BIOCHEMICAL CHARACTERISTICS OF CUCURBITACEAE SALT-SOLUBLE PROTEINS. Item Type text; Dissertation-Reproduction (electronic) Authors AL-KANHAL, MOHAMAD AHMAD. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 04/10/2021 12:40:04 Link to Item http://hdl.handle.net/10150/186471 INFORMATION TO USERS This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted. 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Zeeb Road Ann Arbor, MI48106 8319723 AI·Kanhal, Mohamad Ahmad BIOCHEMICAL CHARACTERISTICS OF CUCURBITACEAE SALT·SOLUBLE PROTEINS The University of Arizona PH.D. 1983 University Microfilms International 300 N. Zeeb Road. Ann Arbor, MI48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark_,/_. 1. Glossy photographs or pages _ V"/' 2. Colored illustrations, paper or print ~/ 3. Photographs with dark background __ 4. Illustrations are poor copy __ 5. Pages with black marks, not original copy __ 6. Print shows through as there is text on both sides of page __ 7. Indistinct, broken or small print on several pages 1// 8. Print exceeds margin requirements __ 9. Tightly bound copy with print lost in spine __ 10. Computer printout pages with indistinct print __ 11. Page(s) lacking when material received, and not available from school or author. 12. Page(s) seem to be missing in numbering only as text follows. 13. Two pages numbered . Text follows. 14. Curling and wrinkled pages __ 15. Other____________________________ _ University Microfilms International BIOCHEMICAL CHARACTERISTICS OF CUCURBITACEAE SALT-SOLUBLE STORAGE PROTEINS by Mohamad Ahmad Al-Kanhal A Dissertation Submitted to the Faculty of the COMMITTEE ON AGRICULTURAL BIOCHEMISTRY AND NUTRITION (GRADUATE) In Partial Fulfillment of The Requirements for the Degree of DOCTOR OF PHILOSOPHY In the Graduate College The University of Arizona 198 3 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Mohamad Ahmad Al ~Kanhal entitled Biochemical characteristics of Cucurbitaceae salt..-.soluble storage proteins and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy Date ~ < /1,/qf'3 Date 0 7 Da"!!7J {'1 / J ? Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced detree·at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate ack­ knowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: DEDICATION TO NORAH iii ACKNOWLEDGMENTS I would like to express my gratitude to all the people who have helped me during phases of this study. In particular, I want to thank professor J. W. Berry, my major professor, for his helpful suggestions and patience throughout this study. Thanks are also due to professors W. F. McCaughey, B. L. Reid, R. L. Price and J. W. Stull for serving on my doctoral committee. I am very grateful to Dr. R. E. Allen, for the use of equipment in his laboratory. Also, thanks are due to Mr. A. Watkins, for his assistance during the two­ dimensional analysis. Special thanks are due to all of the wonderful people in our laboratory who made my task very enjoyable. My deep gratitude is expressed to all of my family members in Saudi Arabia, for their emotional support and constant encouragement. My special thanks go to my wife Monirah and to my lovely daughter, Norah, who introduced happiness to my life. iv TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS viii · '/ LIST OF TABLES '/ . ix ABSTRACT .. I • xi INTRODUCTION 1 LITERATURE REVIEW 3 • I Seed Proteins 3 • 1 Seed Oil 12 • 1 Starch 13 Other Products 14 MATERIALS AND METHODS 16 Seeds Source 1 16 Proximate Analysis 16 Moisture . 1 16 Lipid Extraction 17 Crude Protein 18 Acid Detergent Fiber 20 Ash .... 1 ••• 21 Isolation of Albumin and Globulins 22 Defatting the Samples 22 Albumin and Globulins Separation I 23 Lowry Method 1 • • • • 24 Standard Curv, . 24 Albumin Sampl, Analysis 26 Salt Extract Analysis 27 Electrophorettc Ahalysis of Globulin an4 Albumin Fractions . 27 Detergent Gel System . 27 Staining and Oestaining of the Gel Column I •• • • • • 30 v vi TABLE OF CONTENTS--Continued Page Molecular Weight Determination of Albumin and Globulin by Gel Filtrations ...... 31 Two-Dimensional Electrophoresis of the Salt Extracts 33 First Dimension 33 Equilibration of the Gels 37 Freezing and Storage of the Gels ...... 37 Staining and Destaining the Gels . 37 Measurement of pH Gradient 37 Second Dimension . 38 Loading the Isoelectric Focusing Gels onto the Slab Gels 41 Electrophoresis in the Second Dimension . 42 Staining and Destaining the Slab Gels . 42 Drying the Destained Slab Gels 43 Amino Acid Analysis Globulin Fractions 43 RESULTS 46 Isolation of Albumin and Globulins . 49 Characterization of the Salt­ Soluble Protein Extracts 50 Isoelectric Focusing . 50 Two Dimensional Analysis 60 Characterization of Albumin and Globulin in Fractions . 60 SDS-Polyacrylamide Gel Electrophoresis . 60 Molecular Weight Determination by Gel Filtration 61 Amino Acid Analysis . 67 vii TABLE OF CONTENTS--Continued Page DISCUSSION . 70 Isoelectric Focusing 70 SDS-Polyacrylamide Gel Electrophoresis . 72 Globulin Fractions . 72 Albumin Fractions 72 Molecular Weight Determination 73 Globulin Fractions 73 Albumin Fractions 74 Amino Acids Analyses 75 SUMMARY AND CONCLUSION 77 APPENDIX A 79 REFERENCES 104 LIST OF ILLUSTRATIONS Figure Page 1. Standard Curve for Protein Determination by Lowry Method . 25 2. Calibration Curve; Logarithmic Plot of Molecular Weight versus Distribution Coefficient (Kav) for Chromatography over Sephacryl S-300 32 3. O'Farrell Isoelectric Focusing Gels of Salt-Soluble Proteins 48 4. Electropherogram Component Pattern of C. digitata Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 53 5. Electropherogram Component Pattern of C. foetidissima Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 54 6. Electropherogram Component Pattern of C. sororia Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 56 7. Electropherogram Component Pattern of C. palmeri Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 57 8. Electropherogram Component Pattern of C. moschata Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 58 9. Electropherogram Component Pattern of A. undulata Salt-Soluble Proteins after Isoelectric Focusing in Disc Gel as Described by O'Farrell 59 viii LIST OF ILLUSTRATIONS--Continued Figu~e Page 10. Savoy SDS-Gel Electrophoresis of Globulin Fractions . 62 11. Savoy SDS-Gel Electrophoresis of Albumin Fractions . 63 ix LIST OF TABLES Table Page 1. Proximate Composition of Seed Embryos . 47 2. Recovery Data for Salt-Soluble Proteins . 51 3. Recovery Data for Albumin and Globulin Fractions . 52 4. Molecular Weight Determination of Globulin Fractions by Gel Filtration on Sephacryl 8-300 . 64 5.