AN. ABSTRACT OF THE THESIS OF Luis Alberto Sayavedra Soto for the degree of Doctor of Philosophy in Food Science and Technology presented on October 3, 1983 . Title: Inhibition of Polyphenol Oxidase by Sulfur Dioxide. Abstract approved: r f Dr. Morris W. MontgomefyT Inhibition of polyphenol oxidase (PPO) by sulfur dioxide (S0„) was studied using three different sources of PPO (banana, mushroom, and pear). Several methods to detect PPO activity were tested due to SO.-, interference in some of the assays. The method using 2-nitro-5-thiobenzoic acid (TNB) to react with the quinones was found to be the most reliable for assaying PPO activity in the presence of S0r> whereas, the oxygen electrode and spectrophotometric methods were not suitable. When PPO was exposed to S0« prior to the substrate addition, it was inhibited irreversibly. Trials to regenerate the PPO activity using extensive dialysis, column chromatcgraphy, and addition of copper salts were not successful. Experiments using Cu(II) and the TNB method to regenerate the activity of the pear PPO apoenzyme that was not exposed to S0„, also showed the turnover between Cu(I) and Cu(II) during the enzyme oxidation of _o -phenols. Increased concentrations of S09 and pH less than 5 enhanced the inhibition of PPO by SO,-,. At pH 4 concentrations greater than 20 ppm completely inhibited 1,000 units of PPO activity almost instantaneously. This suggests SO,, as such as the main foi)rm inhibiting PPO. Kinetic studies confirmed the irreversibility of the inhibition. Purified pear PPO was used to determine binding of 35 S0„ to the enzyme. Column chromatography, extensive dialysis and gel electrophoresis did not show S07 bound to PPO protein. Formation of extra bands on gel electrophoresis in the S0„ inhibited pear PPO fractions was demonstrated. This and other evidence suggests that there was a modification of the protein structure of PPO, with retention of protein integrity. Also it is suggested that this was the main mode of direct irreversible inhibition of PPO by so2. Inhibition of Polyphenol Oxidase by Sulfur Dioxide by Luis Alberto Sayavedra Soto A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed October 3, 1983 Commencement June 1984 APPROVED: Professor of Food Science and Tec,:Hn/6logy id charge of major Head of Department of Food Science and Technology Dean of Graduate'pchool J Date thesis is presented October 3, 1983 Typed by the author Luis Sayavedra Soto Acknowledgemets I wish to thank Dr. Morris W. Montgomery, for his friendship and advise. I would also like to express my gratitude to the members of my commitee, and to the faculty, staff, and classmates of the Department of Food Science and Technology for their help, advise, and friendship. A special thanks to Pedro Wesche for allowing me to use part of his file on PPO. TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 Generalities 3 Multiple Forms and Localization of PPO 7 Extraction and Purification 10 Copper and the Active Site 12 Assay of Polyphenol Oxidase Activity 24 Methods that measure oxygen consumption 25 The chronometric method 26 Spectrophotometric methods 27 Inhibition of Polyphenol Oxidase 29 Types of inhibitors 29 Copper interaction 30 Use of thiols 35 Substrate analogs 37 Use of polymeric compounds 38 Significance of the inhibition of PPO in the food industry 40 Sulfur Dioxide 40 Sulfur Dioxide Analysis 45 Sulfur Dioxide and Enzymatic Browning 47 MATERIALS AND METHODS 53 Enzyme Sources 53 Enzyme Extraction Procedure 53 Enzyme Activity Assay 54 Protein Determination 55 Enzyme Purification Procedure 56 Polyacrylamide Slab and SDS Electrophoresis 59 Detection of PPO Isoenzymes 60 Stain for Protein Visualization in Polyacrylamide Gels 60 Analysis of Sulfur Dioxide 61 Effect of Sulfur Dioxide on Enzyme Activity 62 Preparation of Radioactive Sulfur Dioxide 62 Detection of the Radioisotope of Sulfur 63 Generation of the Apoenzyme 64 RESULTS AND DISCUSSION 65 Enzyme Activity Assay 65 Spectrophotometric method 66 Oxygen consumption 67 The 2-nitro-5-thiobenzoic acid method 68 Effect of Time of Exposure to Sulfur Dioxide 78 Effect of pH on Rate of Inactivation 82 Observed Kinetics of Inhibition 86 Isoenzyme Purification 93 Attempts to Regenerate Enzyme Activity After Sulfur Dioxide Exposure 100 Copper experiments 107 Electrophoresis Experiments Involving Sulfur Dioxide 112 Electrophoresis Experiments Involving Labeled Sulfur Dioxide 127 CONCLUSION 129 REFERENCES 136 LIST OF FIGURES Figure Page 1. Overall reaction catalyzed by PPO 4 2. Structural model of the binuclear copper active site of PPO 14 3. Proposed interaction of the oxy site effective structure with monophenolic and diphenolic substrates under normal enzymatic turnover of PPO 17 4. A likely reaction scheme for PPO 20 5. Proposed mechanism of hydroxylation and oxidation of phenols to form o^ -diquinones by Neurospora tyrosinase 22 6. A scheme for the inhibition of the cresolase and catecholase reactions of tyrosinase by azide 32 7. Extraction and purification procedure 57 8. Deviation from linearity of the TNB asssay for PPO activity at increased concentrations of sulfur dioxide 70 9. The TNB assay for PPO activity compared to the spectrophotometric method 73 10. The activity of PPO measured by the TNB method with no sulfur dioxide, and with sulfur dioxide at zero time and after 60 min 76 11. The effect of the concentration of sulfur dioxide on purified pear PPO activity 79 12. The effect of the pH on purified pear PPO activity at fixed concentration of sulfur dioxide 83 13. The double reciprocal Lineweaver and Burke plots in the presence of different fixed concentrations of sulfur dioxide using mushroom PPO after 60 min incubation time 88 14. A plot of Vmax versus amount of enzyme added to differentiate between an irreversible inhibitor from a reversible one 91 15. Elution profile of a phenyl sepharose CL-4B column chromatography of pear PPO 94 16. Elution profile of a DEAE-cellulose column chromatography of pear PPO 96 17. Polyacrylamide electrophoresis on 7% slab gels of the different isolated isoenzymes of PPO from pear using column chromatography 98 18. Elution profile of a phenyl sepharose CL-4B column of an isoenzyme solution of the first peak of the PS column exposed to radiolabeled sulfur dioxide 103 19.. Elution profile of a DEAE column of an enzyme solution from the main peak of activity of the PS column treated with radiolabeled sulfur dioxide 105 20. TNB trace of the regeneration of the PPO apoenzyme activity by addition of Cu(II) 109 21. Polyacrylamide electrophoresis on 7% slab gels of the PS peak at increased concentrations of sulfur dioxide at time zero 113 22. Polyacrylamide electrophoresis on 7% slab gels of the DEAE 2 peak at increased concentrations of sulfur dioxide at time zero 116 23. Polyacrylamide electrophoresis on 7% slab gel of first peak of the PS column at constant concentration of sulfur dioxide after 30 min incubation at different pH levels. Silver stained gel 118 24. Polyacrylamide electrophoresis on 7% slab gel of the first peak of the PS column at constant concentration of sulfur dioxide after 30 min incubation at different pH levels. PPO activity stained gel 120 25. Polyacrylamide electrophoresis on 7% slab gels of the DEAE 1 peak before and after being completely inactivated by sulfur dioxide 123 26. SDS 10% acrylamide gel and 7% acrylamide gel of peak one and two of the DEAE column before and after being exposed to sulfur dioxide. 125 27. Diagram suggesting the points where sulfur dioxide could inhibit enzymatic browning 134 LIST OF TABLES Table Page 1. Examples of the concentrations of sulfur dioxide used to inhibit enzymic reactions in plant tissues 48 2. Applications of selected inhibitors of enzymic browning to fruits and vegetables 52 INHIBITION OF POLYPHENOL OXIDASE BY SULFUR DIOXIDE INTRODUCTION Prevention of undesirable browning of foods, enzymatic or non-enzymatic, has long been the concern of the food scientist. The research reported here focuses on enzymatic browning and its inhibition with sulfur dioxide (S0„). Enzymatic browning is catalyzed primarily by the enzyme polyphenol oxidase (PPO) and is the result of the oxidation and polymerization of polyphenols to _o -quinones. This is a problem for most food processing industries because of the undesirable changes of vegetables and fruits which accompany browning during handling and processing such as, off flavors, change in the nutritive value and decreased appeal to consumers. The inhibition of PPO can be achieved by heat inactivation, by the exclusion of one or all substrates, or by the use of additives. Sulfur dioxide is an additive used to avoid browning of fruits and vegetables during processing. This additive can be easily handled and generated by using sulfites, it is extremly versatile (inhibits enzymatic browning as well as non-enzymatic browning; can be used as an oxidant, bleaching agent, or antimicrobial agent) and it is effective at relatively low concentrations. Recently S0„ was classified as a health hazard compound for asthmatics and the need for alternatives to 2 this additive is becoming more imperative. In order to be able to suggest alternatives, the mechanisms of reaction of PPO and the mode of action of S0„ have to be completely elucidated. Information in this respect has been relatively limited by the lack of pure sources of PPO, especially if fruits are to be considered. The methodology to isolate pure PPO from pears has become recently available in this laboratory making possible more detailed studies. Some authors had explained the inhibition of enzymatic browning by S0„ (Embs and Markakis, 1965; Haisman, 1974) leaving unexplained the direct action of S0„ on the enzyme itself. The purpose of the research reported here is to contribute to the elucidation of the modes of action of S0„ in the inhibition of enzymatic browning.