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Ann Arbor, MI 48106 SEPARATION OF CYTOKININ NUCLEOSIDASE FROM ADENOSINE NUCLEOSIDASE BY AFFINITY CHROMATOGRAPHY AND THE STUDY OF POSTHARVEST CALCIUM TREATMENTS ON SLICED TOMATO DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jessica J. Tarng, B.S., M.S. ***** The Ohio State University 1992 Dissertation Committee Approved by J.B. Allred M.E. Mangino Adviser A. Proctor Department of Food Science and Technology Copyright by Jessica Jiashi Tarng 1992 To My Parents and Dear Husband Jimmy ii ACKNOWLEDGMENTS I wish to express my sincere gratitude and appreciation to Dr. Grady W. Chism III, as my academic adviser, for inspiring and caring for me throughout my research and life in The Ohio State University. Thanks to the members of my reading committee, Drs. J.B. Allred, M.E. Mangino, and A. Proctor, and to the Department of Food Science and Technology for the financial assistance. To my colleague Madge Player, Daniele Korleskind, Isabelle Laye, and Serena Laroia, thank you for sharing, caring, and encouraging me. Special acknowledgment goes to all the faculty, staff, and fellow students who all have helped in their own way. VITA August 20,1962 Born - Taipei, Taiwan, Rep. of China 1980-1984 ......... B.S., The National Chung-Hsing University, Taichung, Taiwan, Rep. of China. 1984-1986 ......... M.S., The National Taiwan University, Taipei, Taiwan, Rep. of China. 1986-198 7........ Research Assistant, The National Taiwan University, Taipei, Taiwan, Rep. of China. 1987-Presen t.... Graduate Research Associate, The Ohio State University, Columbus, Ohio. Major Field: Food Science and Technology TABLE OF CONTENTS DEDICATION.............................................................................................................. i i ACKNOWLEDGEMENTS........................................................................................ iii VITA......................................................................................................................... iv LISTOFTABLES.................................................................................................... vi LIST OF FIGURES.................................................................................................... vii ABSTRACT................................................................................................................ x i CHAPTER I. ISOLATION OF CYTOKININ NUCLEOSIDASE FROM ADENOSINE NUCLEOSIDASE IN RIPE TOMATO FRUIT BY AFFINITY CHROMATOGRAPHY AND THEIR KINETIC CHARACTERISTICS................................................................................. 1 Introduction................................................................................. 1 Literature Review...................................................................... 3 Materials and Methods .............................................................. 20 Results and Discussion............................................................. 30 II. EFFECTS OF VACUUM INFILTRATION AND DIPPING OF CALCIUM SOLUTIONS ON THE QUALITY OF MINIMALLY PROCESSED TOMATO SLICES.............................................................. 67 Introduction................................................................................. 67 Literature Review...................................................................... 69 Materials and Methods .............................................................. 78 Results and Discussion............................................................. 81 LIST OF REFERENCES..............................................................................................103 v LIST OF TABLES TABLE PAGE 1. The protocol for silver staining.................................................. 29 2. Purification of adenosine nucleosidase in each peak fraction of affinity chromatography from ripe tomato fruit...................................................................................... 43 3. Kinetic constant for cytokinin nucleosidase and adenosine nucleosidase activity in the four peak fractions in Fig. 10 and 11........................................................... 49 4. Acceptability scores for calcium vacuum-infiltrated tomato slices.................................................................................... 102 LIST OF FIGURES FIGURE PAGE 1. Chemical structures, trivial names and abbreviations for some naturally occurring and synthetic cytokinins...................................................................... 4 2. Cytokinin biosynthesis interconversion and degradation in plant tissues: key role of N6-[isopent-2-enyl]adenosine.................................................... 6 3. Enzymes of N6-[isopent-2-enyl]adenosine metabolism........................................................................................ 8 4. The steps of affinity chromatography..................................... 16 5. Chemical structure of bicinchoninic acid .............................. 20 6. The reaction scheme shows how the BCA combines the biuret reaction to form the purple color complex.............................................................................................. 22 7. The comparison of protein recovery percentages attained by the widely used Bradford method and the recently developed BCA protein assay method 32 8. Affinity chromatography of cytokinin nucleosidase on an i6Ado-Agarose column (Elution gradient 1)......... 35 9. Affinity chromatography of adenosine nucleosidase on an i6Ado-Agarose column (Elution gradient 1)......... 37 10. Affinity chromatography of cytokinin nucleosidase on an i6Ado-Agarose column (Elution gradient 2)......... 39 vii 11. Affinity chromatography of adenosine nucleosidase on an i6Ado-Agarose column (Elution gradient 2).......... 41 12. Affinity chromatography of cytokinin nucleosidase on an Ado-Agarose column (Elution gradient 2............. ) 46 13. Affinity chromatography of adenosine nucleosidase on an Ado-Agarose column (Elution gradient 2)............. 48 14. Lineweaver-Burk plot of adenosine nucleosidase activity in peak fraction of NS I against adenosine concentration..................................................................................... 51 15. Lineweaver-Burk plot of cytokinin nucleosidase activity in peak fraction of NS I against cytokinin concentration..................................................................................... 53 16. Lineweaver-Burk plot of adenosine nucleosidase activity in peak fraction of NS II against adenosine concentration..................................................................................... 55 17. Lineweaver-Burk plot of cytokinin nucleosidase activity in peak fraction of NS II against cytokinin concentration..................................................................................... 57 18. Lineweaver-Burk plot of cytokinin nucleosidase activity in peak fraction of i6Ado NS III against cytokinin concentration................................................................. 59 19. Lineweaver-Burk plot of adenosine nucleosidase activity in peak fraction of Ado NS III against adenosine concentration............................................................... 61 20. SDS-polyacrylamide electrophoregram of partially purified nucleosidase attained from i6Ado-Agarose affinity chromatography column in Fig. 10 and ......11 65 viii 21. The calibration curve of the molecular weight versus relative mobility f Rvalue on a semi-logarithmic scale.................................................................. 66 22. Effect of calcium lactate and calcium gluconate vacuum infiltration treatments on the firmness of tomato slices at day zero.............................................................