Mekanisme Pengikatan Bilirubin

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Transcript of Mekanisme Pengikatan Bilirubin

( THE BINDING OF BILIRUBIN, PHOTOBILIRUBIN AND BILIRUBIN MODEL COMPOUNDS BY POLYMERIe RESINS. BRUCE M. S.AILOFSKY A thesis subrnitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy. DepmtrnentofChennstry McGill University Montreal, Canada @Bruce M. Sailofsky September, 1988. BINDING OF BILIRUBIN, PHOTOBILIRUBIN AND BILIRUBIN MODEL COMPOUNDS Ph.D. Chemistry Bruce M. Sailofsky THE BINDING OF BILIRUBIN, PHOTOBILIRUBIN AND BILIRUBIN MODEL COMPOUNDS BY POLYMERIe RESINS. ABSTRACT i The photolysis of bilirubin IX-a in organic solvents leads to both EZ/ZE isomers and lumirubins, with the latter being fonned from the former. The relative yields of the various photoproducts is markedly dependent on the solvent. Small amounts of photoproducts are aIso produced on irradiation of aqueous bilirubin solutions; however, the addition of human serum albumin increases the efficiency of the photoreaction, predominantly by isomerization of bound bilirubin. The fonnation of photoproducts frorn aqueous bilirubm soluuons increases the rate of adsorption onto eholestyramine but not peptide-substituted polyacrylamide resins. The photoproducts, principally lumirubins produced by irradiation of bilirubin in DMSO, are selecuvely adsorbed by cholestyramine. Using a semI-quantitative model it 1S shown that in aqueous solution the binding of derivatives of indoles and pyrroles onto ch('lestyrarnine involves electrostatic interactions as weIl as hydrophobie interactions. The free energy of transfer of the small molecule from water to an organic phase permits an estimation of the role of the hydrophobie interactions. { ( ii RESUME La photolyse de la bilirubine IX-a. dans des solvants organiques produit des isomres EZrz;E et des lumirubines, les luuiirubines tant formes par ces isomres EZIlE. Les quantits relatives des photoproduits dpendent du solvant. Des petites quantits de photoproduits sont aussi fonnes par irradiation des solunons aqueuses de bilirubine. L'addItion de la srum-albumine !mmaine augmente l'efficacit de la photoraction surtout par l'isomrisauon de la bilirubine. La formation des photoproduits de solutions aqueuses de augmente la vitesse d'adsorption par la cholestyramine, mais pas celle des rsines polyacrylamides substitues avec des peptides. Les photoproduits, principalement les lumirubines, produits par irradiation la bilirubine dans le sulphoxyde dImthylique, sont adsorbes de faon slective par la cholestyraIIne. En utIlisant un model semi-quantitatif, on dmontre qu'en solution aqueuse la liaison des drivs d'mdoles et de pyrroles par la cholestyramine implique des interactions lectrostatiques ainsi que des interactions hydrophobiques. L'nergie libre de transfert d'une petite molcule dans l'eau une phase organique permet une estimation du rle des interactions hydrorlIobiques. i ACKNOWLEDGEMNTS My sincere appreciation goes to my research director, Professor G.R. Brown, for his constant guidance and support throughout my years at McGill University. 1 am also indebted to Professc:r L.E. St-Pierre for making my studies mcst enjoyable Wlth his infinite suggestions, Inslghts and anecdotes. 1 would like to thank the McGill University Chemistry Department for financial support and for the use of the laboratories and faeihes. My sincere manks goes to the following people who have drrectly or indirectly lent a helping hand in the preparaon of this manuscnpt: Professor J. Chin, for hlS helpful dIscussIOns on the photobilirubin work and for the use of his stopped flow apparatus and software; Professor T.H. Chan, for the use of his Schoeffel Spectraflow deteetor used in the HPLC work; Professor W.c. Purdy and Dr. S. McClintock, for their HPLC expenence in the scparaon and detectIon of bilirubin and its isomers; Professor D. Patterson and Dr. M. Costas, for helping me sort out the thermodyni:lmics of binding and transfer; Dr. S.-D. Clas for translang the abstract into French; Mr. T. Blahovici and Dr. X.X. (Juhan) Zhu for their computer expertIse; and my eolleagues In the laboratory, bath past and present, who have provided me wlth invaluable help and eneouragemen t. Finally, 1 would like to thank my family for their never-ending support throughout my years in university. ( ( ~ to my wife, Dvorah, myfamily, and in memory of my mother. iv Abstract Rsum TABLE OF CONTENTS , , 1 .1 11 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ili List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xi List of Abbreviations and Symbols ....................... .xix 1. INTRODUCTION 1.1 MET ABOLISM OF BILIRUBIN .................... .1 1.2 GENERAL PROPERTIES ........................ 5 1.2.1 Isomers . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.2.2 Solubility . . . . . . . . . . . . . . . . . . . . . . . . .. 6 1.2.3 Acidity ........................... 8 1.2.4 Spectroscpic ................. 8 1.3 TREATMENT OF HYPERBll..IRUBINEMIA ............ 10 1.3.1 Exchange Transfusion . . . . . . . . . . . . . . . . . . 10 1.3.2 Hemoperfusion ...................... .11 1.3.3 Photothempy ........................ 13 1.3.3.1 Photoblirubin . . . . ................. 14 1.3.3.2 Electronic Excited States .............. 20 1.3.3.3 Side Effects of Phototherapy . . . . . . . . . . . . 28 1.3.4 Sn-Protoporphyrin Administration ........... 30 v ( ~ ( 1.4 BINDING TO ALBUMIN .......................... 30 1.4.1 A Description of Binding Sites and Mechanisms of Blndlng .............................. 36 1.4.2 Binding of Photoproducts to HSA . . . . . . . . . . . 39 1.5 THE PRESENT STUDY . . . . . . . . . . . . . . . . . . . . . . . . . . 40 References 1..' 1 , 42 2. SOL VENT EFFECTS ON THE PHOTOISOMERIZATION OF BILffiUBIN 2.1 INTRODUCTION ............................. 48 2.2 EXPERIMENTAL . . . . . . . . . . . . . . . . . . . ......... .49 2.3 RESUL TS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . .51 2.3.1. Photolysis in Pure Chloroform in the Absence of Oxygen ............................... 51 2.3.2 Photolysis in Chlorofonn/Ethanol in the Absence of Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 2.3.3 Photolysis in Chlorofonn/Ethanol in the Presence of Oxygen . . . . . . . . . . . . . .......... 59 2.3.4 PhotolYSlS in Dimethyl Sulfoxlde in the Absence of Oxygen . . . . . . . . . . . . . .. ....... .60 2.3.5 Photolysis in Dimethyl Splfoxide in the Presence of Oxygen . . . . . . . . . . . . . ........... 63 2.3.6 Photolysis in Dimethyl Sulfoxide/Water ........ 63 2.3.7 Photolysis of Aqueous Solutions Containing Albumin . . . . . 1 t 67 2.3.8 Photolysis in the Presence of Poly-l-lysine ...... 78 VI 2.4 SUMMARY Il 88 References . ,. . . . . . . . . . . . . . . . . . Il , 1 , 89 3. ADSORPTION AND PHOTOADSORPT10N OF BILffiUBIN BY CHOLESTYRAMlNE AND SELECT RESINS 3.1 INTRODUCTION .............................. 91 3.2 EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . ..... 93 3.2.1 Bilirubin Solutions . . . . . . . . . . . . . . . . . . . . 93 3.2.2 Adsorption and Photoadsorption Procedure. . . . . . .93 3.2.3 Preparation of Cholestyramine . . . . . . . . . . . . . 95 3.2.4 Synthesis of Tnmethylglycine-Al3.:3-Support ..... 96 3.3 RESULTS AND DISCUSSION ...................... 99 3.3.1 Photoenhanced Adsorption of Bilirubin From Aqueous Buffer Solution, pH = 7.8, by Cholestyramine . . . . . . . . . . . . . . . . . . . . . . . . 99 3.3.2 Adsorption and Photoadsorption of Bilirubin onto From D:methylsulfoxide ..... .111 3.3.3 Adsorption and Photoadsorption of Bilirubin by Cholestyramine From Dimethylsulfoxide .... , ... 118 3.4 SUMMARY .............. Il Il 121 References 1 Il , 123 v viii 4. THE BINDING OF INDOLE AND PYRROLE DERIVATIVES ONTO CHOLESTYRAMlNE AND LYSs-AI:3nSUPPORT 4.1 INTRODUCTION .............................. 124 4.2 EXPERIMENTAL . . . . .......................... 127 4.3 RESUL TS AND DISCUSSION . . . . . . . . . . . . . . . . . . .... 128 4.3.1 Effect of Ionization . . . . . . . . . . . . . . . . . . . . 128 4.3.2 A Qualitative Description of the Hydrophobie Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 4.3.3 Thermodynamic Model of the Binding Mechanism .. 136 4.4 SUMMARY ................ , . . ............... 152 References .................................... 153 5. CONTRIBUTIONS TO ORIGINAL KNOWLEDGE AND SUGGESTIONS FOR FUTURE WORK 5.1 CONTRffiUTIONS TO ORIGINAL KNOWLEDGE ........ 154 5.2 SUGGESTIONS FOR FUTURE WORK ................ 156 6. APPENDICES 6.1 EXPERIMENTAL DA TA . . . . . . . . . . . . . . .