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Other_______________________________________________________________________ University Microfilms International THE CHEMISTRY AND PHARMACOLOGICAL MECHANISM OF SODIUM NITROPRUSSIDE DISSERTATION Presented in Partial Fullfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jeen-Woo Park/ B.S., M.S. ★ * ★ * ★ The Ohio State University 1987 Reading Committee: Approved by Gary E. Means David H. Ives Richard P. Swenson Department of Biochemistry To My Parents, Wife and Daughter ii ACKNOWLEDGEMENTS With my deepest gratitude to my adviser, Dr. Gary E. Means, for his invaluable guidance, continuous encouragement and care. I wish to express my gratitude to Dr. David H. Ives, Dr. Edward J. Behrman and Dr. Richard P. Swenson for their encouragement and valuable help. I would also like to express my gratitude to Mr. David Chang of Campus Chemical Instrument Center for mass spectral data and Dr. George E. Billman of College of Medicine for help in animal experiment. Special thanks are due to my fellow graduate students for their helpful suggestion and assistance. iii VITA January 12, 1955 .... Born-Taegu, Korea 1978 .................. B.S., Seoul National University, Seoul, Korea 1980 .................. M.S. Biochemistry, Korea Advanced Institute of Science and Technology, Seoul, Korea 1980-1983 ............ Researcher, Korea Ginseng and Tobacco Research Institute, Seoul, Korea 1983-1984 ............ Graduate School Fellow, The Ohio State University, Columbus, Ohio 1984-1987 ............ Teaching and Research Associate, Department of Biochemistry, The Ohio State University, Columbus, Ohio PUBLICATIONS Park, J.W. and Means, G.E. (1987) Inactivation of Angiotensin Converting Enzyme by Sodium Nitroprusside. Biochem. Biophys. Res. Commun., in press. Monera, 0., Chang, M.K., Park, J.W., and Means, G.E. (1986) The Deamination of Lysine Residues of Proteins. Fed. Proc., 45. 1540. Abstract of the ASBC/DBC-ACS Meeting, Washington D.C. Park, J.W. and Means, G.E. (1985) Formation of N-Nitrosamines from Sodium Nitroprusside and Secondary Amines. New Engl. J. Med., 313, 1547. Park, J.W., Kim, S.J., and Lee, H.J. (1983) Active Site Functional Residues of Urokinase as a plasminogen Activator. Korean Biochem. J. 16, 100. iv Kwon, B.M. and Park, J.W. (1983) Study of Hydantoin III. Mass Spectrometric Behavior in the Electron Impact of 2- Imino-4-thiazolidinone Derivatives. J. Heterocyclic Chem., 20, 1725. Kwon, B.M., Oh, D.Y., and Park, J.W. (1983) Mass Spectra of Organophosphorus Compounds II. Unimolecular Reactions of 2- Phenyl-l,3,2-dioxaphosphoran 2-oxide Derivatives in the Gas Phase. Phosphorus and Sulfur 16, 271. Park, J.W. (1982) Analysis of Phenolics in Cigaret Smoke by GC/MS with Multiple Ion Selection Technique. Arch. Pharmacal. Res. 5., 71. Kim, K.R., Zlatkis, A., Park, J.W., and Lee, U.C. (1982) Isolation of Essential Oils from Tobacco by Gas Co- Distillation/Solvent Extraction. Chromatographia 15, 559. Park, J.W. (1982) Direct Analysis of Nicotelline from Tobacco by MS with DADI/MIKE Spectrometry. Arch. Pharmacal. Re3. 5., 25. Park, J.W., Lee, H.J., and Kim, S.J. (1980) Studies on the Anticoagulation Process (Fibrinolysis) by Urokinase. I. Purification and Characterization of Urokinase from Human Urine. Korean Biochem. J. 13. 127. FIELDS OF STUDY Major Field: Biochemistry Studies in Molecular Biology. Professors George A. Marzluf and Lee F. Johnson Studies in Carcinogenesis. Professors George E. Milo and Dorothy E. Schumm Studies in Biophysical Chemistry. Professor Alan G. Marshall v TABLE OF CONTENTS page DEDICATION .................................................. ii ACKNOWLEDGEMENTS ........................................... iii VITA .......................................................... iv LIST OF TABLES .............................................. viii LIST OF FIGURES ............................................ ix INTRODUCTION ................................................ 1 MATERIALS .................................................... 14 METHODS ...................................................... 16 Preparation of N-nitroso derivatives of amines ................................................ 16 Colorimetric method for the detection of N-nitrosamines ...................................... 18 Kinetic studies for nitrosation reactions ........ 19 Effect of blocking agents on the N-nitrosation of piperazine by SNP ................ 19 Modification of angiotensin I, II and ACE by SNP ........................................... 20 Assay of ACE activity .............................. 21 Reaction of SNP with thiols ......................... 21 Sulfhydryl group determination by Ellman reaction.. .................................... 22 Identification of GSNO by ion-exchange column ................................................ 22 vi Preparation of S-nitrosothiols ................... 23 Homololysis of S-nitrosothiols ................... 24 Nitrite determination .............................. 24 Identification of gas product .................... 24 Heterolysis of S-nitrosothiols ................... 25 Transnitrosation of S-nitrosothiols ............. 25 Effect of GSNO on the ADH activity .............. 26 Reaction of GSNO with hemoglobin ................. 27 Reaction of GSNO with HSA ......................... 27 Animal experiments .................................. 28 Gas chromatography .................................. 29 High-performance liquid chromatography .......... 29 Mass spectrometry ................................... 30 Other analytical methods .......................... 30 RESULTS ...................................................... 32 N-Nitrosamines from the reaction of SNP with amines .......................................... 32 Modification of angiotensin I, II and ACE by SNP ........................................... 49 Reaction of SNP with thiols ....................... 52 Preparation and characterization of S-nitrosothiols ..................................... 59 Chemical reactions of S-nitrosothiols ........... 70 Animal experiments ............................... 89 DISCUSSION ................................................... 97 LIST OF REFERENCES ......................................... 129 vii LIST OF TABLES Table page 1. Products obtained from the reactions of SNP with physiologically and pharmacologically important amines .......................... 43 2. Pseudo-first order rate constants for the nitrosation of secondary amines by SNP ............................................... 46 3. Effects of glutathione, cysteine and ascorbate on the reaction of SNP with piperazine .................................... 48 4. Effects of modification of ACE with SNP in the presence of competitive inhibitors.......... 54 5. S-Nitrosothiols from the reaction of thiols with sodium nitrite at pH 1.5 ................... 63 6. Homolysis of 20 mM S-nitrosothiols at pH 7.4 and 37° ..................................... 74 7. Reaction of thiols with S-nitrosothiols ...... 82 8. Equilibria for the