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INFORMATION TO USERS The most advanced technology has been used to photo graph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are re produced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. These are also available as one exposure on a standard 35mm slide or as a 17" x 23" black and white photographic print for an additional charge. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 8907271 Chemical modification of amino groups in proteins: Part I. Deamination of lysine residues in RNAse A. Part II. N-cyanomethylation of amino groups in RNAse A Monera, Oscar D., Ph.D. The Ohio State University, 1988 Copyright ©1989 by Monera, Oscar D. All rights reserved. UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 CHEMICAL MODIFICATION OF AMINO GROUPS IN PROTEINS PART I. DEAMINATION OF LYSINE RESIDUES IN RNAse A. PART II. N-CYANOMETHYLATION OF AMINO GROUPS IN RNAse A. DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Oscar D. Monera, B.S., M.S. ***** The Ohio State University 1988 Dissertation Committee: Approved by Dr. Gary E. Means Dr. Edward J. Behrman Dr. Richard P. Swenson Adviser Department of Biochemistry To My Wife Linda, Daughters Roslyn and Rosmari And To My Parents ii ACKNOWLEDGMENTS I am deeply grateful to my adviser, Dr. Gary E. Means, whose invaluable guidance, support and encouragement made this study possible. My sincere appreciation to Dr. Edward J. Behrman, who literally helped and guided me "from the very start to the very end" of my academic pursuit here at the Ohio State University; and to Dr. Richard P. Swenson and Dr. Harold Schecter for their guidance, suggestions and critical evaluations. I would like to especially express my thanks to Mr. David Chang of the Campus Chemical Instrumentation Center for his unselfishness, patience, and invaluable help in the interpretation of the mass spectral data. My sincere appreciation to my fellow graduate students, K. Ampon, J-W. Park, Y.Q. Song and K. Miller, for their encouragement, delightfulness and humor when nothing seemed to come out right. To the Filipino Student Association, whose graduation gift came a year too early. To my whole family, for their inspiration and support. VITA October 11, 1952 .... Born in Looc, Plaridel, Misamis Occidental, Philippines. 1969-1973 ............ B. S. Chemistry, Silliman University Dumaguete City, Philippines 1973-1977 ............ Researcher/Instructor, Department of Chemistry, Silliman University 1977-1982 ............ Instructor (Dept. Head, 1981-82) Dept, of Ag. Chem. & Food Science Visayas State College of Agriculture Leyte, Philippines 1978-1980 ............ M. S. Biochemistry, University of the Philippines at Los Banos College, Laguna, Philippines 1982-1983 ............ EDPITAF-World Bank Fellow Ohio State University Columbus, Ohio 1983-88 .............. Teaching and Research Associate Department of Biochemistry Ohio State University Columbus, Ohio PUBLICATIONS Monera, O.D., M.K. Chang, J-W. Park and G.E. Means. (1986) The Deamination of Lysine Residues of Proteins. Fed. Proc., 45, 1540. Abstract of the ASBC/BDC-ACS Convention, Washington, D.C. Monera, O. D. and E. J. del Rosario. 1982. Physico-chemical Studies on the Stabilization of Coconut Milk Emulsion. Annals of Tropical Research 4:47-54. TABLE OF CONTENTS DEDICATION ............................................. ii ACKNOWLEDGEMENTS ......................... ............. iii VITA ................................................... iv LIST OF TABLES ........................................ viii LIST OF FIGURES ....................................... ix LIST OF ABBREVIATIONS .............................. xiii PART I. DEAMINATION OF LYSINE RESIDUES IN RNAse A. PAGE Introduction ........................................ 1 Materials ............................................ 17 Methods Reaction of n-octylamine with sodium nitrite ... 19 (a) Effect of pH ............................. 19 Identification and analysis of products . 20 (b) Effect of the length of reaction ......... 22 (c) Effect of temperature .................... 23 (d) Effect of sodium nitrite concentration .. 23 Reaction of n-octylamine with sodium nitro- prusside ...................................... 24 (a) Effect of p H ............................. 24 (b) Effect of temperature .................... 25 Reaction of Na-CBZ-L-Lysine and sodium nitro- prusside ...................................... 26 v HC1 hydrolysis and amino acid analysis ......... 27 Derivatization for GC-MS ........................ 27 Reduction of the products of the reaction of Na-CBZ-L-Lysine and SNP by Pc^-H ......... 28 (a) Preparation of palladium black column ... 28 (b) Pd2-H r e d u c t i o n ....................... 29 Purification of RNAse A ...................... 30 Preparation of nitroprusside-modified RNAse A .. 31 Separation of the reaction products ............ 31 RNAse A assay .............................. 32 Gel filtration of native and nitroprusside- modified RNAse A .......................... 33 Amino acid analysis ............................. 34 Results Reaction of n-octylamine with sodium nitrite.... 35 (a) Effect of p H .............................. 43 (b) Effect of the length of reaction ......... 48 (c) Effect of temperature .................... 48 (d) Effect of sodium nitrite concentration .. 52 Reaction of n-octylamine with sodium nitro- prusside ...................................... 54 (a) Effect of p H .............................. 54 (b) Effect of temperature .................... 57 Reaction of sodium nitroprusside with Na-CBZ-L-Lysine ............................... 58 vi Reactions occuring under the conditions similar to those of the HCl hydrolysis of proteins .. 66 Time-course for the reaction of HCl with 2-amino-5-hexenoic a c i d ...................... 76 Reaction of sodium nitroprusside with RNAse A .. 80 Discussion The deamination reactions ....................... 91 Reactions occuring under the conditions similar to those of the HCl hydrolysis of proteins .. 99 Reaction of sodium nitroprusside with RNAse A .. 108 PART II. N-CYANOMETHYLATION OF THE AMINO GROUPS IN RNAse A Introduction ........................................ 113 Materials ............................................ 117 Methods Reaction of n-butylamine with CH2 O and NaCN ... 119 N-Cyanomethylation of RNAse A ................... 120 Gel filtration of native and N-cyanometylated RNase A ....................................... 121 Performic acid oxidation of RNAse A ............ 122 Trypsin digestion ................................ 122 Peptide mapping and isolation by HPLC .......... 123 Preparation of dinitrophenyl derivatives ....... 123 Separation of DNP derivatives by HPLC .......... 124 Carboxymethylation of Ne-DNP-lysine ............ 125 Results Reaction of n-butylamine with CH 2 O and NaCN .... 126 N-cyanomethylation of RNAse A .................... 129 Gel filtration ................................... 132 Identification of the modified lysine residue .. 138 Discussion .......................................... 144 List of References.................................. 152 viii LIST OF TABLES TABLE PAGE 1 Summary of the gas chromatographic and mass spectral data employed to identify the products of the reaction n-octylamine and sodium nitrite .............................. 37 2 Product ratios of the reaction of sodium nitrite and n-octylamine at different pH values ........................................ 46 3 Product ratios of the reaction of sodium nitroprusside and n-octylamine at different pH values ........................................ 57 4 Elution positions of homologous lactones from the amino acid analyzer ......................... 72 5 RNAse A activities of the different fractions from the reaction of RNAse A and sodium nitroprusside .................................... 83 6 Comparison of the amino acid composition of native RNAse A and the different products of its reaction with sodium nitroprusside ......... 89 7 Product distribution of the reaction of sodium nitroprusside and Na-CBZ-L-Lysine ...... 101 8 Distribution of products from 2-amino-5- hexenoic acid after hot HCl treatment .......... 103 9