AN ABSTRACT OF THE THESIS OF

Pedro A.E. Wesche-Ebeling for the degree of Doctor of Philosophy in Food Science and Technology presented on August 30, 1983

Title: Purification of Strawberry and its Role in Anthocyanin Degradation.

Abstract approved: .,.,_... Dr. Morris W. fmptgomery|

Polyphenol oxidase (PPO) from strawberries was purified, partially characterized, and used to study its role in the degradation of anthocyanin pigments. The extraction method included the use of a buffered solution containing the phenolic binders Polyclar AT and Amberlite XAD-4. Addition of Triton X-100 resulted in an increase of the .extracted activity. Higher levels of PPO activity were obtained using citrate rather than acetate in the buffer. This was possibly due to the capacity of citrate to chelate calcium ions, and, therefore, to inhibit crosslinking of the pectic polymers which would trap the . Using a Phenyl Sepharose CL-4B (PS) hydrophobic column two fractions of the enzyme, Fl and F2, were separated from the pectic material and the bulk'of the 280 nm absorbing material. PP0-F1 obtained from the PS column was less hydrophobic than F2, and showed a molecular weight of 111,000 by gel filtration on Sephacryl S-300. Form Fl eluted in the void volume of an anion exchange column and did not penetrate into the running gel during electrophoresis. The binding of PP0-F1 to a Concanavalin-Agarose (ConA) column demonstrated the presence of carbohydrate associated with the enzyme. PP0-F2 showed a molecular weight of 34,500 and could be resolved by anion exchange chromatography into one large peak and a smaller one at its shoulder. Fraction F2 of the enzyme resolved into two bands during electrophoresis, and did not bind to the ConA column, Both fractions of the enzyme showed multiple banding on lithium dodecyl sulfate -- polyacrylamide gradient gels. Both fractions of the enzyme obtained from the PS column showed very high activity in the presence of D-. The Michaelis constants for D-catechin for PPO fractions Fl and F2 were 0.50mM and O.AlmM respectively, and the maximum velocity 82,700 and 18,800 nmoles ■? f 0^ per min per ml respectively. D-catechin in the presence of PP0-F1 was rapidly oxidized and the resulting solution had a maximum absorption at 390 nm. When D-catechin and PP0-F1 were combined in model systems with either pure cyanin or pelargonin, an absorbance peak at 390 nm was noticed with increased absorbance in the region of the anthocyanin pigments. After 24 hr, 50% of the pelargonidin and 60% of the cyanin was destroyed and a brown precipitate was formed. PPO-Fl seemed to oxidize cyanin at a very slow rate but did not oxidize pelargonin. It was suggested that the anthocyanin pigments were destroyed by either the direct oxidation by the formed from D-catechin by PPO, or that the anthocyanin pigments were cc-polymerized into the brown polymeric pigment, tannin, formed from D-catechin- . Purification of Strawberry Polyphenol Oxidase and its Role in Anthocyanin Degradation.

by

Pedro A. E. Wesche-Ebeling

A THESIS submited to Oregon State University

in partial fulfillinent of the requirements for the degree of

Doctor of Philosophy

Completed August 30, 1983

Commencement June 1984 APPROVED:

«-* * • Professor of Food Science and TechnoQ/gy in change of the major

-' Head of departmentT of Food Science and Technology

Dean of Graduateie) School Q

Date thesis presented August 30, 1983 ACKNOWLEDMENTS

I wish to express my appreciation to Dr. Morris W.Montgomery for his interest, help, encouragment, and guidance during the course of this investigation. Appreciation is expressed to Dr. W.David Loomis for his helpful suggestions and advice concerning this research, and to Dr. Ronald E. Wrolstad and Dr. Daryl G. Richardson for their help and for serving in my graduate committee, as well as for the opportunity to continue working with them in post-doctoral research. I also wish to thank Luis Sayavedra, Kim Wissemann/ Bonnie and John Weber, Lauren Mack, Ed Gaines, Richard, Brenda and Flora, Scottie, Susan and Michael, Rich Daniels, Fred Pfeil, Flo Leibowitz and many others for their friendship and for making me feel at home in Corvallis. Special thanks go to Daryl Monk for having made hi-tech available for the preparation of this thesis. I would also like to express my greatfulness to CONACYT for funding my graduate studies at O.S.U. Very special appreciation goes to my family and to Isa Roemer for their unfailing support throughout all these years. THE LOW ROAD by Marge Piercy

What can they do to you ? Whatever they want.

They can set you up, they can bust you, they can break your fingers, they can burn your brain with electricity, blur you with drugs till you can't walk, can't remember; they can take away your child, wall up your lover; they can do anything you can't stop them doing.

How can you stop them ? Alone you can fight, you can refuse. You can take what revenge you can but they roll right over you.

But two people fighting back to back can cut thro' a mob a snake-dancing can break a cordon, termites can bring down a mansion. Two people can keep each other sane, can give support, conviction, love, massage, hope.

Three people are a delegation, a committe, a wedge. With four you can play bridge and start a collective. With six you can rent a whole house, have a pie for dinner with no seconds and make your own music.

Thirteen makes a circle, a hundred fill a hall. A thousand have solidarity with your own newsletter; ten thousand, community and your own papers; a hundred thousand, a network of communities; a million, your own world.

It goes one at a time. It starts when you care to act. It starts .when you do it again after they say no. It starts when you say we and know who you mean; and each day you mean one more. TABLE OF CONTENTS

Page INTRODUCTION 1

LITERATURE REVIEW 3

Polyphenol oxidase 3 Physiological Roles 3 Subcellular Location 10 Molecular Properties 13 Latency 14 Molecular Weight and Multiple Forms 14 Primary and Secundary Structure 16 and the 17 Reaction Mechanisms 21 Specificity 27 Effects of the Polyphenol Oxidase Reaction 30 Inhibition of Enzymatic Browning 32 Physical Methods 32 Effects of the Source of PPO 33 Removal of Phenolic Compounds 35 Removal of Quinones 36 Inhibition of PPO 36 The Strawberry System 41 Strawberry Structure 41 Chemical Composition 42 Strawberry Cell Wall 46 Morphological Changes in the Cell Wall 47 Cell Wall Structure 49 Mechanisms of Cell Expansion 51 Anthocyanin and Color Degradation Mechanisms 54 Effects of pH 55 Effects of Sugar Hydrolysis and Light 60 Effects of Heat and Oxygen 62 Effects of Sulphur Dioxide 63 PaEe

Effects of Heavy Metals 63 Effects of Non-enzymatic Browning 65 Effects of Enzymatic Browning and Phenolic Compounds... 68 Endogenous PPO Substrates 69 Mechanisms of Anthocyanin Degradation 70 Non-covalent Interactions of Anthocyanins 70 Formation of Tannins 71 Covalent Interactions of Anthocyanins 76 STRAWBERRY POLYPHENOL OXIDASE: PURIFICATION AND CHARACTERIZATION 83 ABSTRACT 84 INTRODUCTION 85 EXPERIMENTAL 86 Enzyme Extraction 86 PPO Activity Measurement 87 Hydrophobic Chromatography 87 DEAE-Cellulose Anion Exchange Chromatography 88 Concanavalin-Agarose Chromatography 88 Molecular Weight by Gel Filtration 89 Electrophoresis 89 Detection of Proteins 90 Detection of PPO 90 Protein and Pectin Content Determination 90 RESULTS AND DISCUSSION 91 Optimization of the Extraction Procedure 91 Separation of the Pectic Material 93 Phenyl Sepharose CL-4B Chromatography 95 Electrophoresis and Ion Exchange Chromatography 97 Molecular Weight Determination 98 Concanavalin-Agarose Chromatography 98 LDS-Electrophoresis 100 CONCLUSIONS 100 APPENDIX 102 REFERENCES 114 Page

STRAWBERRY POLYPHENOL OXIDASE AND ITS ROLE IN ANTHOCYANIN DEGRADATION 119 ABSTRACT 120 INTRODUCTION 121 EXPERIMENTAL 122 PPO Extraction and Purification 122 Substrate Specificity and Kinetic Studies 123 Studies of the Interaction between PPO, D-catechin, and Anthocyanin Pigments 124 RESULTS AND DISCUSSION 125 Importance of the PPO-D-Catechin Reaction 127 CONCLUSIONS 134 REFERENCES 143 BIBLIOGRAPHY 147 LIST OF FIGURES

Figure Page

I. 1 Biosynthetic Relationships Between the Major Groups of Phenolic Compounds 7 I. 2 Effect of pH on Anthocyanins Pigments 58 I. 3 Some Proposed Paths of Anthocyanin Degradation 61 I. 4 Reactions of Pro- and Leucoanthocyanidins 75 I. 5 Some Mechanisms and Products of Anthocyanin Co-polymerization 80

II. 1 Phenyl Sepharose CL-4B Chromatography of Strawberry Polyphenol Oxidase 106 II. 2 Binding of PPO Form F-1 to Phenyl Sepharose CL-4B and its Elution with and without the Presence of Phenyl- alanine in the Elution Buffer 108 II. 3 DEAE-Cellulose Chromatography of Strawberry PPO Form F2 Obtained from Phenyl Sepharose Chromatography 109 II. 4 Molecular Weight of Strawberry PPO Forms Fl and F2 by Sephacryl S-300 Superfine Gel Filtration 110 II. 5 Concanavalin-Agarose Chromatography of Strawberry PPO Form Fl obtained from Phenyl Sepharose Chromatography Ill II. 6 Polyacrylamide Gel Electrophoresis of Different Fractions of Strawberry PPO 112

III. 1 Continuous Scanning of the Reaction Between PPO (Form Fl), D-Catechin, and Cyanin of Pelargonin 140 III. 2 Absorption Spectra of Selected Models Containig Combinations of PPO (Form Fl), D-Catechin, and Cyanin and Pelargonidin after 24 hr of Storage at 20oC 142 LIST OF TABLES Table Page

I. 1 Polymeric Phenolic-Binding Agents 38 I. 2 Agents used for the Removal of Quinones 39 I. 3 Methods and Agents used for the Direct Inhibition of PPO 40 1.4 Chemical Composition of Strawberry Fruits 43

II. 1 The Efffects of Different Extraction Procedures on the Activity of Ripe Strawberry PPO 103 II. 2 The Effect of the Type of Buffer and of the Precipitation of Pection with Calcium Acetate on the Activity of Ripe Strawberry PPO 104 II. 3 Purification of the Polyphenol Oxidase from Unripe Strawberries 105

III. 1 Activity of Strawberry PPO Measured Using Phenolic Compounds Found Naturally in the Fruit 137 III. 2 Kinetic Characteristics of the Two PPO Forms Obtained after Chromatography on Phenyl Sepharose Columns 138 III. 3 Effects of PPO (Form Fl) and D-Catechin on both Cyanin and Pelargonin after 24 hr of Storage at 20oC.... 139 PURIFICATION OF STRAWBERRY POLYPHENOL OXIDASE AND ITS ROLE IN ANTHOCYANIN DEGRADATION

INTRODUCTION

Enzymatic browning is a major cause of losses during the harvesting, handling, storage, and processing of fruits. Browning usually leads to changes in the organoleptic, nutritional, and color qualities of fruits and fruit products. Polyphenol oxidase (PPO) is the enzyme responsible for the oxidation of endogenous fruit phenolic compounds leading to the formation of highly reactive quinones and to polymerization reactions that result in the formation of the brown pigment . PPO is found widespread in the Plant Kingdom and has been found in all orders of the Angiosperms studied so far (Mayer and Harel, 1979). In order to be able to effectively inhibit the action of this enzyme it is important to know the molecular and biochemical characteristics of the enzyme from each source. This can only be done using purified enzyme. The purification of from plant tissues is confronted with the presence of high levels of endogenous phenolic compounds, low levels of protein, and in some cases, high levels of soluble pectin as in strawberries. With the advent of effective phenolic-binding agents it has been possible to reduce the probability of the tanning of enzymes by the phenolic compounds and quinones. The purpose of this dissertation was to purify the PPO from strawberries and to use the purified enzyme to study its involvement in the degradation of anthocyanin pigments. The purification steps included procedures that protected the enzyme from the action of phenolic compounds and , increased solubilization of bound enzyme, and the separation of the enzyme from the pectic material. The enzyme was purified using Phenyl Sepharose affinity chromatography, DEAE anion-exchange chromatography, and Concanavalin-agarose affinity chromatography. The molecular weight of different forms of the enzyme was determined by gel filtration. The purified forms of the enzyme were used in model systems containing D-catechin, cyanin, and pelargonin, all of which are found endogenously in the strawberry fruit. This dissertation is divided into three sections. The first section, the Literature Review, includes a thorough review of both PPO and the strawberry fruit, and should be considered as an extension of the discussion of the results and observation described in the other two sections. This Literature Review was written to complete the preceeding review (Wesche-Ebeling, 1981). The second section deals with the purification and characterization of the PPO, and the third section deals with the study of the involvement of PPO in the degradation of anthocyanin pigments. LITERATURE REVIEW

Polyphenol Oxidase

Polyphenol oxidase (PPO) is an enzyme that is widely distributed in all living organisms including fungi, plants, and animals. PPO is also known as , _o -diphenol oxidase, catecholase, and phenolase. The Commission on Enzymes originally refered to two enzymes: laccase or _j3 -diphenol:oxygen (EC 1.10.3.2) and PPO or o^ -diphenol:oxygen oxidoreductase (EC 1.10.3.1). The Commission has now revised the nomenclature, lumping both enzymes together as EC 1.14.18.1 monophenol monooxygenase. In this review particular attention will be focused on PPO from higher plants.

Physiological Roles

No specific physiological function has been assigned to PPO. Recent studies concerning the subcellular location of the enzyme have made it possible to relate the reactions catalyzed by PPO to certain metabolical functions in higher plants. It has not been possible to demonstrate the direct participation of PPO in in vivo reactions for which the enzyme's in vitro activity has been clearly demonstrated. It has also not been possible to verify if the physical properties observed for the enzyme in vitro occur in vivo , e.g. latency, substrate specificity, and the /.oxidation activity ratio. In this section, some of the physiological functions of the enzyme for which more recent documentation exists will be described. Shomer et al., (1979) found PPO in chloroplast thylakoids and mitochondria in young olive fruits. The presence of the enzyme in these organelles suggests a participation in metabolic pathways occuring in them. Some suggested roles for the PPO complex are in catalyzing the transfer of electrons from products of the intermediate metabolism (e.g. during the biosynthesis of phenolic compounds) to oxygen (Mason 1955), as a terminal oxidase of systems oxidizing NADH or NADPH (Butt, 1980), or with o^ -quinone in oxidation-reduction during electron transport (Taneja and Sachar, 1974). During some of the proposed activities of PPO o^ -quinones would be formed as products of the reaction, but no quinone reductase has been isolated from any plant tissues, and the existence of a non-specific, non-enzymatic reduction of quinones in vivo is in doubt. The powerful inhibition of PPO by substituted hydroxamic acids, compounds known for their inhibition of the cyanide-insensitive respiration of plant tissues suggests that PPO may have some function in this pathway in mitochondria (Mayer and Harel, 1979). At present there is no proof of PPO's involvement in photosynthesis. PPO has low affinity for molecular oxygen, at least in vitro . There is no data available about the in vivo kinetic characteristics of the enzyme or of the mechanism of control of PPO activity. Changes in pH and in the level of oxygen in the chloroplast and in the intact tissue occur during photosynthesis and during the activity of ion pumps and respiration. These changes in pH and oxygen may regulate the in vivo activity of PPO, which in turn may regulate the concentration of free oxygen in the chloroplasts (Lerner et al., 1972; Mayer and Harel, 1979). One of the most evident manifestations of PPO activity is the formation of a protective layer through "browning" reactions in injured plant tissues. This may occur as a response to factors causing stress or injury such as mechanical damage, freezing and thawing, adverse climate, exposure to chemicals such as heavy metals, ethylene, ozone, or infection by fungi, bacteria or viruses. Each of the different factors may elicit a different metabolic response, but the purpose of all is the protection of the undamaged tissues (Rhodes and Wooltorton, 1978; Vamos-Vigyazo, 1981). The participation of PPO in disease resistance has been widely supported (Bell, 1974; Rhodes and Wooltorton, 1978; Friend, 1981). Phenolic compounds may be involved in barriers present before infection, e.g. , or in those formed in wound-healing reactions, e.g. quinones and tannins. The production of phenolic compounds in infected, as well as in damaged or senescent tissues has been shown to be accompanied by an increase in the enzymes involved in the biosynthesis of phenolic compounds and in PPO activity (Cheung and Henderson, 1972; Butt, 1979; Chattopadhyay and Bera, 1980), and in the increase in the number of subepidermal phenolic-storing cells (Martyn et al., 1979). The increase in PPO may be due to activation of latent enzyme or de novo synthesis (Mayer and Harel, 1979). PPO activity can be induced by pathogen produced enzymes or their degradation products (Farkas and Ledingham, 1959; Deverall and Wood, 1961). Injury or stress can lead to the loss of contents from broken cells, to metabolic responses in cells close to the site of injury, and to the premature death of some cells in the vicinity of the wound or infection, e.g. the hypersensitive response (Rhodes and Wooltorton, 1978). The first response to stress is the oxidation of pre-existing phenolic compounds by PPO. Many phenolic compounds have been shown to be toxic to pathogens, but the highly reactive quinones have a more extensive effect. They may be toxic to pathogens or react with the enzymes produced by them directly or after polymerization (Rhodes and Wooltorton, 1978; Mayer and Harel, 1979). Some pathogens can also use an endogenous PPO-quinone system to detoxify the toxic compounds produced by the host (Farkas and Ledingham, 1959). During the hypersensitive response there is widespread damage to the membranes of subcellular organelles (Bell, 1974). Freezing and thawing, ozone, and senescence can also lead to membrane breakdown, the release of phenolic compounds from the and of hydrolytic enzymes from the lysosomes, i.e. lipases. Recently it has been found that free fatty acids could activate latent PPO (Golbeck and Cammarata, 1981). The role of PPO during the response of plant tissues to stress does not seem to be only in the oxidation of endogenous phenolic compounds, but it seems now that PPO has an active role in their biosynthesis, specifically in the hydroxylation of phenolic compounds (Mason, 1955,1956). The contents of those derived from the hydroxycinnamate and the shikimate pathways are increased as a response to stress, as well as the activities of PPO, peroxidase and enzymes involved in these biosynthetic pathways (Bell, 1974). The phenolic compounds synthesised are either accumulated in the or deposited in the cell wall (Friend,1981; Fry, 1983). The site for the biosynthesis of cinnamic acids has been suggested to be the choloroplasts (Mueller and Beckman, 1978; Vaughan and Duke, 1981). Lignin, suberin and cinnamic acid derivatives are found in cell walls of healthy and in higher quantities in injured plant tissues. All of these polyphenols are derived from cinnamic acid monomers. Suberin contains cinnamic acids covalently bound to long-chain fatty acids and lignin contains in its structure covalenty bound cinnamic acid alcohols. Cinnamic acids have also been found to be covalently bound to cell wall polysaccharides, e.g. esterified to pectin and forming diferulate or dicoumarate bridges (Rhodes and Wooltorton, 1978, Fry,1983). The peroxidase-catalysed oxidative coupling of phenolic compounds in the cell wall leads to the strengthening of the wall strucure, forming a barrier against pathogens or making the cell wall polysaccharides sterically inaccesible to pathogenic enzymes (Friend, 1981). The biosynthetic pathways leading to the synthesis of cinnamic acid and derived phenolic compounds is shown in Figure 1.1. Most of the enzymes involved in the biosynthetic reactions have been isolated and studied, except for p -coumarate-3-hydroxylase, reaction 3, and the hydroxylation of to 5-hydroxyferulic acid, reaction 8, which has not yet been enzymatically characterized (Rhodes et al., 1981). Both reactions 3 and 8 as well as the hydroxylation of £ -coumaroylquinic acid to have been proposed as PPO catalyzed reactions. Vaughan and Butt (1972) demonstrated the conversion of _£ -coumaric to by spinach beet PPO and proposed that the enzyme is stabilized for hydroxylation in vivo possibly by the presence of NADPH or of reduced hydroxypteridine. It was also proposed that this enzyme had three -binding sites, one for the _o -dihydroxy phenol acting as in the hydroxylation reaction, and one each for mono- and £ -diphenol substrates (Mclntyre and Vaughan, 1975). Figure I.l BIOSYNTHETIC RELATIONSHIP BETWEEN THE MAJOR GROUPS OF PHENOLIC COMPOUNDS (RHODES AND WOOLTORTON, 1978).

A. Enzymes involved: 1. AMMONIA 2. CINNAMIC ACID HYDROXYLASE 3. £ -COUMARATE HYDROXYLASE (PPO?) 4. O-MEIHYL 5. HYDROXYCINNAMATE CoA 6. HYDROXYCINNAMYL CoA REDUCTASE 7. NADP REDUCTASE 8. UNCHARACTERIZED ENZYME (PPO?) 9. PEROXIDASE

B. Biosynthetic Pathways: See next page. PHENYLALANINE M o-COUMARIC ACID — CINNAMIC ACID

CINNAHIC ACID GLYCOSIDES p-COUMARYL ~L p-COUMARYL JL p-COUMARYL JL p-COUMARIC L . ALCOHOL ALDEHYDE CoA ACID 31 ■ / CAFFEIC ACID

9^_ CONIFERYL 7 CONIFERYL ^6 FERULYL ^6 FERULIC ACID » Ar-CH=CH-COSCoA ALCOHOL ALDEHYDE CoA 8| 5-HYDROXY FERULIC ACID :\ \ SINAPYL 7 SINAPYL 6 SINAPYL ^6 SINAPIC ACID ALCOHOL ALDEHYDE CoA CHALCONES FLAVANONES

QUINATE AND SHIKIMATE ESTERS / \ ISOFLAVONOIDS

00 Both _£ -coumaric and ferulic acids have also been reported to be competive inhibitors of PPO during the oxidation of o^ -diphenols (Pifferi et al., 1974; Walker, 1976). In most of the plant tissues studied, the g_ -coumarate-3-hydroxylase activity of PPO could not be separated from the oxidase activity (Parish, 1972; Patil and Zucker, 1965; Vaughan and Butt, 1975). In sorghum and wheat, the PPO-hydroxylase activity could be separated from the bulk of the PPO oxidase activity (Stafford, 1976; Taneja and Sachar, 1974). In spinach beet seedlings exposed to light there was an increase in JD -coumarate-3-hydroxylase activity in the hypocotyl, but not in PPO-oxidase activity, which was mainly associated with the cotyledons (Bolwell and Butt, 1983). Brassica tissue has been shown to metabolize _g. -coumarate to caff eate but it lacks phenolase activity (Rhodes et al., 1981). Possibly two hydroxylating enzymes, one specific for 4-hydroxy-cinnamates and the other associated with PPO-oxidase activity, each possibly found in a different location within the cell, or that £ -coumarate-3-hydroxylase forms part of a multienzyme complex bound to a membrane (Stafford, 1974). It has been noticed that borate deficiency in plants leads to massive accumulation of quinones and melanitic granules, due to an increase in the oxidase activity of PPO (Dugger, 1973), and to poor over-all lignification (Lewis, 1980). It is also known that borate inhibits PPO activity by complexing with the o-dihydroxyphenol substrates (McVicar and Burris, 1948). Boron seems to play a regulatory role in the hydroxylation of _£_ -coumarate and ferulate through the formation of complexes with the o-dihydroxy intermediates and therefore prevents the formation of o-quinones. Boron may have become essential only after sucrose had been selected as the principal carbohydrate for translocation in the phloem, since sucrose does not contain cis-hydroxyl groups for boron complexing. More inforation is needed on the compartmentation of boron and phenolic acids (Lewis, 1980). PPO does not seem to be involved in the hydroxylation of compounds (Schill and Grisebach, 1973; Duke and Vaughan, 1982). 10

Coggon et al., (1973) reported that tea PPO can epimerize tea flavonols at their C-2 position, and that flavonol degallation occured as a consequence of the PPO oxidative activity. The involvement of PPO is doubtful in regulating auxin concentration by oxidation of tryptophan to form indoleacetic acid in vivo (Mayer and Harel, 1979). PPO also appears to be involved in the protection of seeds. One process could be through the melanization and impermeabilization of seed coats (Pierpoint 1970). It has also been noticed that during fruit development higher levels of PPO activity exist during the early stages when the seeds are not yet mature. High levels of PPO offer protection against predators or pathogens. As the fruit ripens, the PPO levels decrease and the enzyme becomes more soluble (Mayer and Harel, 1979). The rise in PPO activity in damaged or senescent cells may contribute to the release and turnover of carbon dioxide and nitrogen. Primary amines and amino acids condense with quinones equimolarly and react further by condensation with one of the carbonyl groups of the quinones. This leads to the release of ammonia and alpha-keto acids which are then decarboxylated (Butt, 1979; Trautner and Roberts, 1950). PPO may also be involved in the formation of humus through catalyzed by quinones (Bate-Smith and Metcalfe, 1957). From the discussion above it can be pointed out that plant tissues may respond to cuts, bruises, freezing and thawing, ethylene, etc. in a similar manner to that described above, that is, with an increase in PPO activity, possible /de_novo/ synthesis of the enzyme, and/or an increase in phenolic compound synthesis and accumulation during storage.

Subcellular Location

The study of the subcellular location of PPO is subject to the same problems encountered during the extraction of plant enzymes (Anderson, 1968; Loomis, 1974; Wesche-Ebeling, 1981). If proper precautions are not taken during tissue homogenization or enzyme 11 extraction procedures, endogenous phenolic compounds and quinones derived from them through PPO-catalyzed oxidation or autoxidation will interact with the enzymes. This "tanning" of the enzymes can lead to the formation of large protein-phenolic complexes and to aggregation with other subcellular components or even organelles (Anderson, 1968). Tanned PPO may retain activity and show characteristics of a 'bound' enzyme. Complexes of PPO with other enzymes, e.g. multienzyme complexes, or with cell structures may not always be artifacts. The tendency of such associations might be reflecting valid in vivo relationships of significance (Stafford, 1974). Inadequate attention has been paid to the presence of PPO in the different parts of the fruit and to the subcellular location of the enzyme, both in the ripe fruit and during fruit development (Mayer and Harel, 1981). PPO seems to be unevenly distributed in tissues or sections of fruits and other plant organs (Vamos-Vigyazo, 1981). In avocadoes, the distal end (blossom) of the fruit contained higher levels of phenolic compounds and of PPO activity (Golan et al., 1977). Dwarf banana PPO shows higher activity in the inner part of the pulp (Galeazzi et al., 1981). The leaves from bean plants do not exhibit dark brown melanotic patches upon wounding due to low PPO levels (Racusen, 1970). The vascular elements of a number of fruits show more rapid darkening than other tissues in the fruit (Joslyn and Ponting, 1951). PPO has been found in the soluble fraction of some plant extracts. A very important experiment by Sanderson (1964) demonstrated that the reportedly 'bound' tea leaf PPO could be made soluble if the phenolic material was removed during the extraction using polycaprolaktam. Kato et al., (1976) used Polyclar AT to bind phenolics during the extraction of PPO from tea leaves and found most of the enzyme in the supernatant. Some PPO in this tissue was also found associated with peroxisomes. PPO has also been found in the soluble fractions of potato tubers (Craft, 1966), sugar cane leaves (Coombs et al., 1974) and in ripe olives (Ben-Shalom et al., 1977a). Peroxidase, IAA oxidase and PPO were found to be released into the medium of peanut cell suspension cultures (Srivastava and van Huystee, 12

1977). Evidence for the occurence of PPO bound to membranes of subcellular organelles is based on data obtained using ultracentrifugation and combined histochemical and microscopic techniques. Using DOPA as a substrate, PPO activity is suggested to cause the formation of electron-dense regions which can be seen under the electron microscope. Using this technique on tobacco and water hyacinth leaves it was found that PPO was localized in the membrane-bound granular component and the thylakoids of chloroplasts (Czaninski and Catesson, 1972; Henry, 1975; Martyn et al., 1979). Differential centrifugation and density gradient centrifugation together with the use of marker enzymes and some compounds, e.g. chlorophyll, are also used as evidence for the subcellular location of PPO. The weight of the evidence seems to indicate that most PPO's are membrane-bound, particularly in the chloroplasts. Tolbert (1973) studied the presence of PPO in a wide variety of plant leaves and found most of the activity to be located in the chloroplasts. PPO was also found in the chloroplasts of spinach beet leaves, and the activity was found to be dependent on the integrity of the thylakoid membranes (Parish, 1972; Lieberei and Biehl, 1978; Meyer and Biehl, 1980, 1982). PPO has also been reported in chloroplasts in green olives (Ben-Shalom et al., 1977), grapes (Harel and Mayer, 1971), wheat (Taneja and Sachar, 1974), and cotton seedlings (Mueller and Beckman, 1978). PPO is associated with vesicles from inner envelopes in young chimera leaves. The envelope may be the site for accumulation or transport of PPO into the (Vaughn et al., 1981). Incorporation of cytosolically-produced PPO into the chloroplasts does not require chloroplast ribosomes or ATPase. It seems that tentoxin, a cyclic pentapeptide produced by Alternaria which induces chlorosis, may affect the transport of PPO from the cytosol to the plastids (Vaughn and Duke, 1981,1982). Association of PPO with other organelles include mitochondria, e.g. spinach beet (Parish, 1972) and green olive fruits (Shomer et al., 1979), and peroxisomes in tea leaves (Kato et al., 1976). 13

Molecular Properties

Most of the information obtained on the molecular properties of PPO have come using Neurospora or Agaricus () as the source for the enzyme. Relatively high yields of pure enzyme needed for the molecular studies can be obtained from these sources. It has been more difficult to obtain pure PPO from higher plants. In these cases, a common source of artifacts is the auto-tanning of PPO by endogenous phenolics and quinones formed during the extraction through enzymatic action or autoxidation (Anderson, 1968; Loomis, 1974). These tanning reactions may change the properties of the enzyme including its apparent molecular weight, electrophoretic mobility, multiplicity, and kinetic characteristics. The use of phenolic-binding polymers and other protective measures during the extraction of the enzyme may prevent or reduce the tanning of the enzyme (Sanderson, 1964; Loomis et al., 1979; Rhodes, 1977). However there appears to be other sources of enzyme modification. Some of the most widely used extraction procedures include the preparation of acetone powders and the 'solubilization' of membrane-bound PPO using detergents, limited proteolysis, etc. These procedures may induce modification in the enzyme structure and/or conformation, as well as activation or inactivation, changes in substrate specificity (i.e. loss of hydroxylase activity), pH optimum, and other properties in the enzyme (Lerner et al., 1972; Walker and Wilson, 1975; Ben-Shalom et al., 1977a; Mayer and Harel, 1979). Other factors that may affect the characteristics of the enzyme besides the conditions of the extraction are the concentration of the enzyme, the length of storage, the age of the fruit or tissue, substrate or inhibitor binding, and the type of tissue. It is not known if the molecular characteristics observed for the enzyme in_ vitro reflect the characteristics in the intact tissue. 14

Latency

It has been reported that PPO exists in a latent state in many tissues. Some of the factors that have been reported to cause the activation of latent PPO are acid or alkaline conditions, anionic wetting agents (i.e. SDS), urea, guanidine chloride, high ionic strength, carboxymethylcellulose, detergents, limited proteolysis, and freezing and thawing (Kenten, 1957a,b; Robb et al., 1964; Mayer, 1966; Sato and Hasegawa, 1976; Ben-Shalom et al., 1977b; Lieberei and Biehl, 1978; Meyer and Biehl, 1981; Butt and Lamb, 1981). Activation of PPO may also be induced by factors such as senescence and light (Meyer and Biehl, 1980), ripening and ageing, frost (Lieberei and Biehl, 1978), loss of chloroplast integrity (Meyer and Biehl, 1980), trace elements in fertilizers and to agents promoting ripening (Vamos-Vigyazo, 1981). The activation of latent PPO may be due to a rearrangement of the tertiary structure of the enzyme and not to the liberation of masking subunits (Robb et al., 1965). A change in the Stoke's radius of the enzyme after activation may indicate involvement of conformational changes (Lerner and Mayer, 1975). Activation may be also due to monomer-oligomer transitions, i.e. association-dissociation processes (Mathew and Parpia, 1971; Lerner et al., 1972), or the dissociation of an enzyme-inhibitor complex (Interesse et al., 1980). It is not known if latency is a phenomenon that occurs in the intact tissue. The increase in PPO activity following stress or at the different ripening stages has been ascribed to de novo synthesis, although this could also be explained by the activation of the existing latent enzyme (Mayer and Harel, 1979).

Molecular Weight and Multiple Forms

Accurate determination of the molecular weight (MW) requires the use of pure enzymes. The most accurate results are obtained using sedimentation equilibrium, but in most of the cases the MW's of the enzyme are based on estimates, e.g. gel filtration and SDS-polyacrylamide gel elecrophoresis (Mayer and Harel, 1979; Robb, 15

1981). The best data have been obtained for mushroom and Neurospora PPO. For mushroom PPO, an acidic enzyme, the MW for the predominant form ranges from 116,000 to 133,000 daltons (Kertesz and Zito, 1962, Jolley et al., 1969). This form seems to be composed of two types of polypeptide chains, light (L=13,400) and heavy (H=43,000), forming a tetramer of quaternary structure L2H2 (Strothkamp et al., 1976). Hydrodynamic studies of the enzyme suggest a globular protein (Sharma and Ali, 1981). Neurospora PPO is slightly basic consisting of only one subunit distributed among a number of forms from monomer to tetramer, ranging in MW from 47,000 to 170,000 (Robb and Gutteridge, 1981). The study of the MW and multiplicity of PPO of higher plants is more complex. Any modification to the enzyme during the extraction may affect its mobility during electrophoresis or gel filtration and therefore the estimated MW. Membrane bound PPO may not behave like globular proteins used as calibration standards in gel filtration and different buffers often change the elution profile during gel filtration due to alteration of the Stoke's radius of the protein (Lieberei et al., 1981). Coombs et al., (1974) report a tetrameric form (MW=130,000) predominating for the soluble PPO from sugar cane leaves with the monomers having a MW of 32,000 using gel filtration. Flurkey and Jen (1979) report the MW for peach PPO of 95,000 and 81,000 using gel filtration, and 100,000 and 90,000 using SDS-electrophoresis. PPO from spinach leaves had a MW estimated by gel filtration of 240,000 and of 43,000 by SDS-electrophoresis (Lieberei et al., 1981). The heterogenicity or multiple forms of PPO has been related to protein association-dissociation behavior of like or different () subunits, to conformational isomerism, or a combination of the three (Jolley and Mason, 1965). Interconversions of multiple forms can be reversible (Sato, 1976) or irreversible (Lerner et al., 1972; Harel el al., 1973). Most of the reported "isozymes" show differences in heat stability, pH optimum, chromatographic and electrophoretic mobilitity, and reaction kinetics (Wong et al., 1971; Rivas and 16

Whitaker, 1973). The presence of PPO of different primary structure, true isozymes, appears to be firmly established in mushroom, Neurospora , and the mammalian melanotic melanocyte (Vanneste and Zuberbuhler, 1974). True isozymes have been reported to occur in wheat (Taneja and Sachar, 1974), grapes (Harel et al., 1973), and in Broad beans (Robb et al., 1965). Spinach leaf PPO shows ten different forms in electrophoresis which are not interconvertible in vitro but were immunologically related (Lieberei et al., 1981). Vamos-Vigyazo (1981) reports an extensive list of multiple forms of PPO from various plant sources. Multiplicity could be a phenomenon occuring in the intact tissues, but it could also result from the release of membrane-bound forms of the enzyme, partial denaturation, fragmentation, proteolysis, activation of latent forms, association with other enzymes, tanning reactions, etc. (Lerner and Mayer, 1975; Srivastava and van Huystee, 1977; Pierpoint, 1970; Mayer and Harel, 1979). 'Isozymes' not originally present in the purified enzyme preparation have been reported to be formed during storage (Coggon et al., 1973). Differences in '' patterns have been reported in connection with subcellular organelles, the stage of development of the tissue, or as a result of attack by pathogens (Samorodova-Bianki and Bazarova, 1971; Mayer and Harel, 1979). Data about the effect of multiplicity on the kinetic characteristics of the enzyme in vivo would be of great significance. More research is required in order to establish if multiplicity is a phenomenon occuring in the intact tissue (Vanneste and Zuberbuhler, 1974).

Primary and Secondary Structures

PPO from diverse sources resemble each other in composition, hydrophobic residues content, the size of the single subunits, and in association/dissociation behavior. The similarities are particularly striking in the basic amino acids and in the content of threonine, serine, glycine, alanine and isoleucine (Vanneste and 17

Zuberbuhler, 1974; Mayer and Harel, 1979). A relatively low content of sulphur amino acids has been reported in PPO's from various sources (Mayer and Harel, 1979), and no cysteine was found in broad bean PPO (Robb et al., 1965). Some of the amino acid sequences and composition that have been reported for higher plants are for spinach beet (Vaughan et al., 1975), grape (Kidron et al., 1977), potato (Balasingham and Ferdinand, 1970) and, pear PPO (Wisseman, 1983). The Neurospora PPO structure has been studied in great detail (Lerch, 1978). It consists of a single polypeptide chain of 407 amino acids and contains an acetylated N-terminal amino acid. The single cystinyl residue is covalently linked via a thioether bridge to a histidyl residue. This bridge is generated in a post-translational modification event and possibly linked to the activation of (latent) pro-PPO. The amino acid sequence indicates the presence of 34% alpha-helical, 15% beta-sheet, 31% beta-turn, and 20% coil regions. The content of hydrophobic residues of PPO seems to be consistently around 30%. This value lies at the crossover point between single chain and multichain proteins (van Holde, 1966), and may explain the tendency for association-dissociation of some PPO (Vanneste and Zuberbuhler, 1974). There are a few reports of association of non-amino acid constituents to the enzyme. Balasingham and Ferdinand (1970) reported RNA accounting for half of the weight of potato PPO and essential for activity. Stelzig et al., (1972) reported RNA also associated with peel PPO. Flurkey and Jen reported that carbohydrate seemed to be non-specifically associated with some of the forms of peach PPO obtained from a hydroxyapatite column. It is very possible that these non-aminoacid components are artifacts formed during the purification of the enzyme.

Copper and the Active Site

PPO was the first enzyme in which the copper was related to catalytic activity (Kubowitz, 1937). Since then, more enzymes have 18 been found to contain copper. These have been classified according to the state in which copper exists. Type 1 (Cull) blue proteins with strong visible absorption and electron paramagnetic resonance (EPR) spectrum include azurin, stellacyanin, ceruloplasmin, laccase, and ascorbate oxidase. Type 2 (Cull) non-blue proteins with low optical absorption and EPR spectrum include superoxide dismutase, diamine oxidase, galactose oxidase, laccase, ceruloplasmin and, carboxypeptidase. Type 3 non-blue proteins with very low optical absorption and no EPR spectrum include ceruloplasmin, laccase, ascorbic acid oxidase, dopamine-beta-hydroxylase, cytochrome oxidase, and PPO (Owen, 1982). Resting PPO is EPR (or electron spin resonance, ESR) non-detectable possibly due to dipolar interaction of Cu(II) with a second Cu(II) or with another paramagnetic metal ion, the presence of diaraagnetic (antiferromagnetically coupled) Cu(II)-Cu(II) pairs, or to the existence of copper in the cuprous (Cul) state (Vanneste and Zuberbuhler, 1974). The determination of the copper content of PPO is complicated by the apparent loss of copper from the enzyme during purification (Mason, 1957a; Mayer and Harel, 1979). Some workers report the presence of one copper atom per chain or subunit: Robb et al., (1965) for broad bean PPO, Kidron el al. (1977) for grape PPO, and Vaughan et al., (1975) for spinach beet PPO. The valence state of the active EPR non-detectable copper of PPO remains unknown (Vanneste and Zuberbuhler, 1974). Kubowitz (1938) reported that PPO contained two copper atoms at its active site and that catalysis involved the copper shuttling between valence states with oxygen binding to the cuprous state (Cul). Mason (1956, 1957a) reported that a pre-requirement for the hydroxylase activity of PPO was the reduction of 2 Cu(II) to 2 Cu(I) (priming of the enzyme) by an o^ -diphenol or other reducing agents. Kertesz (1966) reported that Cu(II) added to apoenzyme was reduced to Cu(I) on reconstitution and that copper always remained monovalent. Deinum et al., (1976) and Aasa et al., (1978) reported that the reaction of native Neurospora PPO with mercaptans lead to the formation of green inactive enzyme 1* complexes between a half-reduced copper-pair and the mercaptan. Recent observations seem to indicate that the functional unit of PPO contains a pair of copper ions. Copper atoms occur in pairs in mushroom PPO (Schoot Uiterkamp et al., 1974), and Neurospora (Lerch, 1976; Deinum et al., 1976). It is not known if the copper pair is attached to a single peptide chain or to two identical or different subunits (Strothkamp et al., 1976). Evidence points to the existence of an antiferromagnetically coupled Cu(II) pair (Robb, 1981). When PPO reacts with hydrogen peroxide in the presence of oxygen, the spectrum of oxytyrosinase appears. Oxytyrosinase is in equilibrium with both free hydrogen peroxide and with oxygen. Resonance Raman spectroscopy of oxytyrosinase suggest that the oxygen is present mainly as a peroxy complex, i.e. two electrons have been donated to the bound oxygen (Eickman et al., 1979). These observations point out the similarity of oxytyrosinase and of oxyhemocyanin. The comparison of the absorption spectra of Neurospora PPO and and the similar luminescence of their CO analogues (Kuiper et al., 1980) suggest a common enviroment for the copper in both enzymes. According to Winkler et al., (1981) the resting form of PPO (met-) can be converted to the oxy-derivative by ligand binding of peroxide:

Cu2* Cu2* O2" Cu* 0 Cu* > +H 0 ^ 2 2 >•! — >-|l > + 02 Cu2* 2 HP Cu2* 0 Cu* 0 Cu* met-PPO oxy-PPO deoxy-PPO

Himmelwright et al., (1980) and Winkler at al. (1981) showed that azide, L-memosine (a structural analog of that inhibits PPO) and peroxide compete for the same at the binuclear copper active site. Ruegg and Lerch (1981) showed that the antiferromagnetically spin-coupled Cu(II) pair could be stoichiometrically substituted by cobalt. The activity was lost and it was shown that the two ions were not in an identical enviroment. A tetrahedral coordination around the cobalt center was suggested. 20

Kertesz and Zito (1962) showed that PPO was not inhibited by 2. -chloromercuribenzoate and therefore was not a sulfhydryl enzyme. The low content or absence of cysteine in PPO reported above seems also to indicate that this amino acid is not involved in the binding of copper in the active site. Photochemical oxidation studies demonstrated that PPO inactivation was pH dependent and enhanced by cyanide. A pH-dependent photo-oxidation is characteristic for histidine and it was suggested that this amino acid was essential for PPO activity, although cysteine is also sensitive to photo-oxidation (Gutteridge et al. , 1977). Lerch (1978) suggested the possible involvement of four out of five histidine residues in Neurospora PPO as ligands to the active site copper as well as in the catalytic mechanism of Neurospora PPO. Using labelled phenol as a substrate and through the process of reaction inactivation, it was found that one of the histidyl residues was lost and the enzyme inactivated. Brooks and Dawson (1966) also proposed the presence of a nucleophilic group in the active site attacked during reaction-inactivation. It is still debated whether the enzyme contains a single active site for both hydroxylation and oxidation or if there are separate active sites on the same or different enzyme subunits. A single active site for both activities has been proposed by Kubowitz (1938), Mason (1956), and in Neurospora by Gutteridge and Robb (1975). The reconstitution of active PPO from apo-PPO and copper was shown to require the addition of copper prior to the addition of substrate. This could be explained as an effect of the substrate on the aggregation of the enzyme and a masking of the copper binding sites (Kertesz and Zito, 1962). Two distinct active sites on the same molecule have been proposed by Dressier and Dawson (1969a,b). In exchange experiments using labelled copper it was noticed that the enzyme exchanged copper at a higher rate during oxidation than during hydroxylation. The presence of two active sites was suggested. It appeared that the copper at the hydroxylation site was non-exchangeable. But the increased copper exchange rate during oxidation could also be explained as being due to the nature of the substrate. The formation of a complex of _o 21

£ -diphenols with copper may facilitate copper exchange (Vanneste and Zubebuhler, 1974). Two distinct sites on different molecules have been proposed by Macrae and Duggleby (1968) for potato PPO and by Stafford and Dressier (1972) for Sorghum PPO. The proposition of distinct active sites on a single or separate protein molecules are usually based on the response of the two different activities to the same inhibitor. These studies fail to take into account the multisubstrate nature of PPO catalysis and the consequent complexity of the observed kinetics. Divergent kinetic effects exerted by an inhibitor should also be observed for a single site (Vanneste and Zuberbuhler, 1974).

Reaction Mechanisms

Enzymes utilizing molecular oxygen (dioxygen) as one of their substrates are divided into two categories: a) , in which the oxygen molecule functions as an electron acceptor only, and is reduced to 0„-, H2 0„, or 2 H„ 0 . b) , which catalyze the reactions in which atoms of oxygen are incorporated into organic substrates. They are subdivided into: 1) Dioxygenases, in which both oxygen atoms are inserted into organic substrates.

A + 02 ~A02 and 2) Mono-oxygenases or Mixed Function Oxidases, in which one atom of oxygen is inserted into an organic molecule and the other reduced to water (Malmstrom, 1982).

AH + 02 + 2 e- — AOH + 02-

The reactions in which the 0-0 bond is cleaved are considered exothermic. The 0^ molecule is kinetically quite inert and exists in a triplet ground state, i.e. a diradical with two unpaired electrons. The direct reaction of a triplet molecule with a singlet to give singlet products is a spin-forbidden process (Hamilton, 1974). 22

Two possible methods of activating CL exist: The first method consists in having the initial reaction of CL occur by a free radical mechanism. It is spin-allowed for a triplet to react with a singlet to give two doublets, i.e. free radicals, and for the doublets to recombine to give singlet products. This process requires the presence of a cofactor or substrate, the reduced state of which gives a highly resonance stabilized free radical by the loss of an electron or the equivalent of a hydrogen atom (Hamilton, 1974). PPO does not fix molecules in the form of hydroxy derivatives by single electron transfer resulting in free radicals or active oxygen formation, but rather by transfer of a pair of electrons which avoids this free radical formation (Kon, 1980). Studying the reaction of PPO with using EPR techniques it was concluded that radicals do not occur in the enzyme process (Mason et al., 1961). The second method consists in the complexing of 0« to a transition metal ion (usually copper or iron) which itself has unpaired electrons. The orbitals of both the 0« and the metal ion overlap and the number of unpaired elecrons of the complex are considered together. The 0„ does not react as a singlet but because it is complexed to a transition metal ion it can react by an ionic, non-radical mechanism. Bimetallic centers in enzymes are able to bind 0„ at higher reduction potentials than monometallic sites (Hamilton, 1974; Malmstrom, 1982). The activation of PPO is assumed to involve electron transfer from the Cu(I) ions to 0„, so that 0„ is really bound in the form of peroxide (Malmstrom, 1982; Himmelwright et al., 1980). There are several factors that make the study of the reaction mechanism of PPO very difficult. The size of the functional unit, the structure of the active site, and the valence state of copper are not clearly known (Vanneste and Zuberbuhler, 1974). It is not always certain what form of the enzyme is being studied in the case where multiplicity is detected. Another complication is the ability of the enzyme to catalyze two different reactions. Since neither the copper in PPO nor the whole enzyme give either an EPR signal or a visible 23

spectrum it is very difficult to follow the reaction using these methods. PPO can also undergo conformational changes induced by phenolic substrates, 0„, and pH, and these changes will affect the observations of the reaction mechanims, number of active sites and substrate dependent Km (Mayer and Harel, 1979). PPO can catalyze two different reactions. First, the hydroxylation of monophenols to yield _£ -diphenols (cresolase or hydroxylase activity). This reaction requires molecular oxygen and an electron donor (AH„ ):

R-^\oH+ 02+AH2- R/yOH+A + H20 — —OH

Second, the oxidation of o -diphenols to _o -quinones (catecholase, dehydrogenase or oxidase activity). This reaction also requires molecular oxygen. Two moles of diphenol are dehydrogenated per mole of 0„ consumed with 0^ undergoing a four electron reduction to water (Mason, 1957b).

2 «/ \OH + Og - 2 R\ \o + 2 H20 —OH 0

The hydroxylase activity is what categorizes PPO as an (Hamilton, 1974). Both activities have been shown to occur together in most PPO's studied. Both are inhibited to the same degree by metal-binding reagents, and substrates for one activity competitively inhibit the other. Each activity has been found proportional to the copper content, and both activities are lost when copper is removed (Mason, 1956). There is still a question of whether an intermediate o^ -diphenol formed during the hydroxylase activity is released from the enzyme or whether the reaction goes directly to give an o_ -quinone, that is, whether PPO could be a dioxygenase of the type: 2 monophenols + 0—» 24

2 o^ -diphenols (Mason, 1965; Vanneste and Zuberbuhler, 1974). It has been proposed by Mason (1956, 1957a) that any disturbance in the protein structure or in the attachment of the copper atoms during the extraction or purification procedures will affect the hydroxylase activity (i.e. binding of 0„) of PPO, but not so the oxidase activity, which is not so dependent on the attachment of the two adjacent copper atoms. The loss of one copper atom would result in loss of hydroxylase activity but not necessarily of oxidase acitivity. Vaughan and Butt (1969) reported that in spinach leaf PPO the hydroxylase activity required some specific enzymatic organization which was unstable, and that the oxidase activity did not require the retention of this arrangement and survived the partial or complete loss of hydroxylase activity. The hydroxylase activity of eggplant PPO decreased with purification (Sharma and Ali, 1980). The hydroxylation of monophenols by PPO in the absence of exogenous hydrogen donor (reducing agent- AH„ ) is characterized by an initial lag period. During this lag period the reducing equivalents needed for the hydroxylation are generated by the autoxidation of monophenols and/or the reduction of o_ -quinones. This lag period can also be overcome by the addition of a small amount of an _o -diphenol, or the addition of an exogenous reductant, resulting in a 'primed' or 'activated' enzyme (Mason, 1956). PPO oxidizes o^ -diphenols more rapidly than the corresponding monophenols (Kertesz and Zito, 1962). The added reducing compounds other than £ -diphenols act indirectly by reducing the accumulation of £ -quinones produced (Mason, 1957a; Vanneste and Zuberbuhler,1974). A competition between _o -diphenols for a reaction site on the enzyme is suggested due to the observation of a prolonged lag period with increasing monophenol concentration (Duckworth and Coleman,1970). It is not clear whether the lag period in the hydroxylase activity is inherent or an artifact due to changes induced in the enzyme during preparation and isolation (Mayer and Harel, 1979). A proposed non-enzymatic hydroxylation hypothesis considers o^ -diphenols to be formed through the action of the quinones on monophenols (Kertesz and Zito, 1962). It is now considered outdated 25 since it fails to explain some observations: the source of oxygen incorporated in the substrate is molecular oxygen and not water, reducing agents reacting rapidly with quinones do not abolish hydroxylation, and high substrate specificity and competitive inhibition by substrate analogs is also observed during the hydroxylation reaction (Vanneste and Zuberbuhler, 1974). Vaughan and Butt (1972) observed that the hydroxylation of £ -coumaric acid to caffeic acid required two electron donation per molecule of 0„ consumed. It was also proposed that reduction by o^ -diphenol is required in every turn of the catalytic cycle of PPO to produce the hydroxylating enzyme species, that is, o^ -diphenols are not activators, but co-substrates of monophenol oxidation (Mason, 1957a,b). Two copper atoms in the enzyme are reduced to the cuprous state (2CuII 2CuI) in the presence of one molecule of o^ -diphenol (Mason, 1956). Activation of the enzyme can also be brought along through the oxygenation of the Cu(I) system (Mason, 1957a). Hydrogen peroxide has been reported to shorten the lag period and tq form an active species of the enzyme: oxy-PPO (Jolley et al., 1974; Mclntyre and Vaughan, 1975). Several reaction mechanisms have been proposed considering both an active site with one copper ion or an active site containing two copper ions. Mason (1957b) proposed a mechanism in which PPO functions as a mixed function oxidase during hydroxylation and as a four-electron transfer oxidase during oxidation. Vanneste and Zuberbuhler (1974) describe mechanisms based on Mason's considering active sites containing one or two copper atoms. Studies of inhibition of PPO by 2,3-naphthalenediol revealed a competitive inhibition. This compound was not oxidized by PPO, and since it cannot undergo keto-enol tautomerism, an electrophilic mechanism of reaction involving ketonization of the _o -phenolic compounds may be involved (Mayer et al., 1964). Mclntyre and Vaughan (1975) studied the kinetics of £ -coumaric acid hydroxylation. They suggested a sequence in which binding of 0^ occured first to reduce the enzyme, followed by binding of £ -coumaric acid, release of 26

caffeic acid, and oxidation of the enzyme. The caffeic acid may be bound again to the cofactor site and the quinone formed and released. The cofactors probably act as electron donors. As stated above, hydrogen peroxide can form an active form of the enzyme: oxy-PPO. This form of the enzyme has an absorption spectrum similar to that of oxy-hemocyanin (Jolley et al., 1974; Himmelwright et al., 1980; Solomon, 1981). From these data the following active site configurations were proposed for monophenols and o^ -diphenols (Himmelwright et al., 1980):

HjO ,0-0, HJOJ I I ;cu(ii) Cu(ll)

oxy-PPO m«t-PPO (resting) f«ff«ctiv«] structure i

0H2

i^ 1/°-°^ Cu(ll)i s* JCudD^q/^Cudl)

Proposed interewtien with monophenolie ( —) and dipheneiic (--J substrates

Winkler et al., (1981) proposed a mechanism of hydroxylation and oxidation of based on the active site configurations described above. Capdevielle and Maumy (1982a,b) studied the oxidation of cuprous phenoxides by molecular oxygen and observed the formation of and _o -benzoquinones. They suggested that the reactions observed provided a mechanistic approach to PPO-catalyzed oxidation and hydroxylation of phenols. It has been observed that during the enzymatic oxidation of certain phenols, the rate of oxidation is maintained for only the first few minutes, after which the enzyme is rapidly inactivated 27

(Smith and Krueger, 1962). It is suggested that the quinone formed undergoes a reaction with a nucleophilic group in the vicinity of the active site before it is released by the enzyme (Wood and Ingraham, 1965; Racusen, 1970). This loss in enzyme activity was termed 'reaction inactivation' and recently the name 'suicide inactivation' has been proposed as correct (Lewis, 1980). Abeles and Maycock (1976) have described the kinetics of suicide (kcat) inhibition of PPO. During suicide inactivation, copper is lost from the enzyme. Ascorbic acid did not prevent this inactivation suggesting the inaccesibility of the quinone to ascorbic acid. Only free quinones are reduced. The more exacting spatial requirements for hydroxylase activity renders it more susceptible to o^ -quinone attack than the oxidase activity (Wood and Ingraham, 1965; Brooks and Dawson, 1966).

Substrate Specificity

The substrate specificity of PPO depends not only on the genus but, to a certain extent, also on the cultivar and on the part of the fruit or vegetable from which the enzyme has been extracted from (Vamos-Vigyazo, 1981). In general fruit PPO has a low affinity (high Km values) for both phenolic substrates (approximately 1 Mm) and oxygen (0.1 to 0.5 mM). The affinity for oxygen depends on the substrate being used and could vary also among different forms of the enzyme isolated from the same tissue (Harel et al., 1964, 1965; Duckworth and Coleman, 1970). The ratio oxidation/hydroxylation activities of PPO were found to range from 1-10 to >40. The ratios may change during isolation, purification or treatment by physical methods (Vamos-Vigyazo, 1981) and hydroxylation activity may be lost completely (Harel and Mayer, 1971). The loss of hydroxylase activity could result from changes in the structure of the protein during its purification (Mason, 1956; Vaughan and Butt, 1969; Harel et al., 1973). Sources in which both activities have been detected are broad bean (Robb et al., 1965), banana (Montgomery and Sgarbieri, 1975), wheat (Interesse et al., 1980), eggplant (Sharma and Ali, 1980) and 28 (Kahn and Pomerantz, 1980). Certain PPO (isozymes) that show apparently only cresolase activity have been reported in wheat (Taneja and Sachar, 1974), sorghum (Stafford and Dressier, 1972) and parsley (Schill and Grisebach, 1973). Many preparations of PPO have been found devoid of cresolase activity: tobacco (Clayton, 1959), cherry (Lanzarini et al., 1972; Benjamin and Montgomery, 1973), pear (Rivas and Whitaker, 1973; Halim and Montgomery, 1978), peach (Wong et al., 1971; Flurkey and Jen, 1980), grape (Cash et al., 1976), olive (Ben-Shalom et al., 1977b), mango (Joshi and Shiralkar, 1977; Park et al., 1980), date PPO (Hasegawa and Maier, 1980), and strawberry PPO (Wesche-Ebeling, 1981). It is very important to check for traces of phenolics left in the extracts that may serve as substrates for PPO. Other phenolics added to the extracts may not be substrates of the PPO but may be reported as such due to the observed oxygen uptake or melanin formation due to coupled phenolic-quinone oxidation reactions (Pierpoint, 1970; Wong et al., 1971; Coggon et al., 1973; Coombs et al., 1974). This effect seems to be also of importance in vivo . The initial rate of browning seems to be related to a special group of endogenous substrates and total browning results as a combination of both PPO activity and secondary oxidation reactions (Vamos-Vigyazo and Nadudvari-Markus, 1982). The substrates with the smallest substituent groups on the phenolic ring were oxidized at a faster rate by banana PPO (Montgomery and Sgarbieri, 1975). The rate of oxidation of substrates can be arranged in the order of the effect of para-substitution on the rate of oxidation as follows: methyl > H? > Cl > CH:CHNH C00H > CNS > C00H > CH0. This indicates that an electrophilic, cationoid type of attack on the dihydric phenol is involved in the oxidation reaction (Mayer, 1962). As the electron-withdrawing capacity of the substituents in the 2_ -position of _o -diphenols increases, the Km and k-cat decrease in the order: H > SCN > C0CH3 > CHO > CN > N02. In order to be a good substrate, the catechol derivative must maintain a density of readily removed electrons around the hydroxyl 29 groups. As the electrons are withdrawn to the para position, binding of the catechol becomes tighter, since saturation of the enzyme occurs at a progressively lower substrate concentration (Duckworth and Coleman, 1970). The presence of electron donating groups in the 2. -position increase, and electron-attracting groups decrease the reactivity of the substrate (Lanzarini et al., 1972). Monophenols are hydroxylated only if they have a para substituted > CH„ group (Vamos-Vigyazo, 1981). For some of the PPO extracted, maximum activity was noticed for an endogenous substrate. Dopamine was the best substrate for banana (Palmer, 1963) and mango PPO (Joshi and Shiralkar, 1977), caffeic acid for bean leaf (Racusen, 1970) and grape PPO (Cash et al., 1976), and chlorogenic acid for sugar cane leaf (Coombs et al., 1974), potato tuber (Abukharma and Woolhouse, 1966), eggplant (Knapp, 1965), and pear PPO (Rivas and Whitaker, 1973). In the case of flavonoid compounds it is probable that the carbon skeleton of the heterocyclic ring alone is important for the ease of oxidation of the catechol side group in a B ring. Glycosidation in the 3-position brings about a complete inhibition of oxidation. Flavanones, (flavan-3-ols), and leucoanthocyanins have non-planarity of the two rings and may serve as substrates for PPO. Catechins have been found to be good substrates for PPO extracted from pears (Tate et al., 1964), tea leaves (Coggon et al., 1973), peaches (Flurkey and Jen, 1980), dates (Hasegawa and Maier, 1980), and strawberries (Wesche-Ebeling, 1981). Flavones, flavonols, anthocyanins, chalcones, and show planarity and do not seem to serve as substrates to PPO. The nature of the bonding between the C-atoms at the 2- and 3- positions of the flavonoids, and the number and position of hydroxyl groups seems significant in determining the role of flavonoids in relation to PPO (Shannon and Pratt, 1967). 30

Effects of the Polyphenol Oxidase Reactions

When the cells in plant tissues are altered mechanically by cutting, bruising or freezing, or by pathological damage in the presence of 0„, browning occurs rapidly. Once the tonoplast is disrupted, the phenolic compounds leach into the cytoplasm and come in contact with PPO with the immediate production of o^ -quinones. 0 -quinones are unstable and highly reactive compounds and undergo a great variety of reactions. They can react with alpha -or epsilon -amino and sulfhydryl groups of amino acids, peptides and proteins, and with other phenolic compounds including flavonoids. After the initial coupling of reactive compounds with the quinones, further oxidation reactions and polymerization occur leading to the formation of the characteristic brown pigments, e.g. melanin. This reaction of the quinones are described in detail by Mason (1955), Mason and Peterson (1965), Pierpoint (1969a,b; 1970) and Wesche-Ebeling (1981). Modification of proteins by phenolic compounds or quinones is also termed 'tanning'. The phenolic compounds themselves can also interact with proteins through hydrogen bonding, hydrophobic interactions, and possibly through ionic interactions at high pH, e.g. pKa of phenol (-0H) is approximately 9.2 (Loomis, 1974; Thompson, 1964). The binding of proteins to tannins has been reported to occur mainly through the formation of multiple hydrogen bonds between the phenolic hydroxyl groups and the carbonyl functions of peptide linkages of proteins (Goldstein and Swain, 1965; Loomis and Battaile, 1966, Haslam, 1974). More recently it has been reported that the protein-tannin interactions occur mainly through hydrophobic interactions (Oh et al., 1980). Loomis (1974) and Loomis et al., (1974) suggested that both hydrogen bond formation and hydrophobic interactions were important, varying with different phenolic compounds. Phenolic compounds are divided into two main groups: flavonoid compounds, including condensed tannins, and the group of C6-C1 and C6-C3 compounds and their derivatives, including the hydrolyzable 31

tannins as well as tyrosine and lignin (Robinson, 1980). Most of the phenolic compounds, including low molecular weight condensed tannins can form quinones, and all of them can participate in polymerization reactions among themselves and/or with proteins (Anderson, 1968; Rhodes, 1977). Unless stringent precautions are taken during extraction and purification of enzymes and proteins from plant tissues, the presence of phenolic compounds and the quinones and polymers formed through the action of PPO can lead to tanning and even inactivation of the enzymes (Robb et al., 1965; Anderson, 1968; Loomis, 1974; van Sumere et al., 1975). The importance of the enzymatic browning reactions is of great significance in foods. These reactions can lead to the destruction or masking of the natural color of fruits by the formation of dark brown or reddish pigments. Undesirable changes in flavor (astringency and bitterness) and odor, as well as loss in the nutritional value, e.g. loss of lysine, methionine, cysteine, and ascorbic acid can also occur (Joslyn and Ponting, 1951; Pierpoint, 1970; Synge, 1975; Davies et al., 1975; Kidron et al., 1977). In some instances enzymatic browning is desirable as in the case of black tea, coffee, tobaco, and dates (Hasegawa and Maier, 1980). In the following sections some of the methods employed for the inhibition of enzymatic browning will be described. The methods can be divided in two groups: those used during the extraction of enzymes from plant tissues, and those employed in food production. In the case of food products, the methods employed for the control and prevention of enzymatic browning must not produce toxic effects, off flavors or change the texture, color or nutritional quality of the treated food. The following methods are used in the food industry: heat inactivation, cold storage, storage in sugar solutions, manipulation of the PPO and phenolic content of the agricultural products, and the use of chemical compounds such as citric acid, ascorbic acid, sulfur dioxide, and cysteine. These methods will be described in more detail in the following section. 32

Inhibition of Enzymatic Browning

Physical Methods

The first step in preventing enzymatic browning is the prevention of bruising or mechanical injury of the fruit or vegetable during harvesting or storage. Mechanical damage will not only cause an almost immediate browning of the affected area, but may also induce the activation of latent PPO, de novo synthesis of the enzyme, and/or synthesis and accumulation of phenolic compounds in the surrounding tissues thus increasing the browning potential of the commodity (see Physiological Roles section above). Since oxygen is one of the substrates of PPO, its exclusion would lower the extent of the reaction. Complete elimination of CL is hardly practicable. Controlled atmospheres can be of immense value in lengthening the post-harvest viability in fresh products (Coombs et al., 1974). Sugar and sugar solutions are used in the freezing preservation of fruit products for sweetening and to exclude direct contact of the tissues with 0^, and reducing the rate of diffusion of the 0« to the tissues (Joslyn and Ponting, 1951). Immersion of the tissues in water may also be used to partially exclude (L. It should be pointed out that prolonged anaerobic storage can lead to the synthesis of abnormal metabolites that can affect the organoleptic characteristics of the commodities (van Buren, 1970). During the extraction of enzymes from plant tissues the use of liquid nitrogen homogenization has shown excellent results. This technique not only provides an oxygen-free atmosphere, but also cools the extracts and allows the fracture of tough tissues (Kelley and Adams, 1977). Both dithionite and hydrosulfite are rapidly oxidized by 0„ and can be used to deplete 0„ in solution (Palmer, 1963). Storage of plant tissues at low temperatures may lower the rate of enzymatic browning. Palavicini (1969) reported a loss of only 16% of the PPO activity in strawberries stored at -20oC for 4.5 months. 33

Jen and Kahler (1974) reported that the PPO activity in peaches stored for a long period at 30C did not decrease below 50%. Pierpoint (1970) reported a decrease in the phenolic content of frozen leaves stored between 0 and -150C and suggested the possibility of the accumulation of _o -quinones and possible modification of endogenous enzymes during this storage period. Freezing and thawing also has the effect of breaking the integrity of the cell membranes thus allowing the contact of PPO with the endogenous phenolic substrates. Heat inactivation or blanching of fruits and vegetables is a method that has been used for a long time for the inactivation of endogenous plant enzymes although it will affect the organoleptic characteristics of the treated tissue. The control of the pH of the medium can also be used to limit the activity of PPO. PPO activity decreases significantly with decreasing pH (van Buren, 1970). Organic acids such as citric, malic, acetic, phosphoric and ascorbic are used during food processing or in buffers to lower the pH. High pH's have an adverse effect in that they increase the ionization of phenolic hydroxyl groups and thus promote autoxidation and the formation of quinones (Loomis, 1974).

Effect of the Source of PPO

The content of PPO and phenolics and thus the browning potential of a fruit or vegetable will vary depending on the source used. Levels of PPO and phenolics vary not only between different species, but also between different varieties and cultivars of the same commodity (Walker, 1962, Amberger and Schaller, 1975). Levels differ also from one tissue to another in the same plant or organ, and even in the different sections of the organ, i.e. fruit (Mottley, 1979; Vamos-Vigyazo, 1981). Phenolic content will also be affected by the site of cultivation and fertilization and irrigation practices (Amberger and Schaller, 1975). The levels of PPO and phenolics vary also with the stage of maturity of the tissue. Fluctuations in PPO activity during growth have been reported for spinach (Sato, 1976), tea leaves (Takeo, 1973) and bean leaves (Racusen, 1970). Changes in PPO activity during 34 ripening of fruits have also been reported for tomatoes (Hobson, 1967); grapes (Cash et al., 1976; Kidron et al , 1978; Wisseman and Lee, 1980); avocadoes (Golan and Sadovski, 1977); olives (Ben-Shalom et al., 1977a); mangoes (Joshi and Shiralkar, 1977); peaches (Chung and Luh, 1972; Flurkey and Jen, 1978); and banana (Mottley, 1979). Growth and ripening seem to bring about a change in the subcellular location (Halim and Montgomery, 1978; Zaprometov et al., 1979), and in the hydrophobicity of the enzyme (Flurkey and Jen, 1978). From the discussion above it can be concluded that the validity of the results obtained for a PPO extract is restricted to enzyme extracted from a given tissue of a fruit/vegetable of a given cultivar grown in well defined conditions as well as to identical experimental procedures (Mihalyi et al., 1978). This helps to explain some of the differences in the results obtained for PPO obtained from the 'same' fruit or vegetable. It has been suggested as a means of lowering the browning potential of fruits and vegetables to select varieties that contain lower amounts of polyphenolic substances or of PPO or both (Mathew and Parpia. 1971), or to select areas where enviromental conditions favor low polyphenol contents (Tate et al., 1964). Other methods suggested for the control of PPO or polyphenol levels are the use of growth substances or other compounds during the development of the commodity. Treatment of the soil with copper caused an increase in PPO levels in beet leaves (Vamos-Vigyazo, 1981). Phosphonic acid derivatives (Ethrel), gibberelic acid, and Ethephon when sprayed on developing fruis, e.g. peaches or depressed PPO activity and prevented browning of the tissues (Paulson et al., 1980; Vamos-Vigyazo, 1981; Arora and Bajaj, 1981). Volk et al., (1978) studied the effect of growth activators and inhibitors on the levels of PPO in suspension cultures of apple cells. Norleucine, a methionine analog, depressed PPO activity by 50 to 70% after 5 days without affecting the growth of tobacco cell suspensions. The effect could be reversed with the addition of methionine and it was suggested that norleucine induced specific loss of some PPO isozymes and was incorporated into others thereby changing 35 their structure and conformation and, consequently, their activity (Bar-Nun and Mayer, 1983).

Removal of Phenolic Compounds

The methods explained below are used mainly during the extraction of enzymes from plant tissues. Organic solvents can be used for the removal of phenolics. One widely used method of extraction is the preparation of acetone powders. Acetone will remove some phenolic compounds, but some difficulties have been observed: difficulty in extracting PPO from the dried powders (Hobson, 1967), the age of the acetone powder affects the yield of PPO (Flurkey and Jen, 1980), and changes in the properties of the enzyme have been observed due to changes in the structure of the enzyme (Ben-Shalom et al., 1977a). Other solvents that have been used are butanol and diethyl ether (Kelley and Adams, 1977). Some phenolic compounds can be modified using specific enzymes in such a way that they no longer serve as substrates for PPO. Catechol-1,2-oxygenase will oxidize an o -diphenol to the corresponding cis.cis-muconic acid (Hayaishi et al., 1955; Kelly and Finke, 1969; Itoh, 1981; Que and Mayer, 1982). 0-methyl transferase will methylate chlorogenic acid and other catechols to the corresponding 3-methyl-ethers (Coombs et al., 1974). Polyphenols containing adjacent hydroxyl groups will form covalent complexes with borate or germanate. The complexes formed can no longer function as substrates for PPO and thus inhibit the browning reactions (Zittle, 1951; Kelley and Adams, 1977). The complex formation between borates and phenolic compounds is favored by an alkaline pH. Sodium meta- and tetraborate were found to be more effective inhibitors of enzymatic browning whereas sodium perborate and boric acid were not effective. The intensity of the borate-phenolic reaction increases with increasing number of adjacent hydroxyl groups (Bedrosian et al., 1959, 1960). Tannins can bind with proteins and enzymes forming complexes 36 stabilized by hydrogen bonding (Loomis and Battaile, 1966) or hydrophobic interactions (Oh et al., 1980) depending on the type of phenolic compound involved (Loomis et al., 1974). The formation of these complexes can be reversed using non-ionic polymers, e.g. polystyrene, non-ionic and cationic detergents (Gray, 1978; Goldstein and Swain, 1965; Loomis, 1969) or using organic solvents (Loomis, 1969) Most of the enzyme extraction procedures used now include the use of polymeric phenolic-binding agents. The most widely used of these agents have been listed and described in detail before (Wesche-Ebeling, 1981). Important reviews describing the use of these agents include Loomis (1969, 1974), Loomis and Battaile (1966), Anderson (1968), Rhodes (1977), and Loomis et al., (1979). A list of polymeric phenolic-binding agents is given in Table 1.1.

Removal of Quinones

0-quinones can be removed by either reducing them to o^ -diphenols or by trapping them through the formation of covalent complexes. The removal of _o -quinones may prevent the tanning of proteins and enzymes, and the formation of polymeric brown pigments. Once formed the quinones will react rapidly not only with agents used to remove them, but also with other compounds present in the extract, e.g. proteins or enzymes, so that complete inhibition of enzyme modification by quinones is almost impossible to achieve. A list of compounds that can reduce or bind quinones is given in Table 1.2, and a detailed description was given before (Wesche-Ebeling, 1981).

Inhibition of PPO

Direct inhibition of PPO can occur through different mechanisms: denaturation or conformational changes of the protein structure, chelation of the copper at the active site, competitive inhibition using substrate analogues, and through natural inhibitors from fungal origin. A list of the principal methods and compounds used as 37 inhibitors for PPO is given in Table 1.3 and a detailed description was given before (Wesche-Ebeling, 1981). 38

TABLE 1.1 POLYMERIC PHENOLIC-BINDING AGENTS.

THOSE BINDING PHENOLICS THROUGH HYDROGEI\ -BOND FORMATION : INERT PROTEINS = BOVINE SERUM ALBUMIN HIDE POWDER GELATIN COLLAGEN CASEIN POLYAMIDES = NYLONS (ULTRAMID, PERLON) POLYCAPROLACTAM POLYETHYLENE GLYCOLS (CARBOWAX) POLYVINYL(POLY)PYRROLIDONE (PVPP, POLYCLAR AT), SOLUBLE PVP POLY-N-MEIHYLACRYLAMIDE DOWEX AG 1 X-8

THOSE BINDING PHENOLICS THROUGH HYDROPHOBIC INTERACTIONS : POLYSTYRENE-DIVINYL BENZENE = AMBERLITE XAD-2 & XAD-4 POLYACRYLIC FORMS = AMBERLITE XAD-7 DOWEX AG 1 & 2 X-8 INERT PROTEINS (BSA)

THOSE BINDING PHENOLICS THROUGH IONIC INTERACTIONS * DOWEX AG 1 & 2 X-8, DOWEX 50 POLYSTYRENE-DIVINYL BENZENE RESINS = AMBERLITE IRA 904 & 938 AMBERLYST 15 & 26A PROTAMINE SULFATE INERT PROTEINS

(Anderson, 1968; Loomis, 1969, 1974; Loomis et al., 1979; Rhodes, 1977; Mayer and Harel, 1979). 39

TABLE 1.2 AGENTS USED FOR THE REMOVAL OF QUINONES.

AGENTS THAT BIND QUINONES COVALENTLY •

INERT PROTEINS BENZENE SULFINIC ACID CYSTEINE ETHYL XANTHATE DIETHYLDITHIOCARBAMATE (DIECA) METABISULFITE

AGENTS THAT REDUCE QUINONES :

ASCORBIC ACID THIOGLYCOLLATE METABISULFITE SULFUR DIOXIDE DITHIONITE GLUTATHIONE CYSTEINE MERCAPTOBENZOTHIAZOLE DITHIOTHREITOL 2-MERCAPTOETHANOL 40

TABLE 1.3 METHODS AND AGENTS USED FOR THE DIRECT INHIBITION OF PPO.

PHYSICAL METHODS : INERT ATMOSPHERES UV LIGHT CRYOMILLING (LIQUID NITROGEN HEAT TREATMENT (BLANCHING) HOMOGENIZING) LOW pH (ACIDS)

CHEMICAL METHODS : AGENTS BINDING TO THE PROSTHETIC GROUP (Cu) OF THE ENZYME = DIETHYLDITHIOCARBAMATE 2-MERCAPTOETHANOL MERCAPTOBENZOTHIAZOLE SUBSTITUTED THIOUREAS THIOSULFATE DITHIOTHREITOL 2,3-DITHIOPROPANOL ETHYL(or)METHYL XANTHATE SALICYLDIOXIME 3,4-DICHL0R0PHENYLSERINE FLUORIDE AZIDE CYANIDE CARBON MONOXIDE HALIDES AND CARBOXYLIC ACIDS EDTA (pH DEPENDENT INHIBITION)

AGENTS AFFECTING THE BINDING SITE OF PPO = RESORCINOL PHLOROGLUCINOL 4-CRESOL 4-NITROCATECHOL 4-CHL0R0PHEN0L FISETIN 3-FLU0R0TYR0SINE 4-NITROCATECHOL PROTOCATECHUIC ACID DERIVATIVES CINNAMIC ACID DERIVATIVES L-MIMOSINE

I AGENTS COMPETING WITH THE H-DONOR AT THE ACTIVE SITE = SUBSTITUTED HYDROXAMIC ACIDS, i. e. SALICYLIC ACID, BENZHYDROXAMIC ACID.

AGENTS THAT CAN SUBSTITUTE Cu AT THE ACTIVE SITE = Hg- Ag- AuCl

FUNGAL PEPTIDE INHIBITORS, i.e. EXPANSIN. 41

THE STRAWBERRY SYSTEM

Strawberries are non-climacteric composite fruits belonging to the genus Fragaria . The majority of the edible portion consists of an expanded and fleshy receptacle carrying achenes, or individual fruitlets. Strawberries have a relatively short life after harvest compared to other fruits, and associated with this short life is a relatively high rate of respiration. Respiration rates increase about 50% from immature to mature stages, and seem to be faster in firm varieties as compared to soft ones (Haller, 1941). It was found that harvested strawberry fruits at the greenish-white stage will develop full color at high temperatures, 25-30°C, in complete darkness. At temperatures below 130C good color development did not occur (Austin et al., 1960).

Strawberry Structure

Five tissue zones can be seen in the strawberry: epidermis, hypodermis, cortex, pith and vascular bundles:

Sepal (Calix)

Epidermal cells

Hypodermis Certieal cells Intracellular spaces

Vascular Thickened tissue cell vails

Achenes 42

The epidermis consists of polygonal cells bearing stomata, thick-walled hairs, and embedded within this layer the achenes. The achenes are connected to the main vascular tissues by a strand of vascular bundles The cells surrounding the achenes usually have very much thicker walls. The bundle zone comprises spiral and annular vessels, e.g. xylem and phloem. The hypodermis consists of raeristematic cells and contains no intercellular spaces. The cortex, or true flesh is composed of rounded parenchyma cells which contain intercellular spaces. The pith zone consists of thin-walled cells which are frequently separated by large cavities (Szczesniak and Smith, 1969; Jewell et al., 1973).

Chemical Composition

A table listing the chemical composition of strawberries emphasizing the Tioga variety was given before (Wesche-Ebeling, 1981) and shown in Table 1.4. Wrolstad et al., (1970a) studied the chemical composition of 13 different selections and 5 varieties of strawberries and obtained the following values: pH 3.49±0.12; total acidity 0.89±0.13 (as citric acid); % soluble solids 9.10±1.01; ascorbic acid 67.0±15.7 mg%; total anthocyanins 5731153 M/g; oxonium salt 137±51 M/g; cyanidin-3-glucoside 64±40 g/g; and % pelargonidin-3-glucoside 88±51. Organic acids content, in decreasing order of concentration is: citric > malic > ascorbic > quinic > succinic » shikimic, glyceric, glycolic, salicylic, gentisic and vanillic acids (Hulme and Wooltorton, 1958; Stohr and Herrmann, 1975). Free amino acids found in high concentrations are aspargine (59 mg%), glutamine (15 mg%), alanine (12 mg%), glutamic acid (8 mg%), aspartic acid (3 mg%), serine, threonine, valine, leucine, isoleucine and cysteic acid (less than 2 mg%) (Tinsley and Bockian, 1959). All values are based on fresh fruit weight. Of the different flavonoid compounds found in nature, only catechins (flavan-3-ols), flavonols, anthocyanins, cinnamic acid derivatives, and leucoanthocyanins can be considered of importance in 43

TABLE 1.4 CHEMICAL COMPOSITION OF STRAWBERRY FRUITS.

pH 3.49±.12 Total acidity 0.891.13 Water content (% w/w) 89.80 Total solids (% w/w) 7.80 Soluble solids (% w/w) 9.1011.01 Insoluble solids (% w/w ) 2.40 Proteins (mg %) 680.00 Lipids (mg %) 470.00 Total carbohydrates (mg %) 8060.00 Pectin (as Ca-pectate, % w/w) 0.54 Reducing sugars (% w/w ) 4.13 Sucrose (% w/w) 0.87 Free amino acids ( mole %) 442.00 Minerals: Calcium (mg %) 16.00 Magnesium (mg %) 11.00 Manganesum (mg %) 7.10 Sulphur (mg %) 13.40 Sodium (mg %) 0.88 Potassium (mg %) 186.00 Phosphorus (mg %) 31.00 Copper (mg %) 0.80 Iron (mg %) 1.30 Zink (mg %) 1.70 Boron (mg %) 1.20 44

TABLE 1.4 Continued.

Organic Acids and Phenolics: Ascorbic acid (mg %) 67.00±15.7 Citric acid (% w/w) 0.92 Malic acid (% w/w) 0.09 Total phenolics (as tannic acid . mg %) 312.00 Total anthocyanins (mM %) 5.73±1.53 (mg %) 27.60 Oxonium salt 1.37±0.51 Cyanidin-3-glucoside (mg %) 6.40±4.00 Pelargonidin-3-glucoside (% of ACN) 88.00±51

Leucoanthocyanins (Abs.Units %) 565.00 Flavanols (Abs.Units %) 87.60 D-Catechin (mg %) 1 - 7.00 L-Epicatechin (mg %) 1.00 Caffeic acid (mg %) 1.00 2. -coumaric acid (mg %) 1 - 1.50 4-hydroxybenzoic acid (mg %) 1 - 3.50 Protocatechuic acid (mg %) 0.60 Gallic acid (mg %) 1 - 4.00 Methyl gallate + ellagic ac: .d (mg %) 1.00 Chlorogenic, neochlorogenic Salicylic, gentisic, and vanill ic acids Traces

( % ) = per 100 g fresh fruit weight. Green, 1971; Adams, 1975; Abers and Wrolstad, 1979; Stohr and Herrmann, 1975; Wrolstad et al., 1970a. 45 foods (Bate-Smith and Metcalfe, 1957). Flavan-3-ols found in strawberries are _D -catechin > _L -epicatechin >> I) -gallocatechin; L -epigallocatechin was not detected (Stohr and Herrmann, 1975). Nine flavonol glucosides were found: -3-monoglucosides and -3-glucuronides of kaempferol (K) and quercetin (Q), K-7-monoglucoside, K-7-glucoside, and Q-3-glucoside (Ryan, 1971, Herrmann, 1976). Anthocyanins (Acn) found are pelargonidin (Pgd) and cyanidin (Cyd) -3-glucosides (Shrikhande, 1976). Anthocyanins are usually present in a ratio of 1:1 Pgd-3-glucoside and Cyd-3-glucoside in some cultivated strawberry varieties, i.e. Fragaria vesca , while a ratio of 1:0.05 was found in other varieties. A ratio of 20:1 is reported for wild strawberries (Wrolstad et al., 1970a). Pgd-3-glucoside content varies from 72 to 95 % in analysis of 5 varieties and 13 selections of strawberries (Wrolstad and Erlandson, 1973). A new anthocyanin was reported present at up to 5% of the total pigment and a furanose or an anomeric form of Pgd-3-glucoside was suggested (Wrolstad et al., 1970b). Cinnamic acid derivatives and other phenolic compounds present in strawberries are, in order of decreasing concentrations: gallic acid, 4-hydroxybenzoic acid > £. -coumaric acid, methyl gallate, ellagic acid > protocatechuic acid » chlorogenic and neochlorogenic acids (Stohr and Herrmann, 1975). Leucoanthocyanidins of varying degrees of polymerization were found in strawberries, the largest fraction being soluble in water, i.e. glycosylated, while the water and alcohol-insoluble, and -extractable fractions where found in lower levels (Co and Markakis, 1968). Leucoanthocyanidins, like procyanidins, can be converted to anthocyanins on heating with acid. Tannins found in strawberries are of the condensed (proanthocyanidin) and hydrolyzable types. The condensed tannins are of the procyanidin and polymeric type, with no prodelphinidin present. The configuration of the monomer units is cis:trans = 74:26. The polymeric chains are terminated by j) -catechin, L^ -epicatechin and D -gallocatechin. The hydrolyzable tannins are of the gallotannin and 46 ellagitannin type. The tannin concentration in strawberries suffers no dramatic loss during ripening (Foo and Porter, 1981) :

OH •*« OH i»* 2,3-cis 2,3-trans

Procyanidins

Strawberry Cell Wall

Some of the characteristics of the cell wall in strawberries can cause a series of problems in enzyme extraction. Strawberries contain high levels of soluble pectin, the levels increasing with ripening of the fruit. Some other fruits also show high levels of soluble pectin, i.e. peaches, apples and pears. Frenkel et al., (1969) noticed that extracts from ripe apples and pears tended to gel. The same was noticed with resolubilized acetone powder extracts from various fruits. In both cases the determination of malic enzyme in gel electrophoresis resulted in poor resolution and showed marked denaturation and the presence of artifacts. Preliminary attempts at separating crude peach PPO preparations by electrophoresis were unsatisfactory due to low protein concentration, poor resolution, as well as to artifacts probably caused by high pectin content (Paulson et al., 1980). Extracts obtained from peach acetone powders showed blocked flow through chromatography columns after resolubilization due to the presence of pectin (Flurkey, 1979). Poor resolution in electrophoresis gels and erratic results during column chromatography were also observed using crude strawberry PPO extracts (Wesche-Ebeling, 1981). Some proteins and enzymes can be precipitated with insoluble cell 47 wall material during extraction, or bound to pectic substances following extraction (Frenkel et al., 1969; Barnes and Parchett, 1976). Removal of pectin can be done through the precipitation of calcium pectate using calcium salts (Wong et al., 1971; Flurkey and Jen, 1980), but in the case of peach extracts one half to one third of the total PPO was lost after precipitation of the pectin (Flurkey, 1979). Degradation of cell wall, cell rupture, and plasmolysis caused by senescence or during frozen storage and processing of strawberries are responsible for softening, loss of crispness and juice release. Strawberries have a relatively low solids content. At about 10% solid matter, strawberries are a solid, whereas whole milk is a liquid at about 13% solid matter. Thus the arrangement of the small amounts of solids in the fruit must be such that they form a very intricate polymeric structure capable of supporting a large amount of liquid (Szczesniak and Smith, 1969). Strawberries that have been canned, frozen or processed to jam retain much of their natural aroma, flavor and most of their color, but suffer a serious textural degradation. The firm, plump, juicy berry loses its turgor and becomes soft and fluid (Szczesniak and Smith, 1969; Jewell et al., 1973).

Morphological Changes in the Cell Wall

Strawberry fruit development can be divided into different stages and counted as days after petal fall (dpf). Stage I includes fruits that are hard green up to when they turn white, approximately 30 dpf. Stage II corresponds to hard fruits with patches of red coloration, and stage III corresponds to firm fruits that are uniformly red or commercially ripe fruits (Wade, 1964; Neal, 1965; Woodward, 1972). The percent composition on a moisture-free basis of the strawberry cell wall reported by Wade (1964) is: glucan, 24%; galactan, 3.9%; mannan, 2.2%; araban, 2.9%; xylan, 3.3%; anhydro uronic acid (AUA), 17.3%; methoxyl, 2.4%; acetyl, 2.0%; lignin, 6.2%; protein, 25.1%; ash, 2.7%. Knee and Hartley (1981) reported the gross 48 composition of strawberry cell wall to be: galacturonic acid, 40.3%; glucose, 31.1%; amino acids, 10.7%; galactose, 7.6%; arabinose, 6.5%; xylose, 1.9%; mannose, 1.7%; rhamnose, 1.1%; and hydroxyproline, 0.1%. There is a three-fold increase in the number of cells in the first 3 dpf. Cell multiplication continues up to 10 dpf, and is followed by a steady increase in the size of the fruits, the cell expansion period. The parenchyma cells increase in size but there is a decrease in area of contact between adjacent cells. This increase in cell size is accompanied by a significant decrease in the weight of cell wall material per fruit. At petal fall the cells have dense walls. At 14 dpf the cell wall shows an outer diffuse and an inner more dense fibrilar layer. The change from dense wall to diffuse fibrils is accompanied by the hydration of existing pectic and hemicellulosic material, and not to increase in cell wall polysaccharides. The swollen material comes to occupy the intercellular spaces (Wade, 1964; Neal, 1965; Knee et al., 1977). There is an increase in cell wall protein level constantly throughout ripening but a hundred-fold less than the increase in cell volume. Sugar content increases with time as does titratable acidity which falls once the fruit begin ripening. The pH decreases from 4.6 at 9 dpf to 3.3 at 35 dpf and then increases to 3.7 at 49 dpf (Woodward, 1972; Knee et al., 1977). Cell wall polysaccharides per fruit increase ten-fold up to 21 dpf after which they remain constant or decrease slightly. From 7 to 21 dpf polygalacturonate, .e.g. pectin, was cell wall bound; one half loosely bound through calcium bridges (EDTA-extractable). A change in the cell wall polysaccharides takes place in such a way that uronide polymers become rearranged to allow for more plasticity in the walls for cell expansion. Smaller quantities of insoluble polyuronides and neutral polysaccharides are left in the cell from 20 to 40 dpf. There is net synthesis of polyuronides but not of neutral polysaccharides during development (Woodward, 1972; Knee et al., 1977). On ripening labeled inositol incorporation into galacturonic acid declines sharply, but 49 labeled methionine incorporation into methyl ester groups is maintained. The esterification of the soluble pectin fraction increases with ripening (Knee ad Hartley, 1981) Pectin does not seem to be covalently bound to wall protein and its solubilization may result from hydrolytic enzyme activity or from loss of calcium-stabilized gel structure due to increase in methylation of the polygalacturonate chain. An increase of xylose, mannose, and glucose residues in the soluble pectin fraction suggests that hemicellulose polysaccharides were being either degraded or released from inter-polysaccharide bonds (Knee et al., 1977). Galactose turnover during ripening may indicate that there is a loss of galactose in the branched pectin fraction. Pectin shows progressive depolymerization with ripening (Knee and Hartley, 1981).

Cell Wall Structure

The primary cell wall consists of cellulose microfibrils embedded in a matrix of other polysaccharides and protein. Hemicellulose may consist of approximately 90% of the continuous amorphous phase of cell walls, and pectinaceous materials, although plentiful in some tissues, i.e. strawberries, may constitute only about 5% in others (Beckman, 1971; Knee and Hartley, 1981). Alberscheim's group have proposed a model for Sycamore cultured cells based on covalent linkages between the different polymers present in the primary cell wall. Xyloglucan (hemicellulose) is hydrogen-bonded to cellulose elementary fibrils. The reducing ends of the xyloglucan are connected to the rhamnogalacturonan through a linear galactan.The rhamnogalacturonan, in turn, is connected to the hydroxyproline-rich protein through a branched arabinogalactan. The order of attachment is then reversed, and the cell wall can thus be considered as a macromolecule (Keegstra et al., 1973). The model described above for the cell wall of dicots is based on the walls of cultured cells, but this model does not include the middle lamella (Labavitch, 1981). The importance of covalent linkages between the cell wall polymers has been questioned, and other theories 50 propose larger involvement of non-covalent interactions in the stability of the cell wall, e.g. calcium bridges (Knee and Bartley, 1981). The presence of phenolic compounds covalently bound to cell wall polymers has been recently reported in both monocots, i.e. wheat (van Sumere et al., 1975) and dicots, i.e. spinach (Fry, 1979). They are cinnamic acid derivatives, i.e. _£. -coumaric and ferulic acids esterified with galactopyranose and arabinopyranose units of polysaccharides (Fry, 1983); and tyrosine found in the cell wall protein (Fry, 1982). These phenols can undergo oxidative coupling reactions leading to the formation of crosslinks between the cell wall polymers i.e. dicoumarate or diferulate in the polysaccharides and dityrosine and isodityrosine in the protein: ,CCM? HP OH PH

R-OC R-OC ^co 0C'.R

Wall-bound diferulate Dityrosine Isodityrosine R'Carbohydrate residue R«Glycoprotein backbone

The presence of phenolic compounds in the cell wall polymers may have different functions. It is possible that cell wall phenolics act as anchored free-radical-initiation sites for the oxidative polymerization of hydroxycinnamoyl alcohols into lignin. It is also possible that through the synthesis of cross-links between the cell wall components by the action of extracellular peroxidase (whose levels are controlled by hormones) may be involved in the control of cell wall extensibility and growth. Crosslinking of cell wall polymers may also occur as a response to attack by pathogens and result in decreasing the accesibility of hydrolyzing enzymes to the polysaccharide substrates (Fry 1979, 1983; Friend, 1981). It has been suggested that acid and neutral pectins of some tissues carry ferulic acid. This could explain the fact that pectin is not always easily extracted from cell walls by non-degrading treatments. The average molecular weight of pectin would be increased 51 by oxidative coupling of ferulic acid leading to the formation of crosslinks between the pectin polymers. This would increase the effectiveness of the non-covalent bonds, e.g. calcium bridges and hydrogen bonds, which hold pectin firmly in the cell wall. Cold diluted alkali will break the diferulate bonds and release ester-linked ferulate (Fry, 1983). Several models of the pectin structure have been proposed (Rees, 1969, 1975; Leeper, 1973; and Walkinshaw and Arnott, 1981 a,b). It is not known if the observed characteristics and proposed models exist in the intact tissue. Observations of pectin in apple cell walls have led to the proposal that pectin polymers may exist as both homogalacturonan and rhamnogalacturonan, and that the heterogeneity of pectins was increased by the attachment of various types of side chains to both types of pectin (Neukom et al., 1980).

Mechanisms of Cell Expansion

As described above, strawberry fruit development is marked by an increase in cell size which is accompanied by cell wall expansion and an observed increase in soluble pectin. Several mechanisms have been proposed through which cell growth is made possible by changes in the characteristics of the cell wall polymers, e.g. cell wall plasticizing. The driving force by which cell wall extension is effected during cell elongation is generally accepted to be the turgor pressure of the cells themselves. Because of the chemical stability of cell walls, any plasticizing process must incorporate two sequential steps: the walls must be first conditioned, followed by processes allowing for cell expansion (Beckman, 1971). Thus far only acidity has been found to bring about cell wall conditioning. Addition of auxin (indole acetic acid, IAA) to plant tissues quickly causes the primary cell walls to be loosened or weakened as a first step in the growth response. The primary growth promoting action of IAA seems to be the triggering of hydrogen ion secretion into the tissue free space. The short latent period between 52 presentation of IAA and a growth response makes it unlikey that de novo protein and polysaccharide synthesis participate in the initiation of cell growth (Labavitch, 1981). The activity of H-ions is presumed to be directly on unidentified, acid-labile wall bonds or on wall degrading enzymes with acidic pH optima. Inhibition of lAA-induced growth by calcium ions has been suggested to be due to the formation of pectic gels in the cell wall or by direct inhibition of cell wall loosening enzymes (Tepfer and Taylor, 1981). The amount of pectin held in cell walls by ionic bonds, i.e. calcium bridges, is quite substantial, i.e. from 69% in cress to 92% in tomato of the uronic acid polymers were water soluble in the absence of calcium. It is not known what kind of bonds retained the fraction of the pectin that could not be extracted with CDTA (cyclohexanediaminetetraacetic acid) and alkali, but it seems to involve covalent bonds. This seems to indicate that the integrity of the pectin molecule does involve covalent bonds, but that these bonds are not as numerous as Albersheim's model suggests (Jarvis, 1982). Both EDTA and Pectasin could accomplish complete maceration of the strawberry fruit leaving the cells with no surrounding pectinous material (Neal, 1965). Tepfer and Taylor (1981) studied the inhibition of IAA growth by several cations and found that there was a good correspondence between the effectiveness of an ion to inhibit growth (Znll > Coll > Call > Mnll > Mgll) and its binding affinity for the cell wall, but poor correspondence between growth inhibition and the ability to induce gelation. These results seem to indicate that pectic gel formation (Rees, 1975) is not involved in cation-induced growth inhibition, but that the major cation binding site in cell wall may be involved in a non-gel forming capacity. Methylation of pectin is related to soluble pectin formation and fruit softening (Knee and Bartley, 1981). Since the degree of methylation is close to the theoretical maximum through all stages in strawberries (Wade, 1964), few salt bonds are needed for cell wall structure stabilization, and small changes in the degree of methylation would bring about large changes in pectin stability (Neal, 53

1965). Cell wall loosening, or fruit softening, may involve new highly esterified pectin with low cohesive properties that needs to be secreted into the middle lamella region. Direct methylation of the pectin in the cell wall may not occur since the immediate precursor of the methyl group, S-adenosyl methionine, would unlikely be exported from the cytoplasm (Knee and Hartley, 1981). Changes in the cell wall properties influencing cell expansion, e.g. cell growth and fruit softening, are accompanied by turnover of cell wall components (Labavitch, 1981). According to Bates and Ray (1981) the soluble pectin seems to be bound at neutral pH to a heat-labile wall component, presumably protein. This interaction is reversed at low pH and by calcium and appears to be of a physical, possibly lectin-like nature. According to the acid-growth theory, IAA causes the lowering of the pH in the cell wall, and the low pH activates cell-wall loosening enzymes (van Volkenburgh and Cleland, 1980). lAA-induced growth can be inhibited by infiltration of the tissues with neutral or calcium containing solutions. The presence of hydrolytic enzymes in the cell wall region has been sometimes difficult to demonstrate. Strawberries were reported to contain exo-polygalacturonase (exo-PG) together with pectin esterase (PE) (Gizis, 1964; Archer, 1979). PE was found to increase from the green to early ripe stages, and to decrease during the senescent "overripe" stage in strawberries (Barnes and Parchett, 1976). Some workers have reported that no polygalacturonase or polymethylgalacturonase activity could be detected (Neal, 1965; Barnes and Parchett, 1976). Endocellulolytic activity was detected only in ripe and senescent strawberries, but this cellulase did not attack insoluble cellulose (Barnes and Parchett, 1976). PE may be involved in the demethylation of the predominantily esterified pectin molecule and participate in an enhanced turnover of calcium linkage zones. This activity together with the methylation of the pectin or the excretion of highly methylated pectin into the middle lamella would allow cell growth and separation to occur. Exo-PG would cause a limited degradation of soluble pectin and cell 54 separation and softening would be more extensive (Knee and Hartley, 1981). Studies of tomato ripening mutants have indicated the importance of both PE and PG in the softening of the ripening fruit (Grierson et al., 1981). It is important to investigate the presence and possible involvement of other hydrolytic enzymes during cell wall expansion, i.e. glucanases, galactosidases. The activity of these enzymes could be detected by observing the turnover of individual carbohydrates in the cell wall polymers (Labavitch, 1981). Keegstra et al., (1973) have proposed an alternative to hydrolytic cleavage during cell wall expansion. Cell wall expansion could occur through the movement of the cellulose fibrils and the xyloglucan (hemicellulose) chains, connected through hydrogen bonding relative to each other, and that this movement could be accomplished by a non-enzymatic creep. They propose high H-ion concentration as the condition that promotes H-bond exchange, e.g. creep. But Valent and Albersheim (1974) suggested that the H-ion concentrations found in the cell wall after IAA treatment was not sufficient to alter hemicellulose binding to cellulose in vitro .

Anthocyanins and Color Degradation Mechanisms

Textural changes are not the only changes that will affect the quality of strawberries or products derived from them. Loss of the characteristic red color is also of great importance, and often more significant than textural losses. The characteristic red color in strawberries is due to the presence of the anthocyanin (Acn) pigments with pelargonidin- (Pgd) and cyanidin- (Cyd) -3-glucosides as the major pigments found in strawberries. Anthocyanins have been reported to be found in anthocyanoplasts. These organelles contain a single tripartite membrane and are found inside the vacuole, and it is suggested that they contain the later enzymes of anthocyanin biosynthesis (Pecket and Small, 1980; Small and Pecket, 1982). 55

Strawberry color is influenced by such pre- and post-harvest factors as variety, maturity, harvest date, irrigation, post-harvest holding time and temperature, and thawing during freezing storage (Sistrunk and Cash, 1968). When considering the whole fruit color loss can occur through several mechanisms occuring alone or in combination: degradation of anthocyanins, formation of brown pigments (enzymatic and non-enzymatic browning), and discoloration through heavy metals or copolymerization with other phenolic compounds (Abers and Wrolstad, 1979). Preventive methods used to avoid excessive loss of anthocyanins include the use of fruit varieties containing higher levels of the pigments, avoiding discoloration (enzymatic browning) during handling, the inhibition of enzymes (blanching), avoiding contact with oxygen, decreasing the pH, use of cans lined with special enamels, the use of minimum heat, and low exposure to direct light (Markakis, 1974). Phenolic compounds, including anthocyanins, are controlled by genes and their concentration can therefore be controlled by breeding (Bate-Smith, 1962). The use of varieties containing higher levels of anthocyanins show much less discoloration after processing or storage. The effects on anthocyanins and color of pH, presence of aglycones, light, oxygen, sulphur dioxide, heat, heavy metals, ascorbic acid, Maillard browning compounds, endogenous phenolics, and PPO and enzymatic browning compounds will be described in the following sections. It should be pointed out that in fruits or food products containing anthocyanins, degradation occurs through a combination of the various mechanisms, although one usually predominates.

Effects of pH

Anthocyanin pigments exist in different forms depending on the pH of the medium and the importance of this phenomenon is that each of the forms is associated with a different color expresion (Figure 1.2).

Formerly it was assumed that the quinoidal base (A) arose by 56 dehydration of the carbinol pseudo base (B); hence it was usually called an anhydro base (Williams and Hrazdina, 1979). The quinoidal base has been found to arise directly by proton transfer from the flavylium cation (AH+). Covalent hydration of the flavylium cation leads to the formation of a carbinol pseudo base (B) which itself exists in tautomeric equilibrium with small amounts of chalcone (C) (Figure I.2,a) (Brouillard and Dubois, 1977; Brouillard and Delaporte, 1977; Timberlake, 1981). At equilibrium and at any pH the quinoidal base is only present to a very small extent. Kinetic comparison between deprotonation and hydration reaction always favor proton loss. At pH lower than 3, the flavylium cation and the carbinol pseudo base exist in equilibrium and only very low levels of the quinoidal base and chalcone exist. At pH higher than 3 the equilibrium tends to shift towards the formation of the unionized or ionized forms of the quinoidal base (A, A-) (Figure 1.2, b & c). The flavylium cation initially present is deprotonated (1) before even a minute fraction is hydrated (2), but the quinoidal base forms are unstable and depending on the pH rearrange into unionized or ionized forms of carbinol pseudo base and chalcone (3) (Brouillard and Delaporte, 1977):

(1) DEPROTONATION acid-base equilibrium= AH — A + H

(2) HYDRATION equilibrium= AH + H20 —*- B + H

(3) RING-CHAIN tautomeric equilibrium= B —*■ C

The equilibrium between the different forms of the anthocyanin is going to depend very much on the characteristics, especially pH, of the medium, e.g. food, and therefore affect the expresion of color. In frozen strawberries, pH was the only observed measurement having a high correlation with color quality. It was calculated that berries should have a pH of 3.51 or lower to have accepatable color after freezing and anthocyanin content should be in the range of 450 to 700 57

/jg/g to have acceptable color quality (Wrolstad et al., 1970). Strawberry puree suffered only gradual loss in red color at pH 3 during storage at 50oC for 20 hours, but adjustment of the pH to 3.8 produced the same change immediately (Sistrunk and Cash, 1968). The ionic form of anthocyanin is by far the most stable and hence the stability of fruits at low pH conditions. The carbinol pseudo base is quite unstable, and the oxidation of anthocyanins in foods during processing or storage is narrowly proportional to the percentage of the pigment in this form (Chichester and McFeeters, 1971). 58

FIGURE 1.2 EFFECT OF pH ON ANTHOCYANIN PIGMENTS :

a) ANTHOCYANIN STRUCTURAL TRANSFORMATIONS WITH pH.

b) EQUILIBRIUM DESTRIBUTION AT 250C OF ,AH+\ 'A1, 'B', AND 'C FORMS OF MALVINIDIN-3- GLUCOSIDE AS A FUNCTION OF pH.

c) PREDOMINANT ANTHOCYANIN CHROMOPHORES IN THE DIFFERENT pH RANGES.

(Brouillard and Delaporte, 1977; Brouillard, 1982). 59

►HtO sOGIc OH AH*-.FLAVYUUM CATION (red) B:CARBINOL PSEUDOBASE jt-H- -H-.^jO jf

3H cyk^.

OGIc OH

A: OUINOIOAL BASE (blue)

(•) con be -H.-OH or -OCHs.

b) Hi ~~^\ t* X-AH^\ OCH, / rsiT0H / ^ 0*^^X^S ^OCH,.

k^O-li-^l \ OH

X-B««v/ X-Cx X;A v. 3 / 1 1 2 J I s „ .

Ct* OrmO tliMM (••nitntiu.

«) Movyliunt mixtur* of Movy- •noutrol mixturo of ion lium cation and i^uinodal noutraf and pr»- Ouinoidal ba

■ i 4 5.iii. 3 1 6 M PH pr,(AH*/A) pr,(A/A-)

FIGURE 1.2 60

Effects of Sugar Hydrolysis and Light

Anthocyanin pigments have an electron deficient flavonoid nucleus that makes them highly reactive and can therefore undergo undesirable color changes (Shrikhande, 1976). They seem to always occur as glycosides in vivo . Hydrolysis of anthocyanins to the aglycones or anthocyanidins may occur due to the presence of beta-glucosidases (anthocyanases) in the fruit or from fungal contamination, or non-enaymatically during processing (Markakis, 1974). Anthocyanidins are highly unstable and can undergo ring fision to yield alpha-diketones which are easily hydrolyzed to acids, i.e. 3,4-dihydroxybenzoic acid (Markakis, 1974; Shrikhande, 1976) (Figure I.3,a). Light does not seem to be a major factor in color loss. The order of stability to light of anthocyanins is: acylated-methylated anthocyanin diglycosides > non-acylated Acn diglycosides > Acn monoglycosides (Markakis, 1974). Acylation occurs through esterification of cinnamic acid derivatives to the carbohydrate units of some anthocyanins. The position of the carbinol-chalcone equilibrium is light dependent. The chalcone is mainly in the trans form, and conversion of the trans- to the cis-chalcone occurs faster in light and was found to favor the cyclization process by decreasing the value of the apparent acidity constant for the flavylium - trans - chalcone equilibrium (Brouillard, 1982). 61.

a) Degradation involvinq hydrolysis

• HO «-*Glc +

OH OH

anthocyanin anthocyanidin

HO^^vOH

k^A^CHO + HOOC OH OH 0

acids S aldehydes ot-diketone

b) No hydrolysis

OGIc OH anthocyanin H0OX * OH glycoside chalcone

Wean be-H.-OH.or-OCH,.

FIGURE 1.3 SOME PROPOSED PATHS OF ANTHOCYANIN DEGRADATION. 62

Effects of Heat and Oxygen

Depending on the pH and temperature conditions, the anthocyanins may be hydrolyzed and/or oxidized of both. At low pH values (pH 1.5 to 3) both oxidation and hydrolysis take place; at pH 5 and above, the main reaction is oxidation. The pigments seem to be more vulnerable to oxidative degradation in both colored states. The least amount of color loss occured in the pH range from 4 to 5 (Hrazdina, 1974).

Lukton et al.t (1956) showed that the rate of anthocyanin destruction both in model systems and in strawberry juice at 45°C was virtually pH independent in the pH range of 2.0 to 4.5 in the absence of oxygen. In the presence of oxygen, however, anthocyanin degradation increased dramatically with pH. The rate of anthocyanin degradation increases with rising temperatures. The rate of equilibrium between the carbinol pseudo base and the chalcone is the slowest among the different anthocyanin forms. At elevated temperatures this equilibrium becomes of greater importance when all forms of the anthocyanin tend to be converted to the chalcone because of the endothermic nature of the (A) ■(AH+) -(B) >(C) sequence reaction. Color will fade not only because of possible pigment degradation but also on account of the changing equilibrium. On cooling colored anthocyanin is reformed after sufficient time (Timberlake, 1981). The chalcone can be degraded further eventually leading to the formation of a brown precipitate (Markakis et al., 1957). Anthocyanins are rapidly oxidized by air and destroyed when they are in the quinonoid form (Robinson, 1980). Another degradation mechanism proposes that the heterocyclic ring of the colorless carbinol pseudo base opens to form a colorless chalcone before the glycosidic bond is hydrolysed, and in the presence of oxygen along with the chalcone, a coumarin glycoside derivative has been found (Figure 1.3, b) (Markakis, 1974). 63

Effects of Sulfur Dioxide

Anthocyanins can be decolorized with sulphur dioxide. No hydrolysis of the 3-glycosidic group, reduction of the anthocyanin or addition of bisulfite to a ketonic or chalcone derivative are involved (Jurd, 1964). Introduction of a methyl or phenyl group in the 4 position (aliphatic ring) of the anthocyanin confers resistance to color bleaching (Shrikhande, 1976), and direct addition at either positions 2 or 4 of bisulfite producing a colorless sulfonic acid derivative has been proposed (Chichester and McFeeters, 1971; Timberlake and Bridle, 1968; Jurd, 1964).

OH H SQ,

CHROMEN-2OII4-SULFONIC ACIDS

The flavylium cation form is the reactive species involved in bisulfite decoloration and the sulphonic acid derivatives formed have similar structure and properties to the anthocyanin carbinol bases. Decoloration can be reversed on acidification or addition of carbonyl compounds, i.e. acetaldehyde (Hrazdina, 1974; Markakis, 1974). The reaction rate is dependent on the carbonium ion (flavylium cation), hydrogen ion (pH) and bisulfite concentrations.

Effects of Heavy Metals

Anthocyanins and anthocyanidins which have a free jo -hydroxyl grouping undergo bathochromic shifts in the presence of metal ions, i.e. Al, Sn, Fe, Cu (Shrikhande, 1976). Discoloration of some canned 64

fruits due to the reaction of the anthocyanins with tin dissolved from the container resulting in the formation of complex metallic compounds was already reported in 1927 by Culpepper and Caldwell. Anthocyanins reacting with tin can act as either cathodic or anodic depolarizers. As cathodic depolarizers, anthocyanins are probably reduced by nascent hydrogen formed in the acid metal-reaction. Anodic depolarizers are anthocyanins with at least two hydroxyl groups in ortho position (Markakis, 1974). Metal chelation is a factor capable of stabilizing the quinonoid base form (blue color) of anthocyanins. Acylation of anthocyanins, e.g. with 2. -coumaric acid, may be important for stable metal-anthocyanin complex formation (Timberlake, 1981). The formation of a co-pigment metal-anthocyanin complex could account for the undesirable "bluish" color of some varieties of frozen strawberries (Wrolstad et al., 1979). In the case of strawberry puree tin chloride seems to stabilize the red color or even enhance it (Cash and Sistrunk, 1970). Strawberry juice not treated with SnCl was browner than the treated sample (Wrolstad and Earlandson, 1973). In this case a red-colored material precipitated which was not bleached by bisulfite (i.e. polymeric in nature) but bleached by hydrogen peroxide. It was proposed that a conjugated (anhydro) quinoidal base which was formed from oxidation of leucoanthocyanins was the entity which formed the purple-pink chelate with tin (Wrolstad and Earlandson, 1973) Other flavonoid compounds, i.e. flavonols and flavones containing a _o -hydroxy group, can react with metal ions and have been found responsible for black discoloration (Herrmann, 1976). Iron and copper complexes of catechol or _o -phenolic derivatives have a latent tendency to undergo electron transfer within their complexes with the formation of the reduced metal and a radical form of the oxidized phenols, i.e. a semiquinone anion (Pollard and Timberlake, 1971). The free radicals can initiate polymerization reactions that can include anthocyanins and further oxidation reactions of phenolic compounds to quinones. Organic acids present in the tissues, i.e. citric, malic, and 65 tartaric, may bind metal ions preferentialy making them unavailable for reaction with anthocyanins or phenolic compounds (Wrolstad et al., 1970a; Pollard and Timberlake, 1971).

Effects of Non-Enzymatic Browning

Considerable loss of anthocyanin pigments and an increase in the non-anthocyanin absorption due to browning reactions occur during the processing and storage of most products containing these pigments. The development of brown colored compounds was found to be preceeded by a major loss of red pigment in the case of strawberry preserve manufacturing (Kertesz and Sondheimer, 1948). Similar reactions have been observed in other products made of athocyanin-containig fruits. The formation of an insoluble red-brown precipitate occuring where browning reactions are observed seems to be the end product of anthocyanin degradation (Abers and Wrolstad, 1979). There are different types of browning reactions that can occur in plant tissues, and during storage and processing of food products. The effects of non-enzymatic browning reactions (Ascorbic acid and Maillard) on anthocyanins and color will be discussed in this section. The effect of phenolic compounds and enzymatic browning reactions will be discussed in the following section. Ascorbic acid (AsA) is present in significant amounts in strawberries, but the levels vary depending on the variety, i.e. Hood and Tioga contain 48 and 19 mg, respectively, of AsA per lOOg fresh fruit weight (Abers and Wrolstad, 1979). The relationship of AsA to pigment breakdown was found to be altered by variety, maturity, cellular constitution, complexing agents, sugars, and active enzymes in frozen strawberries (Sistrunk and Moore, 1971). More than one mechanism seems to be involved in AsA breakdown. In the presence of heavy metals and oxygen, AsA is oxidized to dehydro ascorbic acid (dAsA) with the production of hydrogen peroxide and certain peroxi radicals (Markakis, 1974; Hrazdina, 1974). Both hydrogen peroxide and the peroxi radicals can degrade anthocyanin pigments directly. Jurd (1972) reported that flavylium 66 salts oxidized by hydrogen peroxide may form enolic benzoate derivatives, benzoyl esters of the malvone type, 3-substituted 2-phenylbenzofurans, or ketodiol derivatives. It has been reported that hydrogen peroxide oxidation of malvin under acidic conditions lead to the formation of malvone-type derivatives (e.g. acylated or non-acylated coumarin glycosides) formed through a Bayer-Villiger type oxidation of the quinoidal base form of the anthocyanin by hydrogen peroxide. Anthocyanidins and Acn-3-glycosides do not form coumarin derivatives and apparently rearrange to other, not yet identified products (Hrazdina, 1974). Ascorbic acid can be degraded directly or indirectly by the action of enzymes. Ascorbic acid oxidase directly oxidizes AsA to dAsA in the presence of oxygen and using NADPH as a cofactor. The presence and significance of this enzyme in strawberries needs further study. Indirect oxidation of AsA to dAsA occurs when AsA functions as a reducing agent of the products of some enzymatic reactions, i.e. peroxidase, cytochrome oxidase, and PPO (Wrolstad et al., 1980). PPO produced quinones can directly or indirectly degrade anthocyanins, but in the presence of AsA, the quinones will be reduced and AsA oxidized to dAsA. Thus AsA can temporarily protect anthocyanin degradation by PPO (Sistrunk and Moore, 1971). Both AsA and dAsA can suffer non-oxidative rearrangement and degradation reactions under the storage and cooking conditions of foodstuffs. The mechanisms of these reactions have been described by Kurata and Sakurai (1967 a,b). In the case of dAsA, which can be derived from enzymatic or nonezymatic oxidation of AsA, highly reactive reductones are formed as intermediates. The disappearance of dAsA is usually as fast as that of AsA (Sistrunk and Cash, 1968). In the case of AsA, not only reactive reductones, but also furfural are produced during the degradation reactions (Kurata and Sakurai, 1967 a,b). The degradation of AsA resembles the reactions observed during Maillard browning, or carbonyl (reducing sugars) amine reactions, hence both can be considered together. Non-enzymatic browning is also characterized by the formation of reductones, furfural derivatives, and the formation of brown polymers that cause 67 the masking or destruction of the color of food products. Maillard reactions have been reported not to be prominent in fruit juices because of their aqueous nature and low pH (Pollard and Timberlake, 1971). Fruit products containing high concentrations of sugars, which serve as dehydrating agents, or that may have been subjected to heat usually show greater signs of non-enzymatic browning (Hodge and Osman, 1976). In the presence of furfural derivatives as well as other products of the browning reactions, the anthocyanins are degraded or may enter the polymerized product of the browning reaction as a co-polymer. The fruit product may not show initially a significant change in color since the polymer may visibly resemble the color of the original pigment, but with time the original red color is lost (Chichester and McFeeters, 1971). Fructose, arabinose, lactose, and sorbose were more deleterious to anthocyanin pigments than sucrose, glucose, or maltose, and the presence of oxygen aggravates the destructive effect of these sugars (Markakis, 1974). Debicki-Pospisil et al., (1983) studied the degradation of cyanidin-3-glycoside by furfural and 5-hydroxymethyl furfural and found the reactive species of the pigment to be the quinoidal base form of the anthocyanidins. Considerations that can be taken during the processing of fruits that can diminish the problem of anthocyanin loss through non-enzymatic browning are the use of the lowest possible temperatures during processing, the removal of oxygen, the use of fruit varieties with higher levels of pigment and lower levels of ascorbic acid, and the inactivation of oxidating enzymes (Markakis et al., 1957). Some flavonoid compounds present in fruits, i.e. quercetin, can inhibit the oxidation of AsA due to their ability to chelate heavy metals and to react with free radicals. The aglycones are usualy more effective (Pollard and Timberlake, 1971; Shrikhande and Francis, 1974; Herrmann, 1976). Reducing compounds like thiourea can also decelerate the rate of AsA-catalyzed anthocyanin degradation (Sondheimer and Kertesz, 1953). Flavylium salts condense easily with amino acids, phloroglucinol, catechins, AsA, and other nucleophiles to yield colorless 68

4-substituted flav-2-enes. These derivatives usually undergo further changes, the nature of which depend on the 4-substituent, the net result being the loss of the flavylium pigment (Jurd, 1972).

Effects of Enzymatic Browning and Phenolic Compounds

In fruit and fruit products in which the cell compartmentation has broken down through mechanical damage, or during processing or storage, PPO and the endogenous phenolic compounds will interact and, with time, lead to brown discoloration, i.e. enzymatic browning. In fruits containing anthocyanin pigments the loss of the red color could occur through several mechanisms which will be described below. It was reported over thirty years ago that strawberries did not contain PPO (Joslyn and Ponting, 1951). More recently it was suggested that PPO was of secondary importance in berry fruits (Vamos-Vigyazo, 1981). The presence of PPO in strawberries has recently been demonstrated by several workers. Palavicini (1969) demonstrated that 16% of the PPO activity survived long term storage at -20oC. Cash and Sistrunk (1971) demonstrated PPO activity in acetone extracts of the berries, and Drawert et al., (1974) made electrophoretic studies of 16 strawberry varieties and 7 cultivars that revealed one to seven forms of PPO with DOPA as a substrate. PPO was also found present in strawberries of the Tioga variety (Wesche-Ebeling, 1981). Assessment of the role of PPO in anthocyanin degradation in strawberries and products derived from them usualy involves the comparison of blanched and unblanched samples. It is important to note that the extent to which any initial enzymatic change has been allowed to proceed is likely to be a factor in the appearance and stability of the final product (Pollard and Timberlake, 1971). This could be the case in the period between thawing of frozen or pressing of fresh fruits and the blanching process. Products derived from blanched strawberries, i.e. inactivated PPO, may show no significant difference in discoloration when compared to unblanched products. This observation may indicate that discoloration occurs through non-enzymatic mechanisms, but other 69 mechanisms could be involved. The extent to which PPO was active before blanching is of importance because the quinones and intermediary browning reaction products will serve later as initiators of further browning even if PPO has been inactivated. The heat involved in the blanching operation may also be a source of brown-discoloration due to the fomation of carbohydrate or ascorbic acid breakdown products that may lead to non-enzymatic browning reactions. PPO activity will also be a cause of ascorbic acid breakdown. In the following sections the possible mechanisms of anthocyanin loss involving PPO and endogenous phenolic compounds in strawberries and strawberry products will be discussed.

Endogenous PPO Substrates

Strawberries contain endogenous phenolic compounds that can serve as substrates for PPO, i.e. catechin, leucoanthocyanins, caffeic acid, and gallic acid (see Table 1.4). Substrate specificity studies showed catechin to be by far the best substrate among the endogenous phenolic compounds assayed (Wesche-Ebeling, 1981). Flavonoid compounds other than flavan-3-ols (catechins) do not seem to be good substrates for PPO but they can be oxidized in the presence of transfer substances such as catechin, or other substrates of PPO through indirect coupled oxidation reactions (Froemming et al., 1962; Mathew and Parpia, 1971; Herrmann, 1976). Direct oxidation of anthocyanin pigments by PPO was suggested by Schumacher and Bastin (1965) and Sakamura et al., (1966), but in both cases, anthocyanin was only readily oxidized by the enzyme in the presence of a suitable substrate, i.e. chlorogenic acid, catechin. Ascorbic acid and sulfur dioxide greatly inhibit the degradation of anthocyanin in the presence of PPO and suitable substates therefore confirming the involvement of quinones in the coupled oxidation reactions (Goodman and Markakis, 1965; Sistrunk, 1972). 70

Mechanism of Anthocyanin Degradation

The mechanism of anthocyanin degradation by PPO that has received widest support is the coupled oxidation of the pigments by quinones derived from the PPO activity (Peng and Markakis, 1963; Sakamura and Obata, 1963; Schumacher and Bastin, 1965; Pifferi and Cultrera, 1974):

0. o -DIPHENOL- -DEGRADED ANTHOCYANINS

PPO

H20 •o -QUINONES ANTHOCYANINS

This mechanism does not really describe the fate of the anthocyanin pigments. Anthocyanin pigments may interact with quinones, phenolic compounds, polymers formed during the browning reactions, undergo self-association, or a combination of any of these interactions.

Non-convalent Interactions of Anthocyanins

Indications exist that anthocyanins may undergo self-association in aqueous solutions (Asen et al., 1972). Monomeric anthocyanins in neutral solutions show little optical activity, whereas extraordinarily large molecular ellipticities also observed indicate the presence of anthocyanin aggregates. The driving force for the intermolecular self-association seems to be mainly the hydrophobic interactions among the aromatic nuclei stacked parallel to each other. Glycosidation at the 5-position seems to have an important role in self-association (Hoshino and Matsumoto, 1980; Hoshino et al., 1981a). Intramolecular copigmentation is responsible for the extraordinary stability of the chromophores of acylated anthocyanins. The aromatic residues of the acyl groups are believed to stack with the pyrylium ring of the flavylium cation and, consequently, impede 71 the formation of colorless carbinol pseudobases, i.e. inhibit hydration of the flavylium salts (Brouillard, 1983). From a thermodynamic point of view the intramolecular effect has an important entropic advantage over the intermolecular effect, since the former effect does not need to bring together molecules initially separated in solution (Brouillard, 1983). Anthocyanins can form co-pigment complexes with flavonoids and other compounds at pH's ranging from 2 to 5 (Asen et al., 1972; Scheffeldt and Hrazdina, 1978). Osawa (1982) reviews copigmentation of anthocyanins in vivo . The formation of these co-pigment complexes results in a bathochromic shift (blue shift) in the visible absorption maxima of the anthocyanins and a large increase in the extinction coefficient at pH 3 and higher (Asen et al., 1972). The quinoidal (anhydro) base form of the anthocyanins seem to be involved in the complex formation (Williams and Hrazdina, 1979; Timberlake, 1981). At high pigment levels co-pigmentation is limited by the self-association of the anthocyanin cationic forms (Scheffeldt and Hrazdina, 1978). The most effective co-pigments so far discovered are flavonol 0- and C-glycosides in which the aglycone is quercetin (Brouillard, 1982). Details of the bonding of the anthocyanins to the co-pigment are not known, but hydrogen-bonding is suggested from the fact that co-pigmentation is concentration dependent and that copigment complexes are dissociated by heating (Asen et al., 1972). Hoshino et al., (1980) found that acyl groups of some anthocyanins had an important role in the stability and blueing effect of anthocyanin-flavonoid co-pigment complexes and that hydrophobic interactions were involved in the stability of the complexes. Hoshino et al., (1981b, 1982) propose some models for the anthocyanin-anthocyanin and anthocyanin-flavonoid aggregates.

Formation of Tannins

Catechin is found in significant amounts in strawberries and autoxidation and PPO oxidation of this phenolic leads to the formation of polymers. Polymerization seems to involve a head-to-tail C-C bond 72 formation between the catechol portion (B ring) electrophilic activated sites 2',5' and 6' of a catechin-quinone with the phloroglucinol (A ring) nucleophilic activated sites 6 and 8 of another catechin-quinone or with the 6, 8, 2', 5' or 6' sites of a catechin molecule. The polymer obtained by the enzymatic oxidation has the analytical properties of a tannin (Hathway and Seakins, 1955, 1957a,b):

-in DIHYDRODICATECHIN-B

CATECHIN-QUINONE

TANNINS FORMED THROUGH ENZYMATIC DEHYDROGENATION

Two flavonoid groups of importance are the leucoanthocyanidins (leucoacn) and the proanthocyanidins (proacn) . Known leucoacn posses the structure of a polyhydroxy-3,4-flavandiol differing only in the number and positions of the hydroxyl groups distributed about the atoms 5-8 and 2,-6'. In strawberries they have been reported present in levels amounting to 565 Abs.Units per lOOg fresh fruit weight (Abers and Wrolstad, 1979). In acid solutions, leucoacn exhibit competition between elimination and substituition reactions. In the elimination of water the corresponding anthocyanidins are formed, whereas in the substitution process, the dimeric leucoacn which are formed at first may further react to oligomers with pronounced tannin properties (Weinges and Nader, 1982) (Figure 1.4 a). The ratio of elimination to substitution will depend on the 73 nucleophilicity of the A-ring, the type of solvent, and temperature. At low temperatures and in the presence of moisture the polycondensation of leucoacn occurs at pH 4.5, yielding tannins. Condensation can also occur with other components of the system and it is very difficult to determine the final composition of the tannins which may range in the degree of polymerization from water-soluble tannins to the insoluble phlobaphenes (Baranowski and Nagel, 1983). The term proacn was suggested for all flavonoids yielding anthocyanidins upon treatment with acid. Leucoacn belong to this type of compounds but other flavonoids show this characteristic. Dimeric proacn have been extracted from a wide variety of fruits and found to be composed of two linked flavonoid units. The term proanthocyanidinocatechins has been proposed for this type of proacn. Acid causes the cleavage of the C-4-C-8" bond and the formation of a polyhydroxy-3-flavanol from the 'lower' half and a leucoacn from the 'upper' half which can convert to the anthocyanidin (Weinges and Nader, 1982) (Figure 1.4 b). Dehydrodicatechins was also proposed as a name for dimeric proacn, but dehydrodicatechins that are formed by enzymatic oxidative coupling of catechins possess a structure that does not allow the formation of anthocyanidins upon the action of acid. Dimeric proacn biogenesis does not proceed via the intermediate of a catechin (Weinges and Nader, 1982). Somers and Ziemelis (1972) reported the absence of true leucoanthocyanins (flavan-3,4-diols) from grape juices and extracts and that the dimeric proanthocyanidins that appear in the final stages of growth of the berry are probably biosynthesized by enzymatic (PPO) dehydrogenation of catechin. Robinson (1980) reports that biosynthesis of condensed tannins occurs by polymerization of catechin and/or leucoanthocyanidin monomers. 74

LEUCOANTHOCYANIDIN ANTHOCYANIDIN^-,

CATECHIH !1I CONDENSED H PHLOBAPHENES TANNINS 0

Condensation can be non-oxidative, oxidative-non-enzymatic or catalyzed by a peroxidase or PPO. The mechanism that would predominate would depend on the conditions of the medium. Weinges et al., (1969) and Weinges and Huthwelker (1970) review the formation, constitution and importance of flavonoid-tannins. Creasy and Swain (1965) report the structure of condensed tannins in strawberries (see also Chemical Composition section). 75

a)

Aitfticiuidii

H OH Oiairic Leie*aatktc?aii

b) —Aotdoejioidlfl

OH „

Leieoaitboeyitidia

HO, ^

OH H H OH OH H H ProiitbicjiiKit P«ljlijdr(j|-3-FI»iaBol

FIGURE 1.4 REACTIONS OF PRO- AND LEUCOANTHOCYANINS : A) ELIMINATION AND SUBSTITUTION REACTIONS OF LEUCOANTHOCYANIDINS. B) MECHANISMS OF THE ACID-CATALIZED CLEAVAGE OF PROANTHOCYANIDINOCATECHINS. 76

Covalent Interactions of Anthocyanins

Studies utilizing synthetic flavylium salts indicate that anthocyanins may undergo self-condensation in aqueous media to yield dimeric pigments with spectral characteristics closely related to the original pigments. This dimerization may involve the initial condensation of the flavylium salt and its carbinol base to give a product which then undergoes a series of intermolecular hydride ion transfer reactions. The flavylium dimerization process may be accounted for by initial nucleophilic attack of the 7-hydroxyl of the carbinol base on the flavylium cation or its anhydro base to form salts substituted at position 4 with the corresponding dehydrochalcone nucleus (Jurd, 1972) (Figure 1.5, I). Flavones can be formed as by-products of the reaction which can further participate in the condensation reactions. Naturally occuring anthocyanins would require hydrolysis of the sugar moieties before undergoing these reactions. Hydrolysis of the sugar moieties can occur under mild acidic conditions, i.e. pH 1 to 3 (Jurd, 1967, 1972). Markakis et al., (1957) proposed that brown polymeric pigments could be formed due to the condensation of chalcones formed from the hydrolysis of the pyrylium rings of anthocyanins. Anthocyanins can also condense with flavonoid compounds. Model systems using flavylium salts and catechin showed oxidative condensation involving no atmospheric oxygen between the two compounds. The flavylium salt served as the oxidant and the initial condensation yielded a flavenyl-flavan intermediate. Rapid oxidation by a second molecule of flavylium salt lead to the formation of a flavylium-flavan dimer and a monomeric flav-2-ene (Figure 1.5, III). The flavene formed disproportionates to the flavylium salt and the flavan, and the reaction could continue. If aerobic oxidation was also possible, the levels of flavan would decrease and those of flavylium salt increase (Jurd, 1967, 1972). Malvinidin-3-glucoside and D-catechin condensation reactions show pseudo first order kinetics. At pH 3.5 in an aqueous solution, the equilibrium distribution of malvinidin-3-glucoside is about 75% 77 carbinol base, 8.3% chalcone, 10.4% flavylium ion, and 2.1% quinoidal base (Brouillard and Delaporte, 1977)(see Figure 1.2). At this pH the condensation reactions would shift the equilibrium towards the production of more flavylium ion, thus the reactions would continue over a long period of time. The rate of condensation would increase with decreasing pH (increasing flavylium ion levels) (Baranowski and Nagel, 1983). Timberlake (1980) reported that anthocyanins and phenolic compounds (flavan-3-ols and procyanidins) reacted very slowly, and that the reaction eventualy lead to the formation of yellow xanthylium salts (Figure 1.5, II) that could be glycosylated or not. Condensation could occur via the anthocyanin A position and positions 6 or 8 of the phenolic compounds. Monomeric flavan-3-ols (catechins) may react more readily, but reacting procyanidins are disproportionated into catechins and lower and higher condensed tannins (Timberlake, 1980). The interaction between anthocyanin pigments and tannins has been studied in detail in red wines. Similar interaction may be occuring in the case of strawberries or products derived from them and may explain the presence of a red color even when anthocyanins can almost not be detected. Little (1977) suggested that on processing and during storage of anthocyanin-containing products the pigments become incorporated into the pH non-responsive polymeric moiety resulting in a retardation of pigment degradation and thus an increase in color stability. Heat can induce breakdown of the polymer-anthocyanin bond with the release and degradation of the anthocyanins. Tannins of red wine contain anthocyanin units in their structure and must be regarded as condensed flavonoid pigments (polymeric pigments). These polymeric pigments are much less sensitive to changes in pH and are quite resistant to decoloration by bisulfite. Quinonoid base chromophores derived from anthocyanins and which have been stabilized by substitution with reactive flavans may be present in the polymer. A fraction of the polymeric pigment exists in a colorless form whose level varied depending on the pH suggesting the presence of flavylium chromophores in the polymer (Somers, 1971). The following mechanism was suggested by which tannin pigment 78 materials may be elaborated from the flavylium species of the anthocyanins was proposed by Somers (1971). Electrophilic substitution into a procyanidin component RH by an anthocyanin in the mesomeric carbonium ion form yields a labile flavene which is oxidized by air or by involvment in a redox system to the condensed anthocyanin species. The more stable quinonoid structure assigned to the chromophore of the polymeric pigments is then formed by deprotonation. The substituent (R) is most likely to be a dimeric proanthocyanidin linked at the 6 or 8 positions. Slow increase in the mean molecular size of the polymer could proceed via the species (A+) by further electrophilic substitution into the quinonoid structure or the dimeric proanthocyanidin leading to the formation of polymeric structures (Figure 1.5, IV). This polymerization would be acid-catalyzed. Oxidative polymerization of catechins can occur via autoxidation or PPO to yield tannins (see above) having high absorbance in the 400-500 nm region attributed to the presence of repeated quinone units. Further condensation and cross-linkages would be possible through quinone polymerization mechanisms. Acid-catalyzed and oxidative polymerization may occur simultaneously. Addition or presence of acetaldehyde in a mixture of phenolics and anthocyanins causes a rapid and spectacular color augmentation with shifts toward violet (Timberlake, 1980). The acetaldehyde bridges (Figure 1.5, V) that are formed depend only upon the nucleophilic characteristics of the carbons 6 and 8 of the anthocyanin and so is only slightly affected by the anthocyanin form (Baranowski and Nagel, 1983). Addition of acetaldehyde to strawberry juice lead to an increase in color density, a decrease in anthocyanin content and a decrease in L, a and b values (Hunter colorimeter) characteristic of a lighter red-yellow juice. Addition of catechin with no acetaldehyde lead to a decrease in the L, a and b values probably due to darkening and the formation of browning compounds (Chen and Wrolstad, 1980). Comparison of strawberry juices and concentrates prepared from blanched (microwaved) and unblanched fruits showed a striking difference in chemical composition. Anthocyanins, flavanols, 79 leucoanthocyanins, total phenolics and ascorbic acid levels were higher in blanched samples which also appeared redder visually. The magnitude of the difference between blanched and control decreased with storage, particularly when exposed to oxygen. The red color in blanched samples may be due to non-oxidized polymeric pigments, while the brown color in controls may be derived from oxidized polymers (Wrolstad et al., 1980). Strawberry from the Tioga variety contain greater quantities of the more reactive phenolics, i.e. leucoanthocyanins and catechin, and show greater susceptibility to polymeric browning than berries from the Hood variety. They also have a lower pH (pH 3.5 as compared to pH 3.7 for Hood) and lower levels of ascorbic acid. The higher levels of ascorbic acid in the Hood variety would suggest greater susceptibility towards Maillard browning, but the greater extent of browning in the Tioga variety seems to indicate that anthocyanin degradation and Maillard browning are of less significance than enzymatic browning during color degradation (Abers and Wrolstad, 1979). With the introduction of mechanical once-over harvesting of strawberries, green and ripe strawberries are harvested together. The effect of the immature fruit on strawberry products manufacture, e.g. jam, puree, etc., seems to be more one of dilution of the color. Color is more dependent upon the anthocyanin content and the use of strawberries with high anthocyanin content is suggested for mechanical harvesting. More discoloration was noticed in purees containing green strawberries as compared to those containing only ripe fruit. Discoloration was probably due to several factors including the degradation of anthocyanins, the presence of chlorophyll and phenolic compounds, and of PPO and peroxidase. Green strawberries may also influence flavor due to the absence of a complete enzymatic system for development of flavor compounds (Sistrunk and Morris, 1978; Spayd and Morris, 1981 a,b,c). 80

FIGURE 1.5 SOME MECHANISMS AND PRODUCTS OF ANTHOCYANIN CO-POLYMERIZATION. 81

^1 M niy^s f

AKTHOCTANIK* + ♦ FLAVANOL

Flatttyl-flatai ilaer

H- fl»n»al-*tt>iey»ii» coiiogtatioi.

FlarylUa-flam timn

CH ,^ • H- - CH,-MnH • UTUNII - CHf^H . „„„.„„„ CATESHIN

OM It'frttitaHit)

I-AcataKifc^e irUjt forma tin

FIGURE 1.5 82

ANTHOGYANIN* + ♦ PROCYANIOIN(RH)

OH Fliiin itroetnri

Caidtiui Antbocjioli: ») Fla»>llm tptelei, >) Qalmntid

Pro»ond joljonrle itroetnri.

g-TiBBU-AatliBCHiia eoiiiitatioa. (•) con be -H.-OH or -OCH,.

FIGURE 1.5 Continued. 83

TITLE: STRAWBERRY POLYPHENOL OXIDASE: PURIFICATION AND CHARACTERIZATION.

AUTHORS: Pedro A.E. Wesche-Ebeling and Morris W. Montgomery.

AFFILIATION: Department of Food Science and Technology

Oregon State University

Corvallis, Oregon 97331

RUNNING HEAD: Strawberry Polyphenol Oxidase.

Technical Paper No. from the Oregon Agricultural Experiment Station. 84

STRAWBERRY POLYPHENOL OXIDASE: PURIFICATION AND CHARACTERIZATION.

Pedro A.E. Wesche-Ebeling and Morris W. Montgomery

Department of Food Science and Technology

Oregon State University Corvallis, Oregon 97331

ABSTRACT

A highly active extract of polyphenol dxidase (PPO) from strawberries was obtained using an optimized extraction procedure. The enzyme was extracted at the optimum pH of the enzyme using a buffered solution containing the phenolic binding agents Polyclar AT and Amberlite XAD-4, Triton X-100, and inhibitor. Citric acid in the buffer appeared to inhibit the binding of PPO to the pectic material by chelating the calcium ions. The pectic material was separated from the enzyme using phenyl sepharose chromatography and two fractions of the enzyme, Fl and F2 were separated. Fraction Fl eluted with the void volume of an anion exchange column, had a molecular weight of 111,000 by gel filtration, and did not penetrate into the running gel during electrophoresis. This fraction of the enzyme was bound to a Concanavalin column suggesting that PP0-F1 contains carbohydrate although the mode of association was not be determined. PP0-F2 was resolved into two fractions through anion exchange chromatography. It had a molecular weight of 34,500 by gel filtration, was resolved into two bands during electrophoresis, and did not bind to Concanavalin. All fractions of the enzyme showed multiple banding during LDS-electrophoresis on a gradient gel. 85

INTRODUCTION

Polyphenol oxidase (E.C.I.14.18.1;PPO) is an enzyme that is found widely distributed in plants. No definite physiological role has been assigned to PPO but among those to which this enzyme has been most associated are in electron transport in chloroplasts or mitochondria (Mason, 1955; Butt, 1980); in the melanization and impermeabilization of seed coats (Pierpoint, 1970); the participation in resistance and protection against stress situations such as mechanical damage, adverse climate, exposure to harmful chemicals, or infection by fungi, bacteria, or viruses (Bell, 1974; Rhodes and Wooltorton, 1978; Friend, 1981). PPO has also been reported as being associated in some of the hydroxylating reactions in the metabolism of cinnamic acids (Rhodes and Wooltorton, 1978; Vaughan and Butt, 1972; Stafford, 1974) PPO has been known for a long time to be responsible for most of the browning reactions observed during the handling, storage, and processing of fruits (Mayer and Harel, 1979; Vamos-Vigyazo, 1981; Corse, 1964; Mathew and Parpia, 1971). These browning reactions will affect the nutritional (Synge, 1975), organoleptic (Joslyn and Ponting, 1951), and in the case of anthocyanin-containing fruit, the loss of the natural color (Co and Markakis, 1968; Wrolstad and Abers, 1979). The same reaction catalyzed by PPO that leads to browning will occur during the extraction of enzymes from plant tissues. The presence of high levels of phenolics and of active PPO has been found to lead to the tanning of proteins by the resulting quinones leading to the modification of the characteristics and even inactivation of enzymes (Anderson, 1968; Loomis, 1969, 1974). As a result, the use of special procedures involving the control of these reactions have been proposed and developed (Loomis, 1974; Loomis et al., 1979; Rhodes, 1977). To study the characteristics of PPO and its involvement in browning and decolorization reactions a purified preparation is necessary. The purpose of this work was to purify the PPO from strawberries. The basic extraction procedure developed earlier 86

(Wesche-Ebeling, 1981) was optimized, and the purification of the enzyme was by using different chromatographic procedures. The enzyme was characterized electrophoretically and the molecular weight of the different fractions was determined.

EXPERIMENTAL

Strawberies of the Tioga variety were obtained from Fuji Farms (Troutdale, OR) in the summers of 1981 and 1982. Early work was with ripe fruit, firm and fully red, while unripe berries, firm and green-white, were used for subsequent work. On the same day of harvesting, the berries were washed, decapped, frozen in liquid nitrogen, sealed in oxygen-impermeable plastic bags, and stored at -20oC until used.

Enzyme Extraction The extraction procedure utilized was an optimized procedure of that reported earlier for ripe strawberries (Wesche-Ebeling, 1981). To 160 ml of 0.2 M sodium phosphate - 0.1 M sodium citrate buffer (extraction buffer=XB) pH 5.25 containing 2%(v/v) Triton X-100 (TX) and 5 mM phenylmethylsulfonylfluoride (PMSF). Fifteen grams Polyclar AT (PVPP) and 30 g Amberlite XAD-4 were added and stored overnight for hydration. Both PVPP and XAD-4 had been previously washed as described before (Wesche-Ebeling, 1981). The strawberry nitrogen powder (NP) was prepared by blending the frozen berries under liquid nitrogen. Fifteen grams of the NP was added to the extraction buffer buffer, mixed thoroughly with a stirring rod, and stirred at 4°C for 1 hr. The slurry obtained was filtered through a nylon cloth with pressure and the liquid obtained was centrifuged at 8,500 x g for 10 min at 0oC. The supernatant was filtered through Whatman #1 filter and the crude enzyme extract (Ec) stored at 40C until used. 87

PPO Activity Measurement PPO activity was measured polarographically using an oxygen monitor equipped with a Clark-type electrode (Yellow Springs Instr., YSI Model 53). The reaction chamber was maintained at 250C by a Lauda K2/R (Brinkman Instr.) constant temperature circulating water bath. Standardization of the instrument was with air-saturated water. Oxygen content of air-saturated water at 250C is 258 nmoles oxygen per ml (Cooper, 1977). The activity was measured using 0.1 to 1.0 ml of the enzyme-containing extracts or fractions, and the volume completed to 2.5 ml with XB pH 5.25. After allowing 4 min for temperature equilibration, 0.5 ml of 200 mM 4-methyl-catechol in XB pH 3.50 kept at 250C was added. Only the initial velocities were considered. The activity is reported as nmoles of 0„ consumed per min per ml of enzyme prepartion used.

Hydrophobic Chromatography Hydrophobic chromatography was adapted from the methods developed by Flurkey and Jen (1978) and Wissemann and Lee (1980). Approximately 500 ml of the crude enzyme extract were made to 1 M with ammonium sulfate and then filtered through Whatman #1 filter before application to the hydrophobic column. This column (2.6 x 20 cm) was packed with pre-swollen Phenyl Sepharose CL-4B (Pharmacia Fine Chemicals; PS) equilibrated in an equilibration buffer (EB) composed of 50 mM sodium phosphate 50 mM sodium citrate pH 5.25 containing 1 M ammonium sulfate, 1 M potassium chloride, and 5 mM PMSF. Elution of the enzyme was by decreasing the EB concentration in a stepwise manner, e.g. by diluting the EB to 0.8 x EB ... 0.05 x EB, followed by 50 % ethylene glycol and glass distilled water. Each step consisted of 150 ml of solution and a flow rate of 1.5 ml per min was used. Each of the two peaks of PPO activity obtained was collected separatedly, made to 1 M with ammonium sulfate and applied to a second PS column (0.9 x 20 cm) and eluted in a similar manner. Each elution step consisted of 60 ml of solution and the flow rate used was 1.0 ml per min. Each of the 88 forms of PPO collected was dialyzed against 5 mM sodium phosphate buffer pH 7.0 for 24 hr at 40C with two changes of the buffer. The hydrophobic columns were run at room temperature. The Phenyl Sepharose was regenerated by washing it with 2 volumes of 5 % v/v TX, 2 volumes of distilled water, 1 volume of 95 % ethanol, 2 volumes of n-butanol, 1 volume of 95 % ethanol, and washed with glass distilled water until all the n-butanol was removed. The PS was equilibrated in the EB before packing the column.

DEAE-Cellulose Anion Exchange Chromatography DEAE-Cellulose 32 microgranular (Whatman LTD) was prepared according to the manufacturers specifications for precycling, degassing, equilibration, and removal of fines, and equilibrated finally in 5 mM sodium phosphate buffer at pH 7.0 (EP). The column size was 0.9 x 27 cm and the flow rate used was 1 ml per min. The dialyzed samples from PS were applied, and after washing the column with 50 ml of EP, the enzyme was eluted by a 200 ml linear gradient (5 to 100 mM sodium phosphate pH 7.0), followed by a 100 ml linear gradient (100 to 200 mM sodium phosphate pH 7.0), and a final wash with 50 ml of 400 mM sodium phosphate pH 7.0. The column was run at room temperature.

Concanavalin-Agarose Chromatography Concanavalin-Agarose (Sigma; ConA), 4 ml , was packed in a 5 ml glass syringe with both the bottom and top of the covered with a disk of glass wool filter paper. The column was connected on top to a sample application container and the other end was connected to a pump (Technicon Instr. Corp., Auto-Analyzer Proportioning Pump and Sampler II). The flow rate used was 0.16 ml per min, and the column was run at room temperature. The elution scheme is an adaptation of the procedure described by Nishioka (1978). The column was equilibrated in 5 mM sodium phosphate pH 7.0 containing 0.1% TX (Be). Before use the column was washed with 50 ml of Be containing 1 M NaCl (Bw) and equilibrated with 20 ml of Be. After application of a dialyzed sample from PS the column was washed with 50 ml of Be and the bound enzyme eluted with 30 89 ml of Be containing 1 M of oc-Methyl Mannoside. A final wash was done with 20 ml of Bw.

Molecular Weight by Gel Filtration Sephacryl S-300 superfine (Pharmacia Fine Chemicals) was packed according to the manufacturers specifications in a 2.6 x 100 column (446 ml of resin). The equilibration and elution buffer consisted of 0.1 M sodium phosphate pH 7.0 containing 0.1 M potassium chloride, and 0.01 % v/v Merthiolate. The flow rate used was 0.95 ml per min. The column was calibrated with commercial proteins (all from Sigma): thyroglobulin, glucose oxidase, BSA, ovoalbumin, chymotrypsinogen, and ribonuclease. Approximately 5 mg of each protein was applied, and the elution volume for each determined from the 280 nm absorption peaks. The void volume was determined using Blue Dextran (Pharmacia Fine Chemicals) and equalled 145 ml. Five ml of dialyzed PPO samples were applied and the elution volume obtained by measuring the PPO activity in the 3 ml fractions. The partition coefficient for the standards and samples was calculated from Kav = (Ve-Vo)/(Vt-Vo). A plot of Kav versus the log of the MW was used as a standard curve and the MW of the PPO fractions was calculated from their Kav values. A complete run took 6 hr.

Electrophoresis Polyacrylamide slab gel electrophoresis (PAGE) was performed under discontinuous non-denaturing conditions in a Bio-Rad Protean 16 cm slab gel unit. The gels were 0.15 cm thick, 14 cm wide, and consisted of 12 cm of running gel and 2 cm of stacking gel in which 15 sample wells were formed. The gel and electrode buffer formulation were adapted from Gabriel (1971), and Laemmli (1970), and the slabs consisted of 5.0 or 7.5 % polyacrylamide running gels, and 4 % stacking gels. The electrode buffer consisted of 0.021 M tris, 0.16 M glycine pH 8.3. Enzyme extracts were concentated with Aquacide II (Calbiochem) after dialysis for electrophoresis. To 1.0 ml of concentrated enzyme, 0.25 ml of a 40% sucrose solution containing a very small amount of 90 bromophenol blue was added, and 100 ul of this solution injected per well on the slab gel. The slabs were kept cool during the run by circulating cold water from a Lauda K2/R water bath. The slabs were run at constant current, 15niA per slab. Lithium dodecyl sulfate, LDS, electrophoresis was performed under discontinuous denaturing conditions. The method was the same as for PAGE except for the use of 0.1 % LDS and 1.75 mM EDTA in the upper reservoir electrode buffer, and the use of gradient slab gels, 7.5 to 15 % polyacrylamide running gels (Einerson, 1982; Wissemann, 1983). Bio-Rad low and high MW standards were used. To both the standards and the dialyzed-concentrated enzyme samples 2% (w/v) LDS, 10 % (v/v) glycerol, 5 % (v/v) 2-mercaptoethanol, and 0.001% (w/v) bromophenol blue were added. The samples were placed in a boiling water bath for 3 min and 100 ul of the samples and 20 ul of the standards were applied per well on the slab gels. The gels were run with cooling at 12 mA per slab at constant current.

Detection of Proteins The slabs were stained for proteins using a modification of the silver stain procedure of Wray et al., (1981) developed by Einerson (1982).

Detection of PPO PPO activity was detected by immersing the slabs in freshly prepared 0.2 M sodium phosphate 0.1 M sodium citrate pH 5.25 containig 50 mM of catechol and 0.05 % (w/v) p-phenylenediamine, until the PPO bands became clearly visible. To stabilize the bands, the slabs were rinsed with glass distilled water and immersed in a 1 mM ascorbic acid solution for 5 min and soaked overnight in glass distilled water. The stained slabs were stored in 30 % ethanol

Protein and Pectin Content Determination Protein was determined using the Bio-Rad protein dye-binding assay. Bovine serum albumin was used for the standard curves. Interfering Triton X-100 was removed with the use of Bio Beads SM-2 91

(Bio-Rad) by adding 0.6 g of moist beads per ml of solution and stirring for 2 hr at 40C. The beads were removed by filtering the sample through glass wool. Determination of the total galacturonic acid content, pectin, was done using the method of Blumenkrantz and Asboe-Hansen (1973). Galacturonic acid (Sigma) was used as a standard.

RESULTS AND DISCUSSION

Optimization of the Extraction Procedure The extraction and purification of PPO from strawberries was very difficult due to the presence of phenolic compounds, low protein content, and the presence of high levels of pectic substances. Preliminary work included the preparation of a crude extract using an extraction procedure that overcame some of the problems mentioned above. The extraction method that yielded the best results was using a buffered solution containing the phenolic-binding agents Amberlite XAD-A and Polyclar AT at a 1:1 ratio by weight of the strawberry nitrogen powder used (Wesche-Ebeling, 1981) The use of phenolic-binding agents during the extraction of enzymes from plant tissues has found widespread use since it reduces the possibilities of the tanning of proteins by the phenolics and quinones derived from them (Anderson, 1968; Loomis et al., 1979). Table II.1 shows the differences in extracted PPO activity between different extraction procedures. It can be seen that the use of a buffered solution at the optimum pH for extraction, and the inclusion of the phenolic-binding agents resulted in a significant increase in the extracted activity. PPO activity in strawberries is significantly lower than that found in many other fruits. Using a very similar procedure in the extraction of PPO from unripe pears (Wissemann, 1983) the reported PPO activity in the crude extract was ten times that of a crude extract obtained from unripe strawberries. Initially ripe strawberries were used, but later work was with unripe fruit since twice as much 92 activity was extracted from the same amount of fruit, e.g. 104 ± 25 U for unripe fruit versus 49 ± 17 U for ripe fruit (1 U = 1 nmoles CL per min per ml enzyme extract). Variations in the levels of PPO activity during the development of the fruit have been also reported from many other fruits (Vamos-Vigyazo, 1981; Mayer and Harel, 1979). A large variability in the extracted PPO activity was noticed from batch to batch of the fruit used. Many reports indicate that PPO may be bound to membranes or found inside organelles, specifically in the chloroplasts (Parish, 1972; Ben-Shalom et al., 1977; Martyn et al., 1979). One of the procedures used to release bound PPO has been the use of detergents. Both 2% v/v Triton X-100 (TX) and 1% Lubrol (Sigma; ethylene oxide condensates of fatty alcohols) when used in the extraction buffer resulted in an increase of the amount of PPO activity extracted (Table II.1). Slightly higher levels of activity were extracted with Lubrol, but crude extracts obtained using TX retained more of the original activity during storage. Other workers have also reported increased extracted PPO activity using TX (Sharma and Ali, 1980; Harel and Mayer, 1971; Galeazzi et al., 1980). It has been reported that extraction procedures involving the use of detergents may induce modifications in the physical and chemical characteristics of PPO (Mayer and Harel, 1979). Wissemann (1983) observed no differences between the electrophoretic patterns of PPO extracted in the presence or absence of TX. The electrophoretic patterns of the crude enzyme extracts obtained from strawberries could not be compared since the presence of pectic material interfered. Makino et al., (1973) reported that TX being a mild non-ionic detergent extracted proteins without the distruption of their native conformation or loss of biological activity. The phenolic-binding agents used during the extraction were Amberlite XAD-4 (XAD-4), which binds phenolics through hydrophobic interactions, and Polyclar AT (PVPP), which binds phenolics through hydrogen bonding (Loomis, 1974). It is possible that the presence of TX in the extaction buffer may have interfered in the binding of phenolics. Oh et al., (1980) reported that hydrogen bonding between 93 solutes was favored to occur in hydrophobic solvents while hydrophobic interactions occur primarely in hydrophilic or polar solvents. This may indicate that the presence of TX, which creates an hydrophobic enviroment, the binding of phenolics to XAD-4 may have been reduced, while binding to PVPP may have increased. Preparation of extracts using PVPP only did not differ significantly from those obtained using both PVPP and XAD-4 (Wesche-Ebeling, 1981). An added advantage in the use of TX is the reported ability of non-ionic detergents to revers or inhibit the formation of tannin-protein complexes (Goldstein and Swain, 1965). Foo and Porter (1981) report the presence of both condensed and hydrolysable tannins in strawberries. Another factor that may modify the physico-chemical characteristics of PPO during the extraction is the presence of proteases (Rhodes, 1977). Flurkey and Jen (1980b) reported that the apparent number or multiple forms of PPO could be decreased using protease inhibitors during the extraction and purification steps. PMSF was used in this work to prevent possible modification of PPO by proteases.

Separation of the Pectic Materials Strawberries contain high levels of soluble pectins and the levels increasing with ripeness (Woodward, 1972). Many problems have been associated with the presence of high pectin levels during the extraction and purification of plant enzymes. Among these problems are the gelling of the crude extracts (Frenkel et al., 1969), poor resolution and artifact formation during electrophoresis (Paulson et al., 1980; Wesche-Ebeling, 1981); blocked flow through chromatography columns; and the co-precipitation of the pectic materials during the preparation of acetone powders (Flurkey, 1979). When crude strawberry PPO extracts were electrophoresed, all PPO activity remained on top of the running gel and the stacking gel collapsed (Wesche-Ebeling, 1981). Several methods were tried to separate the pectin from the enzyme. The first was the precipitation of the pectin using calcium acetate as described by Wong et al., (1971). It was noticed that even though a precipitate was formed, it appeared to consist mainly of 94 calcium citrate, since calcium acetate when added to the extraction buffer alone also formed a precipitate. When the buffer was changed to acetate the extracted PPO activity decreased (Table II.2). In both instances, the addition of the calcium acetate resulted in a loss of PPO activity possibly due to co-precipitation of the enzyme. Flurkey (1979) reported that there was a 50 % loss of PPO activity after the calcium acetate precipitation of the pectin and suggested that PPO could have co-precipitated with the calcium pectate. The beneficial effect of the citrate buffer compared to the acetate buffer during the extraction may have been due to the capacity of citrate to chelate metals, possibly binding the endogenous calcium from strawberries and thus preventing the crosslinking of pectic material. Phosphate may also function in this manner. A more loose pectin network may have partially prevented the loss of PPO in the insoluble material. During the preparation of the crude extracts, 80 % of the PPO activity was recovered and 20 % lost with the insoluble material when TX and the phosphate-citrate buffer were used. It seems that the pectin could not be removed completely by precipitation since the extracts obtained did not penetrate the gels when electrophoresed. The degradation of the pectic polysaccharides by treating them with a pectinase preparation (Rohapect D5S, Rohm; 35% P.G., 35% M.E., 30 % T.E.) did not improve the electrophoretic resolution. Next, attempts were made to remove the pectin through chromatographic procedures. Application of a crude extract to a gel filtration column containing Sephadex G-100 (2.6 x 30 cm; run with 0.1 M sodium citrate buffer pH 5.25, 0.5 % v/v TX) resulted in the elution of most of the enzyme in the void volume together with the pectic material. Similarly, when dialized crude extract was applied to a DEAE-Sephadex A-50 ion exchange column (0.9 x 30 cm; eluted with a gradient phosphate buffer pH 7.0), most of the activity came out in the voiud volume accompanied by pectic material. The next procedure tried was the use of hydrophobic chromatography. 95

Phenyl Sepharose CL-4B Chromatography Phenyl Sepharose (PS) chromatography has been recently used for the purification of PPO from peaches (Flurkey and Jen, 1980), grapes (Wissemann and Lee, 1981), and pears (Wissemann, 1983). The procedure used here is an adaptation of thse works. Wissemann (1983) reported that the working conditions that resulted in the best reproducibility were when the PS column was run at room temperature and the sample applied contained TX in it. With the use of similar conditions the elution patterns obtained for strawberry PPO were also highly reproducible. Figure II.1 shows the elution pattern of a sample of crude enzyme extract from ripe strawberries. All of the pectic material was eluted with the void volume and during the washing step with the equilibration buffer. The bulk of the 280 nm absorbing material was eluted together with the pectic material and a second 280 nm absorbing peak eluted after washing the column with ethylene glycol. This second peak seems to constitute the TX bound to the PS and eluted by ethylene glycol. According to Carson and Konigsberg (1981) hydrophobic proteins may bind to PS as a complex with TX. However, in this case, the PPO peaks were eluted before the TX and apparently not as a complex with TX. The same effect was noticed by Wissemann (1983) during PS chromatography of pear PPO. All of the PPO activity was bound to the PS column and two large peaks were eluted, one at 0.4xEB (EB = elution buffer), PP0-F1, and the second at 0.2xEB, PP0-F2. The same elution pattern was noticed for both ripe and unripe fruit and the reproducibility was excellent. Jen and Flurkey (1979) noticed a difference in the elution profiles from PS columns between PPO extracts prepared from peaches at different levels of maturity. PS Chromatography of peach PPO (Jen and Flurkey, 1979), pear PPO (Wissemann, 1983), and grape PPO (Wissemann and Lee, 1980) all resulted in an increase in the specific activity and a recovery of more than 100 % of the applied activity. In the case of strawberry PPO the recovery after PS chromatography was low (Table II.3). During the 96 application of the crude extracts there was an accumulation of some material on top of th PS column, and in the case of unripe strawberries it included a green pigment that seemed to be chlorophyll. Wissemann (1983) observed also the accumulation of green material on top of the PS columns and the analysis of the crude extracts reveled its identity with chlorophyll. The low recoveries from the PS column may have been due to the irreversible binding of part of the enzyme to the material on top of the column that probably included pectin, other polysaccharides, and lipids. It was noticed that repeted use of the PS material, even after washing with ethanol and n-butanol, resulted in a loss of resolution. It seemed that the resin was being saturated with some aterial that could not be washed off using the normal procedure. Only after washing the resin extensively with a 4 % TX solution was it possible to regain the resolution. In order to concentrate the two fractions of PPO obtained from the PS column and to decrease crosscontamination between the two fractions, each was applied to a second smaller PS column. This resulted in a large increase in the specific activity and in the recovery of both fractions. This may have been due to the removal of inhibitory substances (Jen and Flurkey, 1979). The high purification power that PS seems to have for PPO from so many different sources seems to indicate that more than only hydrophobic effects are involved in the binding of the enzyme. Flurkey and Jen (1980b) reported that peach PPO showed a much stronger binding towards PS than to other hydrophobic resins tried. They suggested that the interaction of PPO with PS may have included biospecific interactions due to the complementarity of the molecular contours of the biospecific center of the enzyme (phenol binding) and of the phenyl group in the PS media. Figure II.2 shows the effect on the elution of PP0-F1 from a PS column when phenylalanine (Phe) was included in the media. Phe was chosen because of the similarity of the molecule with the phenyl group of the PS, and because it would not serve as a substrate for PPO and thus would not cause the fouling of the column as has been the case 97 when phenolic derivatives have been used (O'Neill et al., 1973; Gutteridge and Robb, 1975). The addition of Phe resulted in the elution of a portion of the enzyme at 0.6xEB whereas no PPO was eluted at this step when no Phe was included. This confirms the observation that the phenyl group of the PS binds PPO through more than just hydrophobic forces.

Electrophoresis and Ion Exchange Chromatography Electrophoresis on 7.5% polyacrylamide slab gels of the two fractions of PPO obtained from PS revealed two bands with PPO activity with PP0-F2, and that PPO-Fl did not enter the running gel (Figure II.6). It was not possible to obtain clear chromatographs for protein bands using the silver stain. Further purification of the two fractions of PPO included anion exchange chromatography on a DEAE-cellulose column. When PPO-Fl was applied to this column, all of the activity was eluted in the void volume indicating that this fraction of the enzyme did not carry a large enough charge to be retained by the column. On the other hand fraction F2 of the enzyme was resolved into one large peak and a small peak at its shoulder (Figure II.3). The two peaks obtained for PP0-F2 may correspond to the two bands with PPO activity observed during electrophoresis. These results can be related to the attempt done of separating the pectic material from the enzyme through ion exchange chromatography described above. The bulk of the PPO activity that was eluted in the void volume seems to have included the enzyme attached to the carbohydrate material that accumulated on top of the PS column and the PPO-Fl that did not bind to the DEAE column. PPO-Fl showed also a higher storage stability than fraction F2, i.e. 80% of the Fl activity and 68% fo the F2 activity remained after storage at 40C for one month. From the results obtained so far it can be concluded that PPO-Fl and F2 obtained from PS chromatography differ from each other significantly. 98

Molecular Weight Determination The molecular weight (MW) of the PPO fractions was determined using Swphacryl S-300 chromatography. The plot of the log MW versus Kav is shown on Figure II.4. The resolution of the six protein standards was excellent with a correlation coeffiecient of 2 r =0.998 . PP0-F1 was eluted as a single peak at 111,000 daltons + 5% , while PP0-F2 also eluted as a single peak at 34,500 daltons ± 7%. The MW of PPO reported from other plant sources is highly variable. The phenomenon of multipicity has also been observed in plant PPO (Mayer and Harel, 1979). From the data obtained so far it is not possible to know if PP0-F2 is a subunit of PP0-F1, or if the two fractions are different isozymes differing in amino acid composition or in any other molecular characteristics.

Concanavalin-Agarose Chromatography Because of the results obtained for PP0-F1, i.e. it did not bind to an anion exchange column, had a high MW, and did not penetrate into the running gel during electrophoresis, the possibility of the existance of carbohydrate associated with the enzyme was considered. Very few instances exist that report carbohydrate associated with PPO. Flurkey and Jen (1980b) reported that two of the fractions of peach PPO obtained from a hydroxyapatite column appeared to be non-specifically associated with carbohydrate material, and that some, but not all, of this material could be removed after cetrifugation in a CsCl gradient. It is important to note that peaches also contain high levels of soluble pectic material. Stelzig et al., (1972) reported that the carbohydrate moiety of apple PPO was RNAase insensitive, but reacted possitively with orcinol. Recently lectins have been used to separate or purify glycoproteins from other proteins or glycoproteins that do not contain the carbohydrate units for which the lectin has no specificity. Concanavalin bound to agarose (ConA) was used in this work. This lectin is obtained from Jack beans and has high specificity for mannopyranoses and related sugars which are not modified at C-3, C-4, 99

or C-6, i.e. oc-D-mannose, cc-D-glucose, N-acetyl glucosamine, and 2-substituted mannose residues (Clarke and Hoggart, 1982). Both samples of PPO Fl and F2 were applied to a ConA column. PP0-F2 was eluted in the void volume and therefore seem to contain no ConA-interacting carbohydrates bound to it. On the other hand, all of the PP0-F1 activity applied was bound to the ConA column. The addition of NaCl and Tx precluded the possibility of non-specific interactions (Akiki et al., 1980; Nishioka, 1978). Elution of the enzyme was done using o-methyl mannoside, but the recovery was low. General low recovery from ConA columns are usually obtained (Cook, 1976). The low volumes and yields of purified enzyme obtained, both of PP0-F1 and F2, did not allow a more detailed study of these enzymes. It can not be concluded if the carbohydrates are covalently attached to PP0-F1, or if this fraction is an artifact formed during the extraction due to the presence of high levels of soluble pectin and other carbohydrates. This association may be the reason for the lower hydrophobicity of PP0-F1, i.e. it was eluted at a higher elution buffer concentration than PP0-F2. Very little work with PPO has been done that deals with this problem. If the carbohydrate was to be bound covalently to PP0-F1, that is, if Fl is a glycoprotein (assuming that no Maillard-type reactions had occured) a whole new set of questions would arise. It would affect the concepts of multiplicity, subcellular location, physiological roles, and possibly help explain some of the problems encountered during the extraction and purification of PPO from many sources. The other question is on how PP0-F1 and PP0-F2 are related to each other. They may be different isozymes, or PP0-F2 may be the deglycosylated fraction of PP0-F1. In order to answer some of this questions it is important to confirm the mode of attachment of the carbohydrate to the enzyme. It would also be important to check for the existence of glycosylated PPO in other sources. 100

LDS-Electrophoresis All of the different fractions of the enzyme obtained were subjected to reducing conditions and treated with lithium dodecyl sulfate (LDS). Under these conditions the proteins obtain a net negative charge, take an overall conformation close to a random coil, and their separation during electrophoresis will be due to variations in size (Lambin, 1978). LDS electrophoresis was run on gradient slab gels and developed for proteins using the highly sensitive silver stain (Wissemann, 1983; Wray et al., 1981). A series of MW standards were also run for comparison. The slabs obtained show multiple banding for all samples (Figure II.6). This effect has been notices for PPO from varies other sources after gel electrophoresis and electrofocusing (Ben-Shalom et al., 1977; Wissemann, 1983; Benjamin and Montgomery, 1973; Jolley and Mason, 1965). It is very difficult to determine wihich band corresponds to the main fraction of the enzyme applied, or if the sample contained several isozymes. In the case of PP0-F1 and PPO-Fl-ConA the multiple banding may have been due to the presence of carbohydrate associated with the enzyme. It has been reported that glycosylation affects the , thermal stability, resistance to proteases, and the extent of SDS (LDS) binding (Lamport, 1980).

CONCLUSIONS

High PPO activity could be extracted from strawberries with the addition of Triton X-100 to the extraction buffer containing the phenolic binding resins. With the use of Phenyl Sepharose chromatography it was possible to obtain two fractions of the enzyme, Fl and F2, free of pectic material and of the bulk of the 280 nm absorbing material. Phenyl Sepharose has been used with great success in the extraction of PPO from other sources and should be seriously considered by other workers as a first step in the purification of this enzyme. Of the two fractions of PPO obtained, fraction F2 was more hydrophobic than fraction Fl. PP0-F2 had a MW of 34,500 by gel 101 filtration and was resolved from an anion exchange column as a large peak and a smaller one at its shoulder. Two bands with PPO activity were detected after electrophoresis for F2, and this fraction did not bind to a ConA column. PP0-F1 had a MW of 111,000 by gel filtration. It was neither bound to the anion exchange column, nor did it enter the running gel during electrophoresis. Fl was bound to ConA signifying that this fraction of the enzyme had carbohydrate associated with it, although the mode of attachment could not be determined. 102

APPENDIX

STRAWBERRY POLYPHENOL OXIDASE PURIFICATION OUTLINE

EXTRACTION BUFFER STRAWBERY NITROGEN POWDER 1 1 ' STIR FOR 60 min @ 40C I PRESS THROUGH NYLON CLOTH I CENTRIFUGE 8,500 x g FOR 10 min @ 0oC I FILTER THROUGH WHATMAN # 1 FILTER I CRUDE EXTRACT I MAKE TO 1 M AMMONIUM SULFATE I FILTER THROUGH WHATMAN #1 FILTER I APPLY TO PHENYL SEPHAROSE COLUMN I COLLECT THE TWO PPO PEAKS Fl AND F2 I MAKE EACH TO 1 M AMMONIUM SULFATE I APPLY EACH TO A PHENYL SEPHAROSE COLUMN I COLLECT EACH PPO PEAK I DIALYZE EACH vs 5mM NaPi pH 7.0 i ' 1 PP0-F1 PP0-F2 I I CONCANAVALIN AGAROSE DEAE CELLULOSE CHROMATOGRAPHY CHROMATOGRAPHY I \ Fl ConA F2 DE 103

TABLE II.1 THE EFFECTS OF DIFFERENT EXTRACTION PROCEDURES ON THE ACTIVITY OF RIPE STRAWBERRY PPO.

PROCEDURE % MAXIMUM PPO ACTIVITY

WATER EXTRACT 8 BUFFER EXTRACT (B) 25 B + PHENOLIC-BINDING RESINS (R) 76 B + R + 2% v/v TRITON X-100 94 (after 48 hrs @ 40C) 92 B + R + 1% w/v Lubrol 100 (after 48 hrs @ 40C) 68

Extraction Buffer (B) = 0.1 M Sodium Citrate pH 5.25. Phenolic-binding resins = Polyclar AT : Amberlite XAD-4 1 : 1 with strawberry nitrogen powder. 104

TABLE II.2 THE EFFECT OF THE TYPE OF BUFFER AND OF THE PRECIPITATION OF PECTIN WITH CALCIUM PECTATE ON THE ACTIVITY OF RIPE STRAWBERRY PPO.

PROCEDURES % MAXIMUM PPO ACTIVITY

0.1 M K-CITRATE pH 4.5 (Bl) 100

Bl + 1 M CALCIUM ACETATE 61

0.1 M Na-ACETATE pH 4.5 (B2) 40

B2 + 1 M CALCIUM ACETATE 32

The buffer extracts Bl & B2 were adjusted to pH 6.2, made to 1 M with calcium acetate, stirred for 24 hrs at 40C, and the calcium pectate separated by centrifugation at 27 000 x g for 30 min. TABLE II.3 PURIFICATION OF THE POLYPHENOL OXIDASE FROM UNRIPE STRAWBERRIES.

PROCEDURES* VOL UNITS TOTAL PROTEIN UNITS per YIELD PURIFICATION (ml) per ml UNITSW ug / ml mg Prot %

CRUDE EXTRACT 503 168 84,504 65 2,585 100.0 1.0

PS#1 PPO-F1 163 101 16,463 18 5,611 19.5 2.2

PS#1 PPO-F2 105 62 6,510 10 6,200 7.7 2.4

PS#2 PPO-F1 65 302 19,630 7 43,143 23.2 16.7

PS#2 PPO-F2 50 174 8,700 8 21,750 10.3 8.4

Fl ConA 7 89 650 9 9,889 0.8 3.8

F2 DEAE 11 117 1,322 3 35,455 1.6 15.1

(a) PS = Phenyl Sepharose CL 4-B chromatography; ConA = Concanavalin Agarose chromatography; DEAE-cellulose chromatography. (b) 1 Unit PPO activity = 1 nmole 0„ per min per ml of enzyme extract.

o 106

FIGURE II.1 PHENYL SEPHAROSE CL-4B CHROMATOGRAPHY OF STRAWBERRY POLYPHENOL OXIDASE.

Fl & F2 = PPO activity containing fractions. Pectin = expressed as ug anhydrogalacturonic acid per ml (Blumenkrantz and Asboe-Hansen, 1973). PPO activity = as nmoles 0„ consumed per min per ml of extract used. S = Sample. E.B. = Elution Buffer (see Experimental). E.G. = 50% ethylene glycol. 0 O PECTIM (

PPO ACT. (iMlisOt'ouanl) 8 o

■■812 T

T'II aanou

^01 108

A) Phen»l-Seph»roie CL-4B • -O-CH ,-CH-CHj-O-^ \ OH

Phennlalanine

FRACTION NUMBER FIGURE II.2 BINDING OF PPO FRACTION F-1 TO PHENYL SEPHAROSE CL-AB AND ITS ELUTION WITH AND WITHOUT THE PRESENCE OF PHENYLALANINE IN THE ELUTION BUFFER. A)STRUCTURAL SIMILARITIES BETWEEN THE PHENYL SEPHAROSE ACTIVE MOIETY AND PHENYLALANINE. B)rLUTION PROFILE OF PPO FORM Fl. 1.0 EB = Elution buffer, 50 mM NaPi, 50 mM Na citrate pH 5.25, 1M (NH^SO^, 1M.KC1. Column size 2.5 ml. Fraction size, 0.48 ml. Flow rate, 0.16 ml per min. 109

s Sraditits | SBM *_■* 1WWtnnnU Hi HI ™_■ ZOOIBM 400llM 600 • t 1

500 - -— .25

^2

1 1 400 - 1 1 - .20! " ,'1 I'I 8 — — J2 300 "l - .!5 S III

I 200 - 1 - .10 i I 1 1 J > 100 05 111 A A ,, /

1 10 - 20 30 40 50 60 70 FRACTION NUMBER

FIGURE II.3 DEAE-CELLULOSE CHROMATOGRAPHY OF STRAWBERRY PPO FRACTION F2 OBTAINED FROM PHENYL SEPHAROSE CHROMATOGRAPHY. S = sample; elution buffer = 5 to 400 mM NaPi pH 7.0; column size = 0.9 x 27 cm; fraction size = 6 ml; flow rate = 1 ml per min. no

0.60 \ Ribonuclease r2=0.998

0.50 0Chymotrypsinogen

F2 34,500 \ ovalbumin 0.40

Bovine serum albumin kav 0.30 F, 111,000 Glucose oxidase

0.20

0.10 . Thyroglobulin

J L 4.0 4.4 4.8 5.2 5.6 6.0 LOG. MOLECULAR WEIGHT

FIGURE II.4 MOLECULAR WEIGHT OF STRAWBERRY PPO FRACTIONS Fl AND F2 BY SEPHACRYL S-300 SUPERFINE GEL FILTRATION. Kav = (Ve - Vo) / (Vt - Vo). Ill

10 15 20 25 FRACTION NUMBER

FIGURE II.5 CONCANAVALIN-AGAROSE CHROMATOGRAPHY OF STRAWBERRY PPO FRACTION Fl OBTAINED FROM PHENYL SEPHAROSE CHROMATOGRAPHY. Be = equilibration buffer, 5mM NaPi pH 7.0 0.1% Triton X-100; Bm = elution buffer, Be + IM-methyl raannoside; Bw = wash, Be + 1M NaCl; S = sample; column size = 4 ml; fraction size = 0.A8 ml; flow rate = 0.16 ml per min. 112

FIGURE II.6 POLYACRYLAMIDE GEL ELECTROPHORESIS OF DIFFERENT FRACTIONS OF STRAWBERRY PPO. A)ELECTROPHORESIS ON 7.5% POLYACRYLAMIDE GELS UNDER NON-DENATURING CONDITIONS OF FRACTIONS Fl AND F2 OF PPO OBTAINED FROM PHENYL SEPHAROSE CHROMATOGRAPHY. B)LDS-ELECTROPHORESIS ON 7.5 TO 15% POLYACRYL AMIDE GELS UNDER DENATURING CONDITIONS. PPO FRACTIONS Fl AND F2 WERE OBTAINED FROM PHENYL SEPHAROSE CHROMATOGRAPHY, FRACTION F2DE FROM DEAE CELLULOSE CHROMATOGRAPHY, AND FRACTION FIConA FROM CONCANAVALIN-AGAROSE CHROMATOGRAPHY.

IIIII = Zones of multiple banding. A) B) i—i 7.51 SLABS 7.5-157. LOS-PAGE GRADIENT SLABS t—i a> F, ARBS MW Fn F tConA F, F 20E (1103)

— origin

Myosin (a) 200.0

B-6alactotidasef7) 116.2

Hill PhosphorylaseB(6) 92.5

BSA(5) 66.2

OvalbuminU) 4 5.0

mill! s Carbonic 31.0 Anhydrase(3)

Soybean Tryptii 21.5 lnhibltor(2) Lysoiyme(l) 14.4

Gels stained for fieis stained for protein PPO activity U3 114

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TITLE: STRAWBERRY POLYPHENOL OXIDASE AND ITS ROLE IN ANTHOCYANIN DEGRADATION.

AUTHORS: Pedro A.E. Wesche-Ebeling and Morris W. Montgomery

AFFILIATION: Department of Food Science and Technology Oregon State University Corvallis, Oregon 97331

RUNNING HEAD : Strawberry polyphenol oxidase and anthocyanin degradation

Technical Paper No. from the Oregon Agricultural Experiment Station. 120

Strawberry Polyphenol Oxidase and its Role in Anthocyanin Degradation

Pedro A.E. Wesche-Ebeling. and Morris W. Montgomery

Deparment of Food Science and Technology Oregon State University Corvallis, OR 97331

ABSTRACT

Influence of two purified fractions of strawberry polyphenol oxidase (PPO) on D-catechin alone and in combination with either pelargonin or cyanin was investigated through the analysis of model systems. PPO was found to be highly active in the presence of D-catechin, a flavanoid found naturally in strawberies. Km values using D-catechin for the two forms of the enzyme obtained were 0.5 mM and 0.41 mM respectively, while the Vmax values were 82,700 and 18,800 nmoles 0„ per min per mg of protein, respectively. Analyses of model systems containing PPO, D-catechin (0.8mM), cyanin (25.5mM) and pelargonin (26.5mM) alone or in combinations revealed the formation of a yellow-brown pigment from D-catechin that showed a max at 390 nm and had absorbance in the 500 nm region. PPO appeared to oxidize cyanin at a very slow rate and showed no significant activity towards pelargonin. In the presence of D-catechin and PPO, 50 to 60 % of the anthocyanin pigments was degraded after 24 hr at 20oC, and the formation of a precipitate was detected. These results suggest that anthocyanin degradation occurred in part through direct oxidation by the quinones derived from the action of PPO on D-catechin or other phenolic compounds, as well as through the co-polymerization of the anthocyanin pigments into the polymeric brown pigment. Direct oxidation of anthocyanins by PPO seems to play a minor role. 121

INTRODUCTION

The red color in stawberries originates from the presence of varying quantities of anthocyanin pigments. The major anthocyanin pigments present are pelargonidin-3-glucoside (pelargonin) and cyanidin-3-glucoside (cyanin), with pelargonin varying from 72 to 95 % of the total anthocyanin in analyses on 5 varieties and 13 selections of the fruit (Wrolstad and Erlandson, 1973). The level of anthocyanin present in ripe fruits of the Tioga variety used in this work has been reported to be 27.6 mg anthocyanin per gram of fresh fruit (Abers and Wrolstad, 1979). Anthocyanin pigments are very unstable. They possess electron-deficient flavonoid nuclei that makes them highly reactive, and they can, therefore, undergo undesirable color changes (Shrikhande, 1976). In the case of strawberries, color loss has been shown to occur after thawing of frozen fruits, and during processing and storage of strawberry products, i.e. juices, concentrates, wines, preserves, and purees (Wrolstad et al., 1970a, 1980; Abers and Wrolstad, 1979; Sistrunk and Cash, 1970). Loss of anthocyanin pigments can occur through several mechanisms. Some of the factors that have been reported to contribute to the degradation of the anthocyanin pigments include their hydrolysis to the more unstable aglycones (Markakis, 1974; Shrikhande, 1976). A high pH , i.e. higher than 3.6, will lead to a decrease in the levels of the red flavylium forms of anthocyanin and to an increase in the more unstable blue quinoidal bases, carbinol pseudo bases, and chalcones (Brouillard, 1982). At elevated temperatures all forms of the anthocyanin tend to be converted to the chalcone because of the endothermal nature of the quinoidal base — flavylium cation — carbinol pseudo base — chalcone sequence reaction (Timberlake, 1981). Heat also promotes the non-enzymatic browning reactions with the consequent formation of reactive reductones and furfural derivatives (Hodge and Ossman, 1976). Anthocyanin pigments may be degraded by these compounds or enter the polymerized product of the browning reaction as a co-polymer (Chichester and McFeeters, 1971; 122

Debicki-Pospisil et al., 1983). Anthocyanin can also form co-pigment complexes with flavonoids, heavy metals, and other compounds resulting in bathochromic shifts (Asen et al., 1972; Markakis, 1974). More recently it has been suggested that the loss of anthocyanin pigments may be due in part to the presence of an active polyphenol oxidase (PPO) system. The fate of the pigments in such a system is not really known, but it has been suggested that they are not degraded directly by PPO, but by the reactive quinones formed in the PPO reaction (Peng and Markakis, 1963), or that they may follow degradative patterns similar to those described for wine pigments (Wrolstad et al., 1980; Somers, 1971). The purpose of this work was to study the specificity of strawberry PPO using phenolic compounds and anthocyanin pigments found naturally in the fruit. The fate of the anthocyanin pigments in a PPO system was also studied a series of model systems containing different combinations of D-catechin, pelargonin, cyanin, and PPO. The activity of PPO and the levels of anthocyanin pigments were measured, and a series of color analyses were made after allowing interactions in the models to occur for 24 hr at 20oC.

EXPERIMENTAL

Strawberries of the Tioga variety were obtained from Fuji Farms (Troutdale, OR) In June 1982. Unripe fruit in the green-white stage were used for the extraction of PPO. The fruit was washed, decapped, frozen in liquid nitrogen, sealed under vacuum in oxygen-impermeable plastic bags, and stored at -20oC until used.

PPO Extraction and Purification

The extraction buffer consisted of 160 ml of 0.2M sodium phosphate- 0.1M sodium citrate buffer pH 5.25 (XB) containing 5mM phenylmethylsulfonylfluoride (PMSF), 2 % (v/v) Triton X-100, 15 g dry 123

Polyclar AT, and 30 g moist Amberlite XAD-A. Both Polyclar AT and Amberlite XAD-4 were washed before used as described by Wesche-Ebeling (1981). To the XB buffer 15 g of strawberry nitrogen powder was added. The nitrogen powder was prepared by grinding the frozen fruit under liquid nitrogen. After stirring the mixture for 1 hr at 40C, the slurry obtained was filtered through nylon cloth with hand pressure. The filtrate was centrifuged at 8,500xg for 10 min at 0oC, and the supernatant was filtered through Whatman No.l paper to obtain the crude enzyme extract. For hydrophobic chromatography, the crude enzyme extract was made to IM with ammonium sulfate. A phenyl sepharose CL-4B (PS) (Pharmacia Fine Chemicals, Inc.) column (2.6 x 20 cm) was equilibrated in an equilibration buffer (EF) which contained 50 mM sodium phosphate - 50mM sodium citrate pH 5.25, 5mM PMSF, IM ammonium sulfate, and IM potassium chloride. After application of the sample, the column was washed with EB, and the enzyme eluted by decreasing the EB concentration in a stepwise manner, i.e. diluting to 0.8 x EB, 0.6 x EB , etc. Two different fractions of PP0, Fl eluting at 0.4 x EB and F2 eluting at 0.2 x EB, were obtained and each collected separately. To decrease contamination between the fractions and to concentrate the fractions more, each fraction was applied to a second PS column (0.9 x 30 cm) after being made to IM ammonium sulfate and eluted as above but using a lower volume for each elution step. After collection each of the PP0 fractions were dialyzed against 5mM sodium phosphate buffer pH 7.0. The dialyzed extracts were stored at 40C until used. For further details on the purification procedure of strawberry PP0 see the preceding paper.

Substrate Specificity and Kinetic Studies

PP0 activity was measured polarographically using an oxygen monitor equipped with a Clark-type electrode (Yellow Spring Instruments Model 53). The reaction chamber was mantained at 250C by a Lauda K2/R constant temperature circulating water bath (Brinkman 124

Instruments). For activity measurements only the initial slopes of the reaction curves were considered. For substrate specificity studies, 1 ml of the crude enzyme extract was mixed with 1.5 ml of XB. After allowing 4 min for temperature equilibration, 0.5 ml of the desired substrate was added. All substrates were prepared in XB pH 3.5 in order to decrease the possibility of autoxidation, and kept in a 250C until used. The PPO activity is reported as nmoles 0„ consumed per min per ml of enzyme extract used.

Studies of the interaction between PPO, D-catechin, and Anthocyanin pigments

Purified samples of pelargonin and cyanin were obtained from Dr. R.E. Wrolstad (Oregon State University). Other compounds used were obtained from Sigma and used without further purification. The PPO used was the purified fraction Fl obtained from PS chromatography as described above. The first experiment consisted of continuously scanning the reaction of PPO in the presence of D-catechin alone or in combination with pelargonin or cyanin. Scanning was done using a Beckman spectrophotometer model Acta C-III at a speed of 4 nm per sec from 600 to 300 nm and a scale range of 0 to 1.0 absorbance units. The model systems used consisted of 1.4 ml of XB pH 5.25, 0.1 ml of PP0-F1 (117 nmoles 09 per min per ml), and 0.5 ml of 5mM D-catechin in XB pH 5.25 . In the models containing PPO and D-catechin alone, the volumes were brought to 3.0 ml by adding 1 ml of XB pH 3.5 . Model systems containing the anthocyanin pigments were brought to 3.0 ml by adding 1 ml of 26mM cyanin or 1 ml of 27mM pelargonin prepared in XB pH 3.5. The second experiment consisted of the preparation of model systems containing combinations of PPO, D-catechin, pelargonin, and cyanin. PPO activity was measured immediately after preparation of one set of the models, while a second set was left at room temperature, 20oC, for 24 hr. The following volumes and concentrations of the 125 components were used for the preparation of the models: 0.2 ml of PP0-F1 (117 nmoles 0^ per min per ml); 1 ml of 5mM D-catechin in XB pH 5.25 ; 2 ml of 34.5 mg per ml pelargonin in XB pH 3.5 ; or 1 ml of 68.0 mg per ml of cyanin in XB pH 3.5. The volumes were brought to 6.0 ml with XB pH 5.25. Final pH in the model systems was 4.5 . Color analyses of the second set of models were done as described by Wrolstad (1976). Anthocyanin pigment content was measured using the single pH and the pH differential methods. The molar absorbance and wavelength of maximum absorbtion ( Xmax) values used were 22,400 and 520 nm for pelargonin, and 26,900 and 510 nm for cyanin. Degradation index (DI) values were obtained by dividing the value for anthocyanin concentration obtained by the single pH method over that obtained by the pH differential method. Browning was reported as the absorbance at both 390 and 420 nm. Color density, polymeric color, and percent contribution by tannins (% Tannins) were determined from absorbance values obtained from scans (700 to 300 nm) of control and bisulfite bleached samples (0.2 ml of 20% potassium metabisulfite addded to 3.0 + ml of sample): Color density was equal to [(Ai-in/co^ ~ ^7nn^

(A,„„ - A7„n)] x dilution factor for the control samples; and polymeric color was calculated similarly but for the bisulfite-bleached samples; and % Tannins was calculated from the ratio of polymeric color to color density x 100.

RESULTS AND DISCUSSION

The presence of PP0 in strawberries has been reported by Pallavicini (1969), Cash and Sistrunk (1971), and Drawert et al., (1974). Earlier work by the author (Wesche-Ebeling, 1981) involved the purification of strawberry PP0. Purification of the enzyme proved to be difficult due to the presence of high levels of phenolic compounds and pectin, and of general low levels of the enzyme. A method was developed to overcome these problems and a highly stable crude extract, as well as two purified fractions of the enzyme (Fl and F2) 126 were obtained from hydrophobic chromatography and used in this work. Strawberries have been reported to contain endogenous phenolic compounds some of which serve as substrates for PPO. The fruits of the Tioga variety used were reported to contain 312 mg total phenolics as tannic acid, 565 absorbance units of leucoanthocyanins, and 27.6 mg total anthocyanin per 100 g fresh fruit weight (Abers and Wrolstad, 1979). Other phenolic compounds reported are D-catechin at 1 to 7 mg, L-epicatechin at 1 mg, caffeic acid at 1 mg, j) -coumaric acid at 1 to 1.5 mg, gallic acid at 1 - 4 mg, and others at levels lower than 1 mg per 100 g fresh fruit weight (Stohr and Herrmann, 1975). Table III.l shows the activity of PPO (crude extract) on some of the endogenous phenolic compounds. No activity was observed on monophenols, i.e. j). -coumaric acid, but the enzyme showed highest activity towards D-catechin. The activity measured using the other phenolics compounds was much lower. Leucoanthocyanins and gallic acid were not tested, but they may both serve as substrates. Kinetic constants of the two purified fractions of the enzyme were determined from reciprocal plots and are shown on Table III.2 . Both D-catechin and 4-methyl catechol were used as substrates. Fraction Fl showed the highest activity as well as the highest storage stability. Previous work had shown that PPO-Fl was associated with carbohydrate material, and this may explain the higher storage stability observed for this fraction. 4-methyl catechol was used for routine measurement of PPO activity, due to the low solubility of D-catechin. D-catechin was also shown to be a very good substrate for pear (Tate et al., 1964), tea leaves (Coggon et al., 1973), peach (Flurkey and Jen, 1978), and date PPO (Hasegawa and Maier, 1980). 127

Importance of the PPO-D-catechin Reaction

The finding that D-catechin was not only found endogenously in strawberries, but also that it served as a good substrate for strawberry PPO is of great significance. This may help explain some of the results or work related to anthocyanin-color loss in strawberry products during processing or storage. Cash and Sistrunk (1971) used PPO extracted from strawberries in heat-treated strawberry puree and measured the rate of decolorization in the presence and absence of added catechol. They observed some thermal degradation of the pigment and an increase in the decolorization in all the samples adjusted to pH 4.0 as compared to those at pH 3.0 . They also observed that the addition of PPO did increase the rate of decolorization, but that there was no significant difference between samples to which only PPO was added and those containing PPO and catechol. They concluded that PPO seemed to react preferentially with the pigments to the exclussion of added substrate. But the presence of D-catechin, and leucoanthocyanins, in the strawberries and the high activity of PPO towards D-catechin, and low activity toward catechol (9 % of that measured with D-catechin), would suggest that PPO would be reacting with the endogenous phenolic compounds. These reactions would explain the browning and decolorization of the samples and the addition of catechol would have very little significance. Spayd et al., (1982) studied the influence of PPO from mushroom on strawberry puree prepared from ripe and unripe fruit and observed that the addition of the enzyme had no effect on the color or phenolic content of the purees after 48 hr. It is possible that mushroom PPO had low specificity for the phenolics in stawberries and that the endogenous PPO had been active during the preparation of the purees and the addition of exogenous PPO did not have a significant effect. PPO activity would probably occur only for a short time and the enzyme would be inactivated through suicide inactivation. The activity of PPO would not influence the levels of flavonoids or of total phenolics significantly but would be enough to initiate oxidation and 128 polymerization reactions. A storage period of 48 hr may not be sufficient for these reactions to become evident. An important question still remains, and that is, how are the anthocyanin pigments degraded in the presence of a PPO system? Some of the mechanisms of color loss in anthocyanin-containing samples by a PPO-system that have been proposed include: the masking of the red color by the formation of brown melanin pigments originating from PPO activity (and perhaps from non-enzymatic browning); the direct oxidation of the anthocyanin by PPO (Sakamura et al., 1966); and the oxidation of anthocyanin by the quinones originating from the PPO and secondary oxidation reactions (Peng and Markakis, 1963; Pifferi and Cultrera, 1974), or the co-polymerization of the anthocyanin with the polymer formed from the browning reactions (Somers, 1971; Jurd, 1967, 1972). Strawberries contain leucoanthocyanins, mainly of the water-soluble type (Co and Markakis, 1968), and condensed tannins of the procyanidin and polymeric type whose chains are terminated by D-catechin, L-epicatechin, and D-gallocatechin (Foo and Porter, 1981). Robinson (1980) reported that the biosynthesis of condensed tannins could occur through non-oxidative, oxidative non-enzymatic, or PPO-catalyzed polymerization of D-catechin and/or leucoanthocyanidin monomers. Hathway and Seakins (1955, 1957a,b) studied the formation of tannins formed through the autoxidation and PPO-catalyzed oxidation of D-catechin. They observed formation of polymers exhibiting bands at 270 and 310 nm and a shoulder at 500 nm, but this shoulder was less intense with the polymer formed through autoxidation. The formation of these tannins followed by further polymerization would lead to the formation of brown pigments (melanin, phlobatannins) that in the case of strawberries would mask the red color. Leucoanthocyanidins could also undergo polymerization forming tannins, and even copolymerize with other tannins forming complex co-polymers (Weinges and Nadel, 1982). To study the formation of polymers from D-catechin by purified strawberry PPO, a model system was set up, and the formation of the colored polymer followed spectrophotometrically (Figure III.l.A). The 129 polymer formed differed from that reported by Hathway and Seakins (1957a,b) in that only one peak at 390 nm was noticed. The reaction occured rapidly and after 40 min the solution was dark yellow-brown. Similar models were set up with PPO, D-catechin and either cyanin or pelargonin. The spectra obtained with either anthocyanin pigment were very similar (Figure III.2). The browning reaction occured at a slightly slower rate, which may be due to competition between D-catechin and the anthocyanin pigments for the active site on PPO. The same peak at 390 nm was noticed the tail of which extended into the absorbance region of the anthocyanin pigments, i.e. 510-520 nm. After 30 min it was still possible to see some redness in the preparations but after 60 min the solution became yellow-brown. Control models containing D-catechin, PPO, and the anthocyanin pigments alone or in combinations other than those described above showed no spectrophotometric changes after 1 hr. From these results it is not possible to know what the fate of the anthocyanin pigments. To study this another set of models was prepared (Table III.3). The models contained PPO, cyanin, pelargonin, and D-catechin alone or in combinations. The pH of the models was 4.5 and a series of standards were prepared to account for possible autoxidation of D-catechin or degradation of the anthocyanin pigments. This pH is higher than that found in strawberries which is approximately 3.5, but was used to allow for higher PPO activity and for shorter storage periods. Activity of PPO was measured immediately after the preparation of each of the models. Reaction of PPO with D-catechin occured very rapid, whereas, when both D-catechin and the anthocyanin pigments were present together the reaction rate was reduced. This reduction, as mentioned above, could have occured because of competition between the anthocyanin pigments and D-catechin for the active site on the enzyme. When PPO was allowed to react with either pigment only a small uptake of oxygen could be detected, slightly more with cyaninthan pelargonin. It is possible that the enzyme can react, although at a very slow rate, with the anthocyanin pigments, especially with cyanin which contains an £ -hydroxyl group on the B-ring of the molecule. 130

Also the enzyme or anthocyanin preparations may not nave been completely devoid of phenolic compounds which could have served as substrates for PPO and indirectly degraded the pigments. A second set of model systems was stored for 24 hr at 20oC. The levels of anthocyanin pigments remaining, as well as a series of color analyses were made after this period. There was essentially no loss of anthocyanin pigments in the control models containing only the anthocyanin pigments or in those containig anthocyanin and D-catechin. (Table III.3: #2,3,6,7). No significant loss of anthocyanin could be detected in the model containing PPO and pelargonin (#8), while the model consisting of PPO and cyanin (#4) showed a loss of about 20% of the pigment. These results indicate that PPO appears to be able to oxidize cyanin, while pelargonin does not appear to serve as a substrate for PPO, i.e. it does not contain an £ -hydroxyl group in the B-ring of the molecule. The levels of anthocyanin pigments that had suffered hydrolytic cleavage of their carbohydrate moieties was not measured, but it has been reported that glycosidation of flavonoids makes them unsuitable as substrates for PPO (Vamos-Vigyazo, 1981). Specificity of PPO towards anthocyanin and anthocyanidin pigments needs a more detailed study, although the direct oxidation of anthocyanin by PPO does not appear to be the main route of anthocyanin degradation. The degradation index (D.I.) values for all the models mentioned were essentially the same. High D.I. values indicate the presence of larger quantities of material absorbing light in the same region as the anthocyanin pigments. The model systems containing PPO, D-catechin, and either pelargonin or cyanin showed different results (Table III.3: #5,9). About 50% of the pelargonin and 60% of the cyanin was lost after 24 hr. No cyanin could be detected using the pH differential method and the D.I. value could therefore not be calculated. The D.I. value for the pelargonin-containing model (#9) was twice that found in the other pelargonin-containing models (#6,7,8). The visible spectra of the pelargonin-containing models after 24 hr of storage are shown on Figure III.2 .The models containing 131 pelargonin alone, or in combination with D-catechin or PPO show the characteristic peak at 510-520 nm and a second peak at 360 nm. The models containing PPO and D-catechin with and without pelargonin show the same peak at 390 nm seen in Figure III.l . Similar results were obtained with the cyanin-containing models. The model containing pelargonin and D-catechin showed an overall increase in absorption when compared to the model containing pelargonin alone (Figure III.2) indicating that condensation between the two compounds may have occured (Jurd, 1967; Baranowski and Nagel, 1983). Color analyses involved the comparison of samples treated with bisulfite with untreated samples. The method takes advantage of the resistance of polymeric tannin pigments to bisulfite bleaching (Somers, 1971). Color density is the sum of the absorbance at the Xmax of the anthocyanin pigment and the absorbance at the ^ax of the polymeric pigment for the untreated samples. Polymeric color values are obtained in the same way but using the bisulfite-treated samples. The max usually used to represent the polymeric pigments is 420 nm, although in this work the peak at 390 nm better represents the polymer formed from D-catechin (even in. the presence of anthocyanin pigments). Spectra of the models containing PPO, D-catechin, and either of the anthocyanin pigments showed that the absorbance at 390 nm of the bisulfite-treated samples was higher than that of the untreated samples. Both samples had the same absorbance at 410 nm and the absorbance was lower for the bisulfite-treated samples at wavelengths higher than 410 nm. Using the absorbance values at 390 nm the values for the percent contribution of color by tannins obtained were greater than 100%, while using the values at 420 nm, the percent contribution of color by tannins was 82% and 87%, respectively, for the cyanin- (#5) and pelargonin- (#9) containing models (Table III.3). The percent contribution of color by tannins is an index of polymerization-browning reactions, but with the model systems used in this work the values for percent contribution of color by tannins obtained will vary depending on the wavelength used to represent the polymeric pigment. It seems, therefore, more valid to use the values of the anthocyanin pigment concentration remaining and the absorbance 132 at the max of the polymeric pigment to represent color loss in the model systems. From the results obtained it can be concluded that PPO participates in the formation of polymeric pigments, i.e. tannins. The building blocks of the polymer include D-catechin and probably leucoanthocyanins and other phenolic compounds. Wrolstad et al., (1980) reported rapid loss of leucoanthocyanidins, flavanols (catechins), and of total phenolics in strawberry juice and concentrate samples during storage. A concurrent loss of anthocyanin pigments was also observed. They observed a faster loss in control samples, i.e. those in which PPO was inactivated, than in blanched ones. The loss of anthocyanin pigments occurs at a much faster rate in the presence of products of the enzymatic browning reactions, i.e. quinones and polymeric pigments. Several mechanisms through which these compounds can degrade anthocyanin pigments have been proposed. One proposal is that the quinones react directly with the anthocyanin pigments degrading them (Peng and Markakis, 1963; Pifferi and Cultrera, 1974). This reaction would be very fast and may be responsible for the rapid initial loss of pigment observed during processing and storage of anthocyanin-containing products, i.e. strawberry juice and concentrate (Cash and Sistrunk, 1971; Wrolstad et al., 1980). Many products prepared from anthocyanin-containing fruits maintain a red color for a long period of time, but in many cases, no monomeric anthocyanin can be detected and the red color seems to be derived from the polymeric phenolic material. It has been reported that anthocyanin and flavonoid compounds, including D-catechin, can undergo oxidative condensation and that some of the products of condensation retain a red color derived from the presence of flavylium moieties (Jurd, 1967, 1972; Little, 1977; Timberlake, 1980, Baranowski and Nagel, 1983). Anthocyanin pigments can also react with leucoanthocyanidins or procyanidins, and can even be incorporated into tannins. These reactions have been studied in detail by Somers (1971) in the case of 133 condensed flavonoid pigments (tannins) present in red wines. A fraction of these polymeric pigments seems to contain flavylium chromophores and their color expression will vary with pH. But in general the polymeric pigments were quite insensitive to pH changes and to bisulfite bleaching, and they appear to contain quinonoid base chromophores derived from the anthocyanin pigments. Polymeric pigments can be derived from catechin or lecuoanthocyanidin polymerization which could occur through either acid catalysis, autoxidation, or PPO-catalyzed oxidation (Robinson, 1980). Some polymeric pigments contain repeated quinone units that would be responsible for high absorbance at the 400 to 500 nm region (Somers, 1971). Anthocyanin pigments would be co-polymerized during the formation of polymeric pigments. The red color expression of the polymeric pigments would be lost through the degradation of the incorported anthocyanin moieties either on the polymer itself or if released again into the medium. Anthocyanin pigments may also be degraded by PP0 through another mechanism. Strawberries contain high levels of ascorbic acid (AsA), i.e. 18.8 . and 48.2 mg AsA per g fresh fruit, respectively, for the Tioga and Hood varieties (Abers and Wrolstad, 1979). In the presence of an oxidative system, i.e. PP0, AsA will reduce the products of the oxidation reactions, i.e. quinones, and will in turn be oxidized to dehydro-ascorbic acid (dAsA). dAsA will be further degraded and form intermediate reductones and furfural derivatives (Kurata and Sakurai, 1967a,b). This breakdown products of AsA have been proven to degrade anthocyanin pigments (Debicki-Pospisil et al., 1983). AsA and dAsA degradation products are very similar to those formed through Maillard-type browning reactions. Both lead to the formation of brown polymers, i.e. melanin, and anthocyanin pigments may also be incorporated into these polymers and show similar characteristicas to those seen in tannin-type polymeric pigments (Chichester and McFeeters, 1971). It has been recently reported that juices and wine prepared from mold-contaminated strawberries showed an increase in viscosity of the juice, a reduction in the fermentation rate, and an acceleration in 134 color degradation (Pilando et al., 1983). This indicates that not only endogenous PPO but also PPO originating from mold contamination can contribute to color loss.

CONCLUSIONS

From the results obtained in this work it can be concluded that strawberry PPO does play a role in the degradation of anthocyanin pigments. It does not appears that direct oxidation of the anthocyanin pigments by PPO is the main route of decolorization, although it could oxidize cyanin at a very slow rate. The reaction of PPO with D-catechin, a flavonoid found in strawberries, occured at a rapid rate with the formation of a polymer absorbing at 390 nm. It is suggested that anthocyanin may be oxidized by the quinones originating from the PPO action on D-catechin or other phenolic compounds. Another mechanism through which anthocyanin may be degraded is through their co-polymerization into the polymeric pigments, tannins, formed as a product of the polymerization of quinones and phenolic compounds. These co-polymers would show red color derived from the incorporated anthocyanin pigments. With time all of the anthocyanin pigments would be destroyed and all the color would be derived from the brown polymeric pigments. The fate of anthocyanin pigments in anthocyanin-containing products will depend very much on the composition, conditions, and processes to which the product is subjected. If proper caution is not taken against the presence of active PPO, any of the processes described above may occur. The proposed use of once-over harvesting of strawberries is of great importance in this respect since it would mix unripe fruit containing higher levels of PPO with the ripe fruit. The processing of both types of fruit together may result in a faster degradation of the color due to the presence of higher levels of PPO (Sistrunk and Morris, 1978; Spayd and Morris, 1981; Wesche-Ebeling, 1983). The use of mold-contaminated strawberries should be avoided since it has been demonstrated that molds contain enzyme systems that 135

can lead to color loss (Pilando et al., 1983). Inhibition of PPO before the processing of fruits, e.g. through blanching, as fast as possible is important. The period between the thawing of frozen fruit, or crushing of fresh ones, and the blanching process is most critical. The extent to which any PPO activity is allowed to occur will influence the characteristics of the final product by allowing the formation of quinones or small-size polymers that would serve as initiators of further oxidation and polymerization reactions during processesing or storage. No PPO activity would be expected in fruits as long as the tissues are kept intact. Therefore, proper handling of the fruit is important, as well as avoiding freezing and thawing of the fruit during storage. PPO has been reported to be highly stable and to resist long periods of frozen storage (Palavicini, 1969; Wesche-Ebeling, 1981). The addition of ascorbic acid may serve only temporarily for the protection of the pigments by reducing the quinones produced by PPO or through autoxidation. But the accumulation of oxidized and degradation products of ascorbic acid will contribute to the decolorization through the degradation of the anthocyanin pigments. In those processes where heat and high sugar are involved, Maillard-type browning reactions could occur (Hodge and Osman, 1976). The browning compounds formed would also contribute to the breakdown of the anthocyanin pigments. Hydrolysis of the anthocyanin pigments to the more unstable aglycones would also lead to the loss of red color. Hydrolysis could occur during processing or storage, or be catalyzed by hydrolyzing enzymes found endogenously or from fungal origin. The significance of this path of anthocyanin degradation needs further studies. In previous work with purified strawberry PPO it was noticed that there was a rapid decrease in activity with a decrease in pH (Wesche-Ebeling, 1981). By keeping the pH as low as possible the enzyme would be inhibited significantly although not completely. A low pH is also beneficial in that it would keep the majority of the anthocyanin pigments in the stable flavylium ion form (Brouillard, 1982). Acidification and blanching-inhibition of PPO appears to be the 136 processes that would result in a more stable product, as long as the heat applied does not promote extended non-enzymatic browning reactions. 137

TABLE III.l ACTIVITY OF STRAWBERRY PPO MEASURED USING PHENOLIC COMPOUNDS FOUND NATURALLY IN THE FRUIT. CRUDE ENZYME EXTRACT USED.

SUBSTRATE CONC. ( mM) % MAXIMUM ACTIVITY

D-CATECHIN 10 100

CHLOROGENIC ACID 5 11

CAFFEIC ACID 10 13

PROTOCATECHUIC ACID 10 k

£ -COUMARIC ACID 10 0 138

TABLE III.2 KINETIC CHARACTERISTICS OF THE TWO PPO FRACTIONS OBTAINED AFTER CHROMATOGRAPHY ON PHENYL-SEPHAROSE COLUMNS.

PPO FORMS SUBSTRATES Fl F2 Km* Vmax* Km* Vmax*

D-CATECHIN 0.50 603(a) 0.41 150(a) 82,700(b) 18,800(b)

A-METHYL 2.71 337(a) 3.73 141(a) CATECHOL 46,100(b) 17,600(b)

(*) Michaelis Constant, Km, in mM. Maximum Velocity, Vmax, in: (a) nmoles 0„ per minute per ml of enzyme extract (b) nmoles 0„ per minute per mg protein. 139

TABLE III.3 EFFECTS OF PPO (FRACTION Fl) AND D-CATECHIN (CAT) ON BOTH CYANIN (CYD) AND PELARGONIN (PGD) AFTER 24 HR OF STORAGE AT 20oC.

MODELS % PPO ANTHOCYANIN(mg/ml) DEGR. BROWNING PPO CYD PGD CAT MAX.ACT SINGLE pH pH DIFF INDEX A A

1 + - - + 100 - - - 1.23 1.03

2 _ + _ _ _ 11.8 11.0 1.07 0.02 0.01 3 - + - + - 11.5 10.3 1.12 0.06 0.05 k + + - - 5 9.6 8.6 1.12 0.05 0.03 5 + + - + 83 4.4 0.0 - 1.27 1.06

6 _ _ + _ _ 11.7 10.3 1.14 0.02 0.01 7 - - + + - 11.8 10.6 1.11 0.06 0.00 8 + - + - 2 11.2 10.2 1.06 0.05 0.04 9 + - + + 83 7.6 3.2 2.38 1.15 0.99

MODELS COLOR POLYMERIC % CONTRIB. PPO CYD PGD CAT DENSITY COLOR BY TANNINS

5 + + - + 0.54 0.41 82 9 + - + + 0.55 0.48 87 140

FIGURE III.l CONTINUOUS SCANNING OF THE REACTION BETWEEN PPO (FRACTION Fl), D-CATECHIN, AND CYANIN OR PELARGONIN. EACH CURVE REPRESENTS 10 min OF REACTION TIME. 300 soo S00 400 X(«m) 600 300 V* A dm) 600

FIGURE III.1 142

X (nm)

FIGURE III.2 ABSORPTION SPECTRA OF SELECTED MODELS CONTAINING COMBINATIONS OF PPO (FRACTION Fl), D-CATECHIN, AND CYANIN AND PELARGONIN AFTER 24 HR OF STORAGE AT 20oC. THE MODELS CORRESPOND IX) THOSE DESCRIBKH IN TABLE 111.3. 143 REFERENCES

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