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ETD Template STUDIES OF COPPER(II) COMPLEXES OF BIOACTIVE PEPTIDES by Rong Meng BS, University of Science and Technology of China, 1997 MS, University of Science and Technology of China, 1999 Submitted to the Graduate Faculty of Art and Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2006 UNIVERSITY OF PITTSBURGH FACULTY OF ARTS AND SCIENCES This dissertation was presented by Rong Meng It was defended on Apr. 27th, 2006 and approved by Billy Day Adrian Michael Stephane Petoud Stephen G. Weber Dissertation Director ii STUDIES OF COPPER(II) COMPLEXES OF BIOACTIVE PEPTIDES Rong Meng, PhD University of Pittsburgh, 2006 Separation-based determinations of peptides hold the promise of measuring the concentrations of many peptides and their metabolites. However, typical chromatographic methods bear disadvantages in their selectivity and/or sensitivities. Thus, means for lowering detection limits are needed. The biuret reaction, the coordination of Cu(II) and peptides in basic solutions, provides a sensitive detection method based on the reversible Cu(III)/Cu(II) electrochemistry in peptide-bonded states. Thyrotropin-releasing hormone (TRH) is an important neuropeptide that undergoes the biuret reaction. The cationic form of TRH is not retained in a typical reversed-phase column. We have modified the common acidic mobile phase for peptide separation with a surfactant to retain the cationic TRH. A surfactant-preconditioned packed-bed capillary serves as the sample loop to preconcentrate TRH through sequential loading. The preconcentration has improved the detection limit for TRH 50-fold in the capillary HPLC-EC system. TRH lacks the N-terminal amine and C-terminal carboxylate that are usually required in the biuret reactions of Cu(II) and tripeptides. The unique structure of TRH affects the electrochemical behavior of the complex. We have utilized various electrochemical and spectroscopic techniques to determine the stoichiometry of the complex and to identify the binding sites in the peptide. Our data reveal that the interactions between Cu(II) and TRH are also possible under physiological pH. iii The unusual structure of TRH complicates the electrochemistry of Cu(II)-TRH complex. Multiple chemical reactions accompany the redox processes. We have investigated the kinetics of the coupling reactions using a rotating ring-disk electrode. The results suggest deprotonation of the ligand and inter-molecular interactions between complexes. Using microelectrode voltammetry, we have discovered that the octarepeat peptide of the prion protein is involved in Cu(II)/Cu(I) electrochemistry at physiological pH. This observation is relevant to the copper toxicity: Cu(II) may be reduced in vivo to Cu(I), which reacts with hydrogen peroxide to produce damaging oxidative radicals. The octarepeat peptide stabilizes Cu(I) by altering its redox potentials. Thus, the prion protein may have a function of anti-copper- toxicity. iv TABLE OF CONTENTS ACKNOWLEDGEMENT ...............................................................................................................x PREFACE...................................................................................................................................... xi 1. Introduction: Neuropeptides and Their Interactions with Cu(II).............................................1 1.1. Neuropeptides and Biuret Method for Peptide Analysis .................................................2 1.2. Biuret complexes: structure and electrochemical implications .......................................5 1.2.1. Regular peptides.......................................................................................................5 1.2.2. Peptides containing His residues .............................................................................7 1.3. The octarepeat peptide of the prion protein and its interaction with Cu(II) ..................10 1.3.1. PrP and prion diseases. ..........................................................................................10 1.3.2. PrP as a metal binding protein. ..............................................................................11 1.3.3. Cu(II)-octarepeat coordination...............................................................................11 1.4. Goals ..............................................................................................................................14 BIBLIOGRAPHY..........................................................................................................................15 2. Online Preconcentration of Thyrotropin-Releasing Hormone (TRH) By SDS-Modified Reversed Phase Column for Microbore and Capillary High Performance Liquid Chromatography (HPLC)...............................................................................................................21 2.1. Abstract..........................................................................................................................22 2.2. Introduction....................................................................................................................23 2.3. Experimental..................................................................................................................26 2.3.1. Microbore HPLC ...................................................................................................26 2.3.2. Capillary HPLC .....................................................................................................27 2.3.3. Brain tissue samples...............................................................................................28 2.4. Results and discussion ...................................................................................................29 2.5. Conclusion .....................................................................................................................38 ACKNOWLEDGEMENTS...........................................................................................................39 BIBLIOGRAPHY..........................................................................................................................40 3. Binding of Copper Ion to Thyrotropin-Releasing Hormone (TRH) and Its Analogs............44 3.1. Abstract..........................................................................................................................45 3.2. Introduction....................................................................................................................46 3.3. Experimental..................................................................................................................48 3.3.1. Chemicals...............................................................................................................48 3.3.2. Instruments and procedures ...................................................................................48 3.4. Results and discussion ...................................................................................................51 3.4.1. The binding stoichiometry .....................................................................................52 3.4.2. The equatorial donors ............................................................................................56 3.4.3. The effect of pH.....................................................................................................62 3.5. Conclusion .....................................................................................................................67 ACKNOWLEDGEMENTS...........................................................................................................68 BIBLIOGRAPHY..........................................................................................................................69 4. The Rotating Ring-Disk Electrochemistry of Copper Complexes of Thyrotropin-releasing Hormone ........................................................................................................................................73 4.1. Abstract..........................................................................................................................74 v 4.2. Introduction....................................................................................................................75 4.3. Experimental..................................................................................................................78 4.3.1. Chemicals...............................................................................................................78 4.3.2. Instruments and procedures ...................................................................................78 4.3.3. Simulation..............................................................................................................81 4.4. Results and discussion ...................................................................................................82 4.5. Conclusion .....................................................................................................................96 BIBLIOGRAPHY..........................................................................................................................97 5. The Electrochemistry of Cu(II) Complex of the Octarepeat Peptide of the Prion Protein....99 5.1. Abstract........................................................................................................................100 5.2. Introduction..................................................................................................................101
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