
Hydrophobic amino acids 35 20 19 Charged amino acids acidic 24 38 34 basic 13 16 7 total 36 54 41 98
The major amino acids found in the three fractions were aspartic acid (plus asparagine), glutamic acid (plus glutamine), and glycine; representing 40, 50 and 60% of the three respective fractions.
Alanine was high in HA 1 and serine was high in DEAE 3.
The presence of an unknown peak was noticed in HA 2 and DEAE 3, but not in HA 1. The identity of this peak was not determined, but it eiuted towards the end of the run with the basic and hydrophobic amino acids; specifically, between histidine and lysine.
Protein-Phenolic Interactions
The interaction between the purified PPO extracts, DEAE 2 and 3, and two different phenolic compounds, (2,3-dihydroxybenzaldehyde,
DHBA, and chlorogenic acid, CA) were studied at pH 7.0 and 5.6. The results of this experiment are detailed in Table 4. The activity measurements were recorded immediately following the application of the fractions on the electrophoresis gels. Controls were stored at the same pH without the added phenolic compounds.
At pH 7.0 slight activity lost was observed in the extracts that had been incubated at 40C with the phenolics. There was even a slight increase over the controls for DEAE 3. Results with DEAE 2 plus 3 mM chlorogenic acid showed that after standing for 20 hr this fraction had developed five additional enzyme forms that had PPO activity
(Figure 17b, sample 2). When this mixture was allowed to react at room temperature for 30 minutes before electrophoresis no apparent increase in the number of PPO forms was observed (Figure 17b, sample
3). Figure 17b also illustrates the results when DEAE 3 was reacted Table 4. Protein-phenolic interactions with d'Anjou pear PPO.
Chlorogenic Acid [3 mM] 2-3,Dihydroxybenzaldehyde [5 mM] % of original Color of Number of % of original Color of Number of enzyme activity Extract Bands enzyme activity Extract Bands
pH 7.0
DEAE 3a 105% clear 1 105% Light tan
DEAE 2 90 light 6 76 tan brown
pH 5.6
DEAE 3 75 clear 1 8 tan 1
DEAE 2 4 brown 0 6 tan 0
All of these extracts were held at 40C for 20 hr before applied onto a 7% acrylamide electrophoresis slab gel. 100
Figure 17. Polyacrylamide slab gel electrophoresis (7%) of PP0- Phenolic interactions. Both gels are stained for polyphenol oxidase activity. All samples were incubated at pH 7.0.
17A. Samples: 1. DEAE 2 2. DEAE 3 3. DEAE 3 + 5mM 2,3 dihydroxybenzalde- hyde (DHBA) at 40C for 20 hr. 4. DEAE 3 + 0.5mM DHBA at 40C for 20 hr. 5. DEAE 2 + 5mM DHBA at 40C for 20 hr. 6. DEAE 2 + 0.5mM DHBA at 40C for 20 hr.
17B. Samples: 1. DEAE 2 2. DEAE 2 + 3mM chlorogenic acid (CA) at 40C for 20 hr. 3. DEAE 2 + 3mM CA at 250C for 30 min. 4. DEAE 3 5. DEAE 3 + 3mM CA at 40C for 20 hr. 6. DEAE 3 + 3mM CA at 250C for 30 min. 7. PS 1 8. PS 1 + 3mM CA at 250C for 30 min. 101
12 3 4 5 6 B
12 3 4 5 6 7 8 Figure 17 102 0 with chlorogenic acid for 20 hr at 4 CS and 30 min at room temperature; and when PS 1 was allowed to react for 30 min at room temperature. In all of these trials there was no increase in the number of PPO bands as compared to the control samples.
Figure 17a is a photograph of the electrophoresis of the two enzyme extracts to which 5 mM and 0.5 mM DHBA was added and allowed to for 20 hr at 4°C. No additional PPO bands appeared, although DEAE 2 plus 5 mM DHBA did cause streaking on the gel.
The next set of experiments were run at pH 5.6 for 20 hr at 40C; no trials were run for 30 min at room temperature. The results shown in Table 4 indicate that enzyme activity was decreased under these conditions when compared to the controls. Electrophoresis of the samples revealed no new PPO bands and, with DEAE 2, the original band was missing. DEAE 2 plus 3 mM chlorogenic acid was very brown after
20 hr at 40C suggest that the loss of activity was due to protein-phenol interaction. 103 DISCUSSION
Enzyme Extraction
Detergent extraction, using Triton X-100, considerably increased the amount of polyphenol oxidase activity that could be extracted from d'Anjou pears. Several other PPO researchers have reported similar results (Sharma and Ali, 1980; Galeazzi et al_., 1981; Walker and
Hulme, 1966; Hare! and Mayer, 1971). The increase in enzyme activity has been attributed to an activation due to the detergent (Galeazzi et al., 1981), and to the disruption of chloroplast membranes to release more enzyme (Parish, 1972; Hare! ert al_., 1965). The natural ripening of fruit with the concomitant breakdown of tissue, appears to render
PPO in a more soluble form than is observed in the green fruit (Kidron et_ al_., 1978). The pears used in this study were green and unripe. Therefore, it is possible that the detergent treatment was necessary to release enzyme that was associated with membranous material.
There has been some concern over the use of Triton in the isolation of PPO due to its ability to extract other membrane components besides the enzyme. There is also the possibility that it may alter the structure and properties of the enzyme molecule (Rhodes,
1977; Mayer and Hare!, 1979). However, Triton is a mild non-ionic detergent and generally extracts proteins without disruption of their native conformation or loss of biological activity (Makino et al.,
1973). The detergent creates a hydrophobic environment which is favorable to the enzyme and, once the unbound detergent is removed, it is possible to study the physiochemical properties of the protein 104
(Furth, 1980). To observe any changes due to the use of the detergent two samples were compared by electrophoresis; one in which Triton was used in the initial extraction procedure and one in which Triton had not been used. The results revealed no differences between the electrophoretic patterns of the two samples.
The Triton X-100 did also extract the green pigment from the pear skins. This was also noted by Walker and Hulme (1966) and Parish
(1972) with their respective work on apple and spinach beet PPO. To further study this green pigment an ether extract was prepared and an absorption spectra was run between 700 and 300 nm. The results revealed peaks at 660, 470, and 430 nm, suggesting that the pigment is a mixture of chlorophyll derivatives. Robinson (1980) indicated that chlorophyll a had one absorption maxima at about 660 nm and another at
430 nm. This correlates well with the results observed here.
Robinson also reported that chlorophyll b had absorption bands at 640 and 460 nm. The ratio of a to b in higher plants is usually about 3:1
(Robinson, 1980). Although the fractions were not quantitated in this work it can be seen from Figure 2 that there was more chlorophyll a present than the b derivative. However, the occurrence of chlorophyll derivatives in tissues is more complex than four specific absorption maxima. Six different forms of chlorophyll a have been separated on the basis of absorption maxima ranging from 663 to 700 nm (Robinson,
1980). Interaction of pigments with protein or lipid molecules will also influence the position and shape of the absorption bands.
Goedheer (1966) reported that Triton X-100 extracted a chlorophyll-protein complex from spinach chloroplasts. For this 105 reason it was of interest to know whether the green pigment from pears was complexed with PPO. But when the extract was applied onto a hydrophobic column all of the pigment remained attached to the top of the column, while the enzyme could be eluted in several clear fractions; indicating that the enzyme was not in a tight complex with chlorophyll. Vaughan et al_. (1975) recommended the use of cetyltrimethylammonium bromide (Cetavlon), a cationic detergent, in the extraction of PPO from spinach beet since it did not liberate the chlorophyll pigments.
The use of the anionic exchange resin, AG 2-X8, was used as a phenolic scavenger to improve the efficiency of separating the unwanted endogenous phenols from PPO (Smith, 1980). The adsorption capacity of this resin is due to hydrophobic, ionic, and hydrogen bonding (Moore and Stein, 1951; Loomis et al_., 1979). Therefore, it was possible that the Triton could have interfered with the efficiency of phenolic removal. Unfortunately, due to the absorption peak of
Triton at 275 nm, it was not possible to determine a 280/260 ratio on the crude extract (Smith, 1980). However, as mentioned before, the electrophoretic pattern of the enzyme did not differ in the presence or absence of the detergent.
Flurkey and Jen (1980c) reported that the apparent number of multiple forms of PPO could be decreased when protease inhibitors, trasylol and phenylmethylsulfonyl fluoride (PMSF) were used during each step of the purification of peach PPO. No differences in enzyme multiplicity were seen in this work either with or without the use of protease inhibitors. However, it was decided to adopt their 106 recommendations, and protease inhibitors were used throughout the extraction and purification of d'Anjou pear PPO.
Purification of Polyphenol Oxidase
The purification of PPO from higher plants has typically been a difficult task and the trials with d'Anjou pears were no exception.
Several problems were encountered, some of which have already been discussed in the preceeding section. A major obstacle in the purification scheme was the variation in the elution patterns during chromatography. Several different elution conditions were tried with each column before good resolution and reproducible results were obtained.
The effect of temperature on the elution of d'Anjou pear PPO from the Phenyl Sepharose, DEAE, and hydroxylapatite columns was an interesting phenomenon. Generally, when working with active enzyme preparations it is important to work at low temperatures to avoid alterations in enzymic or molecular properties. However, when chromatography of PPO was carried out at low temperatures it was not possible to obtain good resolution or reproducible results. At 40C the enzyme could not be eluted from the Phenyl Sepharose column, while on the other columns PPO came out in the void volume. These results are in contradiction to the normal behavior of proteins. Low temperatures generally favor adsorption to ion-exchange columns
(Peterson and Sober, 1962) and decrease adsorption in hydrophobic interaction chromatography (Hjerten et al_., 1974). However, Hjerten
(1973) was not able to find a strong temperature effect on the release 107 of the protein, phycoerythrin, from hydrophobic Sepharose columns; apparently other parameters affecting hydrophobicity were more important in the protein binding to the column.
It is possible that at the low temperature there is a slight unfolding of the protein molecule which would expose more of the interior hydrophobic groups. This would cause the enzyme to bind very tightly to the Phenyl Sepharose column. The ratio of nonpolar to polar amino acids would also be greater which would cause a decrease in the affinity of the protein towards the charged resins. Ogamo j?t al. (1981) reported a dramatic increase in the affinity of mucopolysaccharides for Phenyl Sepharose CL-4B at 40C versus at room temperature. The amount of heparin retained on the gel markedly increased as the temperature was lowered. Several researchers have implicated that carbohydrate material may be associated with PPO
(Stelzig et aj_., 1972; Balasingham and Ferdinand, 1970; Flurkey,
1979). Using Con A affinity chromatography Nishioka (1978) has found that tyrosinase of human melanoma was a glycoprotein. If pear PPO is also found to have carbohydrate material covalently attached to its protein structure, this could help to explain the behavior during column chromatography at lower temperatures.
Hydrophobic chromatography, using Phenyl Sepharose CL-4B resin, provided a rapid partial purification of d'Anjou pear PPO. There was excellent reproducibility when the column was run at room temperature and with Triton X-100 in the crude enzyme extract. The enzyme was eluted in a single major, protein peak, clearly separated from the bulk of the other 280 nm absorbing material. The results indicated an 108
increase in the specific activity and a recovery of more than 100% of the PPO activity from the crude extract for pear PPO. Occasionally, the column yield was approximately 200%. These observations are consistent with those reported by Wissemann and Lee (1980b) and
Flurkey and Jen (1978) for grape and peach PPC respectively, purified on a hydrophobic column. These investigators found yields of PPO activity of over 180% in comparison with the crude extract and attributed this to the removal of some inhibitory substance.
Neither of the above researchers found the use of Triton X-100 necessary. However, with d'Anjou pear PPO the results were more consistent and the resolution was better when Triton was present in the crude extract before Phenyl Sepharose chromatography. The use of
Triton X-100 in conjunction with Phenyl Sepharose chromatography has
been successful in the isolation of chlorophyllase (Shimokawa, 1982) and human brain membrane proteins (Carson and Konigsberg, 1981). The
latter researchers reported evidence that the membrane proteins bound
to the Phenyl Sepharose as a complex with the detergent. However with
pear PPO the Triton was eluted after the enzyme peak, when the ethylene glycol was used as an elutant and not as a Triton complex.
The use of hydrophobic chromatography is also another method to
remove residual phenolics, if they had not already combined with the
protein. Raymond et al. '{19BI) reported that the hydrophobic proteins from a sunflower extract were bound to the Sepharose matrix while 98% of the chlorogem'c acid present in the precolumn fraction passed
straight through the hydrophobic column. 109
To test the hydrophobicity of PPO Flurkey and Jen (1980a) studied the adsorption characteristics of peach PPO with twenty-four various hydrophobic media. They found that their enzyme preparations would adsorb to Phenyl Sepharose, 4-phenylalanine-TETA-alkyl Sepharose and octyl- and decyl-agarose columns. It was noticed that the extent of adsorption of peach PPO was greater to the Phenyl Sepharose resin than to the Cg or C10 agarose medias. The hydrophobicity of Phenyl Sepahrose is equivalent to a 4 to 5 carbon straight alkyl chain, therefore, they concluded that forces other than hydrophobic interactions were probably involved in the attraction. There was good separation and purification, 24 fold, of d'Anjou pear PPO in this one step and it appears that this resin was the key to the successful purification of d'Anjou pear PPO.
The remainder of the purification of pear PPO was performed using
DEAE-cellulose and hydroxylapapatite chromatography. An overall purification fold of 148, 103, and 7 was achieved for HA 1, HA 2, and
DEAE 3, respectively. The overall yields for each of the respective fractions were 28%, 7%, and 10%. The low purification fold for DEAE 3
is a reflection of the low specific activity of this fraction. HA 1 and HA 2 had over 1000 units of enzyme activity per yg of protein, while DEAE 3 only had 68 units/pg. The observation that different enzyme forms of PPO have different specific activities has been previously reported (Golbeck and Cammarta, 1981; Rivas and Whitaker,
1973). Several other methods for the purification of PPO have been
reported with varying degrees of success. Rivas and Whitaker (1973)
reported a purification procedure for Bartlett pears that involved 110
Sephadex gel filtration, DEAE-chromatography and HA chromatography, that yielded two enzyme fractions that were homogeneous by electrophoresis, however, the extracts were tanned. A purification fold of over 900 was obtained by Go!beck and Gammarata (1981) with spinach PPO. They used sonication, ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. A 340-fold purification with a 35% yield was found for apple PPO by Hare! et a!.
(1965), when a long combination of Triton X-100 extractions, ammonium sulfate precipitations, and two DEAE-cellulose columns were used. But generally the purification values are closer to 100 or even lower, as reported by Patil and Zucker (1965) for potatoes, Anosike and Ayaebene
(1981) for yam tubers, Sharma and Ali (1980) for eggplants, and
Flurkey (1979) for peaches.
The values for specific activity and purification fold are based on protein levels and unless similar methods for protein determination are used by all researchers it is difficult to compare the various purification procedures based on purification fold. Unfortunately, the phenolic compounds that also cause problems in purifying native proteins from plants also interfere in several protein assay procedures; including the commonly used assay by Lowry et aj_. (1951).
Loomis (1974) pointed out the problems of determining the protein content in plant extracts and recommended the use of the Kjeldahl procedure applied to a trichloroacetic acid precipitate or the measurement of turbidity upon addition of sulfosalicyclic acid. Robinson (1979) suggested the use of the Bradford dye binding procedure (Bradford, 1976) for rapid determination of protein in plant Ill extracts because polyphenolics do not interfere. However, even the protein-dye binding methods are not thoroughly acceptable, since they are subject to variations in the response of different proteins to the dye (Peterson, 1979).
Purity and Stability of Pear PPO
Polyacrylamide slab gel electrophoresis was used to determine the purity of the pear PPO preparation. HA 1 and DEAE 3 were shown to be pure even after the use of the sensitive silver stain for protein. HA
2 was free from other contaminating proteins, except for a trace of HA
1 PPO. The silver stain can detect as little as 0.01 nanogram of protein per square millimeter of gel (Merril e_t al_., 1981) and is 2 orders of magnitude more sensitive than the Coomassie brilliant blue stain (Wray ei^ aj_., 1981). The Coomassie blue and Amido black stains lack the sensitivity to detect proteins present in low or trace concentrations (Merril et aj_., 1981). This work with pear PPO is the first to use the silver stain to study the purity of a PPO preparation.
Few reports have been published on PPO preparations from plants that have been purified to apparent homogeneity (Patil and Zucker,
1965; Coggon et aj_., 1973; Vau'ghan et aK, 1975; Golbeck and
Cammarata, 1981; Sharma and Ali, 1980; Anosike and Ayaebene, 1981), and only three groups used fruits (Rivas and Whitaker, 1973; Kidron et al_., 1977; Flurkey, 1979). However, only Vaughan et al_. (1975) was able to show that their enzyme isolate was homogeneous by the criterion of SDS polyacrylamide electrophoresis and sedimentation 1.12 velocity. Their purification procedure involved several steps using the detergent Cetavlon, ammonium sulfate fractionation, carboxymethyl cellulose chromatography, and hydroxylapatite chromatography.
Purified pear PPO was found to be stable for up to 4 months at
40C in a solution of 36 mM phosphate, 0.72 M potassium chloride, pH 7.
Twenty-five percent of the enzyme activity was lost initially, but after 15 days the activity stabilized and remained at that level for
120 days. When the enzyme extracts were stored at -40oC, 50% of the original activity was lost after a 2 month storage period. These results are in direct contrast to those reported by Smith (1980) for a crude d'Anjou pear PPO extract. In her study the enzyme was most stable at pH 5 and lost no activity when stored for three months at
-40oC. She indicated that the sample lost 11% of the initial activity in 11 days at 40C before the experiment had to be stopped due to microbial growth. The purified extract showed no signs of microbial growth until after the 120 days of storage. Flurkey and Jen (1980b) found no significant loss in PPO activity when their purified preparations were stored for 2 months at -150C in 20% glycerol, sucrose, or ethylene glycol solutions. They noticed a gradual loss in activity when these samples were stored at 40C.
The ionic strength (0.72 M potassium chloride) was relatively high in the purified pear PPO extract which seemed to help to stabilize the enzyme. Previous work on PPO has shown that its activity increases with an increase in the ionic strength of the buffer (Lerner et al_., 1972; Rivas and Whitaker, 1973). In this thesis it was also noted that when the pear PPO extracts were diluted 113 1:10 or 1:20 a significant amount of activity was lost, even overnight, at 40C. Apparently the more concentrated protein and ionic solution in the purified sample helped to stabilize the enzyme. This was also reported by Balasingham and Ferdinand (1970) for potato PPO.
Their PPO extract when diluted lost 35% of the activity in one day at
0oC and a further 30% after a second day.
Effect of DMSO
DMSO was initially investigated as a possible component of the elution buffer for the hydrophobic column. This solvent has been recommended for proteins requiring a more hydrophobic environment, to decrease the polarity of the solution (Cooper, 1977). As an elutant with the Phenyl Sepharose column, DMSO was not successful, however, some interesting results were obtained. DMSO significantly increased the enzyme activity of PPO over the control. At a level of 5% DMSO in the solution an 80% increase in activity was observed. No other reports on the effect of DMSO on PPO are available, although Rees and
Singer (1956) found that DMSO was one of a few nonaqueous solvents in which several proteins were soluble. This included trypsin which recovered essentially all enzymic activity after being removed from
100% DMSO; indicating that the DMSO did not significantly alter the structure of the enzyme.
DMSO is a dipolar aprotic solvent that has a high dielectric constant, but cannot act as a proton donor (Martin and Hauthal, 1975).
However, it possesses a lone pair of electrons and forms a strong association with proton donors, such as water. The hydrogen bonding 114 between DMSO and water is very extensive and leads to a higher orientation among water molecules (Martin and Hauthal, 1975). This ordering of water molecules plus the high dielectric constant, lowers the attraction between oppositely charged ions and helps to maintain the proteins in solution (Morrison and Boyd, 1973). Other substances which decrease the polarity of the solution are sucrose, glycerol, and ethylene glycol (Cooper, 1977; Hofstee, 1973), all of which increase the stability of peach PPO at concentrations of 20% (v/v) (Flurkey and
Jen, 1980b).
Molecular Weight Determination
A large portion of this thesis was devoted to elucidating the nature of the three multiple forms of d'Anjou pear PPO that were detected by ion-exchange chromatography and polyacrylamide gel electrophoresis. It was of interest to know if these three forms differed due to charge, molecular weight, or amino acid composition.
The multiplicity of PPO has been well documented and can be interpreted in several ways. There has been considerable debate on whether PPO multiplicity represents the existence of true isozymes or artifacts of the purification procedure, such as from proteolysis or tanning by phenolics. Good evidence has been presented by Jolley et al. (1969b) and Horowitz £t al_. (1961) for differences in the amino acid composition of tyrosinase isozymes isolated from mushroom and
Neurospora, respectively. Other reports have also observed various degrees of aggregation of subunits to yield dimers and tetramers that migrate differently on gel filtration columns and during 115 electrophoresis (Hare! and Mayer, 1968; Balasingham and Ferdinand,
1970; Galeazzi et aJL» 1981). However, a search of the literature revealed no information on differences in amino acid composition between various multiple forms of PPO isolated from higher plants.
The molecular weight of each of the three forms of d'Anjou pear
PPO was investigated using Sephacryl S-300 gel chromatography and LDS electrophoresis in 10% and gradient polyacrylamide slab gels.
Sephacryl S-300 superfine is a relatively new gel filtration resin that consists of ally! dextran crosslinked with NjN1-methylene bisacrylamide to give a rigid hydrophilic macroporous gel (Johansson and Lundgren, 1979). This resin has a very wide fractionation range
(MW 10,000 to 1,000,000), with good resolution over the entire range.
One of the main advantages of this resin over dextran or agarose gels is that it is possible to use high flow rates and still obtain good resolution (Hoff and Easterday, 1978). Thus, in cases where slow changes in protein conformation may occur during the normal 18-24 hr elution on Sephadex or Sepharose columns, these changes can be minimized by eluting the protein in 2 hr on a Sephacryl column (Le
Maire et al_., 1980). Hoff and Easterday (1978) indicated that there was good correlation between elution volumes of proteins and their molecular weight. The molecular weight for pear PPO determined by gel filtration on this column was 45,300 daltons ± 5%. All three forms of the enzyme, whether applied to the column individually or combined, had the same molecular weight.
Next the molecular weights of each of the fractions were determined by LDS electrophoresis. LDS or SDS electrophoresis is 116 commonly used to examine the subunit structure of proteins. Native proteins with widely different charge, size, and shape are converted, upon disulfide bond reduction and SDS binding, to SDS-protein complexes of their individual polypeptide subunits (Lasky, 1978). SDS has two main effects on proteins. The first is to give a uniform charge surface density by imparting a large negative charge and, the second, is to provide an overall conformation close to a random coil structure (Lambin, 1978). Thus, with the same charge and shape, the differences in the electrophoretic mobility will be due to variations in size. Molecular weight determinations with SDS electrophoresis can be accurately made in both homogeneous polyacrylamide gels (Weber and
Osborn, 1969; Laemmli, 1970) or in gradient acrylamide gels (Lasky,
1978; Lambin, 1978). Lasky (1978) claimed that the SDS gradient gels yield better band resolution and more reproducible molecular weight determinations. The results obtained for the three fractions of pear
PPO were the same on both the homogeneous and gradient polyacrylamide
LDS gels. A value of 59,600 daltons ± 3% was determined for the molecular weight of each of the three PPO multiple forms.
The greater resolution obtained by LDS electrophoresis also
revealed the presence of several minor contaiminating proteins. As
indicated before, the silver stain is able to detect as little as 0.01 nanograms of protein.
One of the most disturbing phenomenon of this thesis was that the
values for the molecular weight of pear PPO obtained from Sephacryl
S-300 chromatography and LDS electrophoresis were so different. There
is a discrepancy of 24% between the two values. If the 117 electrophoresis values were representative of the subunit, or minimal, molecular weight of PPO, then those values should have been lower than the molecular weight determined for the native protein by gel filtration. There are several possibilities to explain the discrepancy, but, unfortunately, the lack of a more thorough understanding of the nature of plant PPOs makes these only speculations.
Problems which are inherent in the use of gel filtration columns and electrophoresis techniques can lead to abnormal results. The standard proteins used in gel filtration were all globular proteins and although it has been suggested that PPO from fungi also has a globular conformation (Sharma and Ali, 1981; Lerch, 1978), there exists very little structural information on plant PPOs to know how they compare. Variations between the shape and densities among various proteins can affect their elution from gel filtration columns
(Ackers, 1975; Le Marie et al_., 1980) and if pear PPO is not a globular protein it will have a different elution pattern than the standards that were used. Many phenolases are membrane-extracted proteins, and therefore, do not necessarily behave like soluble globular proteins (Lieberei et al_., 1981). Information from this thesis has indicated that pear PPO is a hydrophobic protein and thus may not behave the same as hydrophilic proteins on gel filtration columns. Some proteins, such as lipoproteins and those with prosthetic groups that may increase protein density, have more compact structures in solution and are, therefore, retarded on gel filtration columns, which decreases their apparent molecular weight (Andrews, 118
1970). Lerner and Mayer (1975) also reported that changes in the buffer and pH of the elutant changed the elution pattern of grape PPO on Sephadex G-200 columns.
There are also several limitations in the SDS procedure. In certain cases abnormalities in SDS binding and protein conformation have been reported to lead to slightly increased or decreased electrophoretic mobility (Weber and Osborn, 1975). Highly negatively charged proteins tend to bind less SDS which lowers their net negative charge and mobility to result in higher molecular weight values
(Dunker and Rueckert, 1969). Moreover, SDS binding to proteins is influenced by pH, ionic concentration, and buffer composition (Lambin,
1978). Glycoproteins also react differently on SDS gels since the carbohydrate component may be important for electrophoretic mobility and affect SDS binding. Proteins with high proline content have also been shown to exhibit lower electrophoretic mobility on SDS gels
(Furthmayr and Timpl, 1971). Using SDS electrophoresis and gel filtration to determine the molecular weight of Neurospora tyrosinase
Lerch (1976) reported a 20% higher value with SDS than with gel filtration. He attributed the discrepancy to the high proline content of tyrosinase. Unfortunately, the content of that amino acid was not determined in the amino acid analysis for pear PPO in this work.
The molecular weight value of 45,000 ± 5% determined by Sephacryl
S-300 is in better agreement with other reported values for PPO from higher plants and fungi. Examples of a few of the reported PPO molecular weight values are: spinach - MW 43,000 (Lieberei et al.,
1981; Golbeck and Cammarata, 1981), Neurospora - MW 42,000 (Lerch, 119
1976), and spinach beet - MW 40,000 (Vaughan et a]_., 1975). Lerch (1978) calculated the molecular weight of the Neurospora enzyme to be
46,000 from the amino acid sequence. However, a list of reported molecular weights by Robb (1981), as well as other published data
(Anosike and Ayaebene, 1982), indicate that the values for the molecular weight of PPO are variable.
A common observation by other researchers that was not found in this work is the existence of higher polymeric forms of PPO (Golbeck and Cammarta, 1981; Anosike and Ayaebene, 1981; 1982; Balasingham and
Ferdinand, 1970). These include dimers and tetramers of the approximately 40,000 MW subunit; indicating an oligomeric protein with four active subunits. Two possibilities exist to explain this discrepancy. First is that pear PPO is a single chain enzyme molecule and does not exist in higher polymeric forms. This has been concluded by Vaughan et al_. (1975) for spinach beet PPO. The second, is that the conditions used in this work were such as to cause dissociation of a polymeric form into an active subunit. Dixon et &]_. (1979) have indicated that simple dilution, a change in pH, or an increase in salt concentration can be sufficient to cause dissociation. The protein concentrations of the purified pear PPO extracts were very low (about
2 yg/ml). Furthermore, the pH was changed and the salt concentration increased for application on the Phenyl Sepharose and ion-exchange columns. All of these conditions favored dissociation. Dilution also favors the dissociation of PPO (Robb and Gutteridge, 1981; Golbeck and
Cammarata, 1981) and in the very low protein concentrations required for enzyme assays the monomeric form is predominantly present 120 (Vanneste and Zuberbuhler, 1974). Thus, the enzyme used in the activity assay medium may be in a very different state of aggregation from that which might be present at higher protein concentrations.
From the results obtained by LDS electrophoresis it can be postulated that pear PPO does not consist of two different types of polypeptide chains as was reported for mushroom tyrosinase (Strothkamp et aj_., 1976). They reported the existence of one heavy (MW 43,000) and one light (MW 13,400) subum't, that combined as 1^2 in the tetrameric form. Neither of the two pear PPO fractions, HA 1 or HA 2, had any other predominant protein bands on the LDS gels. DEAE 3 did have one other protein band, corresponding to MW 39,000, which did appear on the gradient gels but it was never noticed on the LDS homogeneous gels. Robb and Gutteridge (1981) have reported that the
Neurospora enzyme only contains one subum't corresponding to MW
46,000.
Isoelectric Focusing
The isoelectric points of the three pear PPO multiple forms were determined to compare the differences due to charge in these fractions. It was found that fractions HA 1 and HA 2, which had been judged as homogeneous by electrophoresis, each contained two active enzyme bands after isoelectric focusing. Apparently, this charga difference was not significant enough to be detected on normal slab gels. One of these bands, pi 4.5, was similar in both fractions, although it was much more active in HA 1 than in HA 2. Only one band was detected with DEAE 3 and that corresponded to a pi of 6.9. 121 Flurkey (1979) also noticed that a homogeneous fraction of peach PPO
separated into several forms during electrofocusing. His results
indicated a pi range between 4.5 to 5.5 for peach PPO. Ben-Shalom £t al. (1977a) reported nine active bands on isoelectric gels, with a
partially purified PPO preparation from green olives, with strong enzyme activity located at pH 5, 5.6 and 7. Examples of other
reported values for the isoelectric point of PPO include: pi 4.1,
6.8, and 7.1 for tea (Coggon et al_., 1973). pi 4.7, 4.8, 4.85, and
4.95 for mushroom (Robb and Gutteridge, 1981), pi 7.8 for Neurospora
(Robb and Gutteridge, 1981), pi 5.2 for banana (Galeazzi et al.,
1981), and pi 4.7, 4.9, 5.1, 5.5, and 6.0 for grape (Dubernet and
Ribereau-Gayon, 1974). It seems interesting that the majority of
these values are in the acidic range with an occassionai pi above 6.0.
Amino Acid Composition
From the results discussed so far on pear PPO it can be concluded
that the three multiple forms are a group of molecular species having
identical molecular weights but differing in their distribution of
charges. In order to determine if this charge difference can be
translated into differences in the amino acid composition among the
three forms an amino acid analysis was performed.
The three fractions of pear PPO did not have the same amino acid
composition. The most noticeable difference was that HA 1 contained a
higher proportion of hydrophobic amino acids than HA 2 or DEAE 3. HA
1 contained more alanine, valine, isoleucine, leucine, and
phenylalanine. The amino acids proline and tryptophan were not 122 determined, both of which are also considered to be hydrophobic amino acids (Lehninger, 1975). This is unfortunate, as the proline content of PPO has been shown to be relatively high in Neurospora (Lerch,
1978), grapes (Kidron et aj_., 1977), and spinach beets (Vaughan et al., 1975). This would significantly add to the hydrophobic nature of the pear enzyme.
HA 2 contained the highest number of charged amino acid residues, and was particularly rich in aspartic acid and/or asparagine. HA 2 also had the highest proportion of basic amino acids.
DEAE 3 had less hydrophobic residues than HA 1 but it also had slightly less charged amino acids than HA 2. The biggest difference between DEAE 3 and the other two fractions was its higher content of serine, glycine, and glutamic acid. The content of basic amino acids
(lysine, arginine, and histidine) was not unusually high in DEAE 3, in fact it was actually lower than for HA 1 and HA 2. To have the most basic isoelectric point (pi 6.9) one would expect the protein to have a preponderance of basic over acidic amino acids. In contrast, DEAE 3 had the highest ratio of acidic to basic residues; a ratio of 4
(acidic:basic) versus a ratio of 2 for HA 1 and HA 2. It is possible that DEAE 3 is folded in a conformation that exposes more of the basic amino acids and thus raises its isoelectric point or that this protein has a higher basic charge density that is not reflected in these amino acid profiles because the ratio of aspartic and glutamic acids to their amides is unknown. A different ratio of aspartic acid to asparagine or glutamic acid to glutamine in DEAE 3 could be responsible for the higher pi. 123
During the amino acid analysis of HA 2 and DEAE 3 an unknown peak was noticed that eluted between histidine and lysine. The area of this peak was very similar to the area of lysine. This peak was never seen in the HA 1 analysis. The identity of this peak was not determined; although the compound was probably either basic or hydrophobic in nature due to its position of elution. Modification of amino acid residues during or after protein synthesis is not uncommon and modifications can lead to changes in their elution from amino acid columns. Possibly this peak represents a glucosamine derivative of asparagine or serine. In their paper on the fluorimetric method of amino acid determination Roth and Hampai (1973) indicated that amino sugars, such as glucosamine, react in a similar manner to amino acids and can be fractionated on this column.
The major amino acids found in the three fractions were aspartic acid and glutamic acid, and/or their amides, and glycine. This was also noted for PPOs isolated from other sources (Duckworth and
Coleman, 1970; Balasingham and Ferdinand, 1970; Jolley el a_l_., 1969a).
Table 5 is a comparison of pear PPO amino acid analysis to PPOs isolated from other higher plants. The amino acid values previously presented in Table 1 were converted to fractions to facilitate the comparison. The amino acid composition of pear PPO HA 1 shows considerable similarities with the profile for the enzyme isolated from spinach beet, grape, and potato. The similarity is particularly noticeable in both the basic and hydrophobic residues. Also the threonine and serine content are in good agreement. The pear enzyme is richer in glutamic acid and/or glutamine, plus glycine and alanine. 124 Table 5. Comparison of pear PPO amino acid analysis to PPO from other higher plants.
Fraction of total amino acids present Pear PPO Spinach Grape Potato Amino Acid HA 1 beet PP0a PP0D PP0c
Cysteic acid .01 .02 None .02
Asparagine & Aspartic acid .11 .12 .13 .10
Threonine .06 .06 .06 .05
Serine .06 .07 .08 .07
Glutamine & (alutamic acid .13 .06 .09 .10
Glycine .14 .08 .07 .08
Alanine .12 .07 .08 .08
Valine .07 .08 .06 .07
Methionine None .02 None .02
Isoleucine .04 .04 .06 .05
Leucine .08 .09 .07 .09
Tyrosine .03 .03 .06 .03
Phenylalanine .03 .05 .04 .04
Histidine .03 .02 .02 .02
Lysine .05 .06 .06 .06
Arginine .04 .03 .04 .04 _d Proline .08 .09 .05
Tryptophan - .01 - - aVaughan et a]_. (1975) bKidron et aK (1977) ^Balasingham and Ferdinand (1970) not determined 125
A very low cysteic acid content was reported for all of the enzymes and no methionine was found in pear and grape PPO.
In comparison with the results obtained by Lerch (1978) from the amino acid sequence of PPO from Neurospora, the similarities are also
remarkable. The only noticeable difference is more serine and
phenylalanine in the Neurospora enzyme while pear PPO has more glycine, alanine, and glutamic acid.
Fractions HA 2 and DEAE 3 differ more significantly from the other isolated PPOs. HA 2 is much richer in asparatic acid, glutamic
acid, and glycine and has less threonine, valine, isoleucine, leucine,
and phenylalanine. These differences are also true for DEAE 3 which
is also high in serine residues. The conclusion from the amino acid work is that pear PPO HA 1 has an amino acid profile similar to other PPOs isolated from higher
plants. This fraction was found to have a higher proportion of
hydrophobic residues and less total charged residues than either HA 2
or DEAE 3. The three multiple forms of pear PPO have the same molecular weight but differ in their isoelectric points and amino acid
profiles.
Protein-Phenolic Interactions
Protein chemists working on the purification of active enzyme
forms cannot dismiss the possibility that these forms are not true
isozymes but instead are artifacts of the purification procedure.
This is particularly true in the extraction of plant enzymes. Several
researchers have warned the plant enzymologist to beware of the 126 interaction of endogenous phenolic compounds with proteins (Loomis and
Battaile, 1966; Loomis, 1969, 1974; Van Sumere et al_., 1975;
Pierpoint, 1966; Anderson, 1968). Phenols can combine with proteins reversibly by hydrogen bonding or irreversibly by oxidation to quinones followed by covalent condensations of the quinones with reactive groups of proteins (Loomis, 1969). The latter reaction can lead to brown polymers of melanoproteins. One good indication that a plant protein has been modified by quinone additions is the browning of the plant extract (Loomis, 1969). In these cases the changes in molecular weight and net charge resulting from protein-phenolic interactions can cause changes in the electrophoretic and chromatographic patterns of protein (Fieldes and Tyson, 1973; Bendall and Gregory, 1963; Smith, 1980). Moreover, according to Loomis
(1969), if only a few or a single quinone group adds to an enzyme molecule, the damage may not be noticeable by a "tanning" of the extract. But the effect could be sufficient to slightly change the charge or some other physical property of the protein, and thus an artifact, not an isozyme, is created.
To observe the effect of two phenolic compounds on the purified extract of pear PPO the following study was undertaken. Chlorogenic acid was chosen since it is one of the major phenolic compounds present in pears (Sioud and Luh, 1966; Ranadive and Haard, 1971). It was of interest to see if the addition of chlorogenic acid to the purified fractions would result in the appearance of other PPO multiple forms and, furthermore, if any of these newly created forms coincided with the three major forms of pear PPO that were studied in 127 this thesis. The results did show an increase in the number of PPO bands on electrophoresis gels after the incubation of PPO fraction,
DEAE 2, with 3mM chlorogenic acid for 20 hr at 40C at pH 7.0. None of the new enzyme forms were similar to the other two forms of PPO. When this same mixture was allowed to react at room temperature for 30 min before electrophoresis no increase in the number of PPO bands was observed. At pH 5.6 and 40C, DEAE 2 plus 3mM chlorogenic acid, was dark brown and had very little enzyme activity after 20 hr.
In an effort to understand these results it should be noted that the pH optimum for d'Anjou pear PPO is 5.0 (Smith, 1980). It was found in this work that the enzyme is five times more active at pH 5.0 than at pH 7.0. Thus, at pH 5.6 PPO is more active and will oxidize the added chlorogenic acid very rapidly to form the corresponding quinones. The quinones then condense with the enzyme to inhibit PPO activity. Reaction inactivation has long been associated with PPO
(Miller and Dawson, 1941; Robb, 1981). At pH 7.0 the enzyme is much
less active and thus loses little activity due to reaction
inactivation. Another factor involved is the ionization state of the
phenolic compound. The pKa for £-diphenols is approximately pH 9.5
(Merck Index, 1976) and, therefore, at pH 7.0 more of the diphenol will be in the ionized form than at pH 5.6. Loomis and Battaile
(1966) have pointed out that in the ionized form the phenols undergo
autooxidation more readily than in the unionized form. These
compounds could then react with the enzyme to yield the additional
bands seen at pH 7.0. Moreover, in a purified system such as this,
the enzyme molecule is the major compound in the solution to which the 128 phenol/quinone can react. The extract was tanned but not dark, and the enzyme had lost little activity; indicating that the quinone concentration was not high enough to inhibit the enzyme.
Leatham e£ al_. (1980) have shown that oxidized chlorogenic acid in a model system was capable of crosslinking proteins at low concentrations to form dimers, trimers, and higher polymers. They indicated that once initiated the crosslinking reaction was slow with chlorogenic acid and required at least 15 hr before changes in molecular weight were noted by SDS electrophoresis. These workers hypothesized that the reaction rate was dependent on two competing reactions that were dependent on the quinone concentration. One was the reaction of the quinone with the protein and the other was the condensation of two quinones. At low quinone concentrations the quinones were limiting in that there were not enough to react with all of the protein.
Another interesting aspect of their work was the discovery of two unknown ninhydrin-positive peaks that eluted during amino acid analysis of the crosslinked extracts (Leatham et aj_., 1980). A decrease in lysine and sulfur amino acids in the quinone-protein mixtures and the appearance of two new peaks between the aromatic amino acids and lysine were reported. They postulated that these new peaks may represent altered amino acids involved in covalent quinone-protein crosslinks. The implications of these observations to the work with pear PPO are intriguing since an unidentified amino acid peak was also seen in fractions HA 2 and DEAE 3 that eluted just before lysine. Also the content of lysine was less in these two 129 fractions than in HA 1; in fact the lysine content of DEAE 3 was less than half of what was found in HA 1. It is certainly possible that the unknown peak represented an altered amino acid involved in a covalent quinone-protein interaction. There were no noticeable changes in the electrophoresis pattern or the color of the extract at either pH 7.0 or pH 5.6 when chlorogenic acid was added to PPO fraction DEAE 3. Either the enzyme was not active enough to produce any reactive quinones or the available lysine groups on the enzyme molecule were already covalently attached to phenolic molecules.
Fields and Tyson (1973) have indicated that covalent bonding between oxidized phenolics and protein tend to decrease the net positive charge of the protein and cause it to migrate further on electrophoresis gels. DEAE 3 did have the largest relative mobility of the three PPO fractions.
Hurrell et al_. (1982) have recently studied the effect on the lysine of casein which had been reacted with caffeic acid aerobically oxidized under alkaline conditions or enzymically oxidized with PPO.
Lysine and tryptophan were the only amino acids severely decreased by the reaction of casein with the oxidized phenol. Their results suggested that each bound lysine unit was associated with several quinone units and proposed the pathway illustrated in Figure 18. From their work and Pierpoint's (1966) the primary point of substitution of lysine was probably into position 6 of the quinone ring. Again, according to Hurrell et al_. (1982) this type of linkage would not tend to release free lysine on acid-hydrolysis. The subsequent reactions of the covalently modified protein-bound lysine was dependent on 130 quinone concentration to form the brown polymerized products. When the quinone concentration was low, further browning may not occur, but the protein is still modified by the attached quinone.
The sulfhydryl groups of cysteine have also been implicated as possible points of attachment for oxidized phenols (Loomis and
Battaile, 1966; Pierpoint, 1966; Synge, 1975; Van sumere et al.,
1975). However the low concentration of cysteine in PPO (only one residue was found in the amino acid sequence of Neurospora PPO by
Lerch (1978) and that was found to be involved in a thioether linkage with a histidine residue) makes it more likely that the quinones would be reacting with protein-bound lysine in PPO.
The second phenolic compound investigated was
2,3-dihydroxybenzaldehyde (DHBA). Sato (1976) reported that DHBA was capable of forming crosslinks with spinach PPO to convert the monomeric form into a dimer without loss in enzyme activity. The presence of DHBA might be able to overcome the dilution effect on PPO and form higher molecular weight species of the enzyme. However, there were no significant changes in the multiplicity of the PPO fractions, DEAE 3 and HA 2, when either 5 or 0.5 mM DHBA were added.
The appearance of a new molecular weight form of PPO was not observed under the conditions described by Sato (1976). OH •O- OH Polyp hcnolic acid
101. HUyrne or ilkall I • 0H 0 OH Neutral and 1 1 ' l" alkaline pH 101 0 _yoH -> Oulnone — 0 NH, " -^1 -O- «< >.o ♦ LYS NH- ■LYS^ NH-LYS^ \ NH-LYS^ /\ Complexes «NH,-LYSs Quinonc Protein-bound between lysine and lysine .YS-NH OH polymerized quinoncs 1 1 \ T * quinons ♦I0| NH-LYS / Cro- tsllnkage
Figure 18. Interaction of Polyphenol with Protein - bound Lysine (Hurrell si fill.. 1.982)
ca 132 CONCLUSION
The purpose of this thesis was to examine the molecular structure of d'Anjou pear PPO in the hopes that this information would lead to a better understanding of the mode of action of PPO and how to control its undesirable effects in foods. Although this work was unable to answer many questions about this enzyme, it has contributed a reasonable amount of knowledge concerning the purification and properties of pear PPO.
The extraction of PPO from d'Anjou pears was accomplished with the phenolic binder, AG 2X-8, and Triton X-100. The chlorophyll pigment present in the pear skins was also extracted, but was removed after chromatography on a hydrophobic column. There were no differences in the multiplicity of the enzyme with the presence or absence of Triton X-100 in the initial extraction procedure.
The purification of pear PPO was achieved after chromatography on
Phenyl Sepharose CL-4B, DEAE-cellulose, and hydroxylapatite columns.
An interesting temperature effect was noted during column chromatography and only after the columns were run at room temperature were sharp peaks and good resolution obtained. The greatest increase in specific activity was due to the Phenyl Sepharose column and it appears that this resin is the key to the successful purification of d'Anjou pear PPO. Three separate fractions of pear PPO were homogeneous when stained for protein with the sensitive silver stain on regular slab electrophoresis gels. However, other minor protein bands were noticed during LDS electrophoresis and only one of the three forms was homogeneous by isoelectric focusing. 133
The effect of DMSO on enzyme activity was investigated and found to significantly increase the activity of pear PPO over the control.
The compatibility of PPO with DMSO suggests that the enzyme is active in a hydrophobic environment.
The molecular weight of the three multiple forms of pear PPO were determined by Sephacryl S-300 gel filtration and LDS electrophoresis.
The gel filtration results indicated a molecular weight of 45,300 while the molecular weight by LDS electrophoresis was 59,600 daltons.
The discrepancy between these two values was attributed to problems inherent in the use of these techniques since the molecular weight standards were globular proteins and the conformation of PPO is not known. The existence of higher polymeric forms of PPO were not found in this work nor did LDS electrophoresis reveal the presence of two different types of polypeptide chains as has been reported for mushroom tyrosinase.
The isoelectric points of the three PPO fractions were: HA 1 - pi 4.3 and 4.5, HA 2 - pi 4.5 and 4.7, and DEAE 3 - pi 6.9. The three fractions also did not have the same amino acid composition. HA 1 contained a higher proportion of hydrophobic amino acids and less total charged residues than either HA 2 or DEAE 3. All three forms contained a high amount of the acidic amino acids and/or their amides, plus glycine. During the amino acid analysis of HA 2 and DEAE 3 an unknown peak was noticed that eluted between histidine and lysine.
This peak was not present in the HA 1 amino acid profile. It was postulated that this peak could be a glucosamine derivative of 134 asparagine or serine or it may represent an altered lysine involved in covalent quinone-protein crosslinks.
A study on the effect of two phenolic compounds on the purified pear PPO extracts was undertaken. Chlorogenic acid was rapidly oxidized by fraction HA 2 at pH 5.6 and the enzyme was inactivated.
At pH 7.0 the oxidized phenol combined with the enzyme to yield an increase in the active multiple forms seen during electrophoresis.
There were no detected changes in electrophoresis patterns at either pH 7.0 or pH 5.6 with chlorogenic acid plus DEAE 3. There were also no significant changes in either PPO fraction when
2,3-dihydroxybenzaldehyde was added to the extracts.
The results indicate that the three multiple forms of pear PPO have the same molecular weight but differ in their isoelectric points and amino acid profiles. These differences could suggest that these three forms are in fact true isozymes. However, there does exist the possibility of artifact formation due to protein-phenolic interactions which cannot be excluded as an alternative explanation for the charge differences observed among the three forms of pear PPO. 135
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