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ELECTROCHEMICAL STUDIES OF METAL-LIGAND INTERACTIONS AND OF METAL BINDING PROTEINS A thesis submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY of RHODES UNIVERSITY by JANICE LEIGH LIMSON October 1998 Dedicated to Elaine and the memory ofRalph and Joey ACKNOWLEDGEMENTS My sincerest thanks to Professor Tebello Nyokong, for the outstanding supervision of my PhD. My gratitude is also extended to her for fostering scientific principles and encouraging dynamic research at the highest level. Over the years, Prof Nyokong has often gone beyond the call of duty to listen, encourage and advise me in my professional and personal development. Professor Daya, who has co-supervised my PhD, I thank for initiating research that has been both exciting and stimulating. His infectious enthusiasm for research has propelled my research efforts into the fascinating realm of the neurosciences. I am immensely grateful to both supervisors for their collaborative efforts during the course ofmy PhD. I acknowledge the following organisations for bursaries and scholarships: The Foundation for Research and Development in South Africa. The German DAAD Institute The British Council Algorax (Pty) LTD Rhodes University My appreciation is extended to the following people: My mother, Elaine, whom I deeply respect and admire for her tenacity and many sacrifices in ensuring that I received the best education possible. Her joy and pleasure in furthering my education has without doubt been a driving force. Hildegard for teaching me the alphabet, Aunt Cynthia for patient match-stick arithmetic, Uncle Peter for heavy metals, and my brother, Jonathan, for always providing the spark. My father, Ralph, who taught me the difference between wisdom and knowledge. The staff of the Chemistry Department for their support. Margie Kent and Benita Tarr for their kindness. Joyce Sewry, for Chemistry tutorials in my first year of study. Andre Adriaan, whose skill at creating specialised glassware was of immeausurable value. My research group for kindness, friendship, sharing of ideas and support throughout. Clinton Boyd for many fruitful and enlightening discussions on biochemistry. Graham (PC Support), Shirley (EM Unit) and Brian (Electronic Services) for generously giving of their time and expertise. Rodney, Paula and Guy, I thank for their time and valuable suggestions, while proof-reading sections ofthis thesis. To the friends who have encouraged, supported and beared with me throughout this thesis, my heartfelt thanks. Courtney Dahler for caring. To Ilya Eigenbrot: Spasibo. I am especially indebted to Guy Nelson, for helping me climb yet another tree, for gourmet cooking and a relaxed, happy environment in which to write this thesis. ABSTRACT Electrochemical methods were researched for the analysis of metals, proteins and the identification of metal binding proteins. Adsorptive cathodic stripping voltamrnetry for metal analysis combines the inherent sensitivity of electrochemical techniques with the specificity of ligands for the non­ faradaic preconcentration of analytes at the electrode. The utility of catechol, resorcinol, 4-methylcatechol and 4-t-butylcatechol as ligands was explored for the sensitive analysis of copper, bismuth, cadmium and lead on a mercury film glassy carbon electrode. Metal complexes of lead, copper and bismuth with resorcinol showed the largest increase in current with increase in metal concentration, whereas complexes of these metals with 4-t-butylcatechol showed the lowest current response. Cadmium showed the highest current responses with 4-methylcatechol. The four metals could be determined simultaneously in the presence of resorcinol, although considerable interference was observed between bismuth and copper. The electroanalysis of cysteine and cysteine containing proteins at carbon electrodes are impaired by slow electron transfer rates at carbon electrodes, exhibiting high overpotentials, greater than 1 V vs Ag!Agel . Metallophthalocyanines have been shown to promote the electrocatalysis of cysteine at lowered potentials. Chemical modification of electrodes with appropriate modifiers is a means of incorporating specificity into electroanalysis, with applications in electrocatalysis. A glassy carbon electrode was modified by electrodeposition of cobalt (II) tetrasulphophthalocyanine [Co(II)TSPct to produce a chemically modified glassy carbon electrode (CMGCE). The CoTSPc-CMGCE catalysed the oxidation of cysteine in the pH range 1 to 10. The significance of this electrode is an application for analysis of proteins at biological pH's. A biscyanoruthenium(II) phthalocyanine CMGCE catalysed the oxidation of cysteine at 0.43 V vs Ag/AgCl a significant lowering in the overpotential for the oxidation of cysteine. Metallothionein, a metal binding protein, is believed to be involved in metal homeostasis and detoxification in the peripheral organs of living systems. A method for the quantitative determination of this protein utilising its high cysteine content was presented. At pH 8.4 Tris-HCl buffer, and using a CoTSPc-CMGCE modified by electrodeposition of the modifier, the anodic peaks for the oxidation of metallothionein was observed at 0. 90 V vs Ag/AgCI. Ferredoxin is a simple iron-sulphur protein. One tenth of its residues are cysteine. Ferredoxin is involved in simple electron transfer processes during photosynthesis and lV respiration. Electrochemical studies of spinach ferredoxin were conducted at a CoTSPc-CMGCE. Anodic currents for the oxidation of the cysteine fragment of ferredoxin was observed at 0.85 V vs Ag/AgCl in HEPES buffer at pH 7.4, representing a new method for analysis of this protein. Voltammetric studies of its ferric/ferrous transition have shown quasi-reversible waves atE~ -0.62 V vs Ag/AgCl only in the presence of promoters. At a CoTSPc-CMGCE, a cathodic wave attributed to the reduction of Fe(III)/Fe(II) was observed at Epc -0.34 V vs Ag/AgCl. This represents an alternative method for voltammetric studies of the ferric/ferrous transition at significantly lowered potentials. Melatonin, a pineal gland hormone functions m setting and entraining circadian rhythms and in neuroprotection as a free radical scavenger and general antioxidant. Using adsorptive cathodic stripping voltammetry, the binding affinities of melatonin, serotonin and tryptophan for metals, were measured. The results showed that the following metal complexes were formed: aluminium with melatonin, serotonin and tryptophan; cadmium with melatonin and tryptophan; copper with melatonin and serotonin; iron (III) with melatonin and serotonin; lead with melatonin, tryptophan and serotonin, zinc with melatonin and tryptophan and iron (II) with tryptophan. The studies suggest a further role for melatonin in the reduction of free radical generation and in metal detoxification and may explain the accumulation of aluminium in Alzheimer's disease. v TABLE OF CONTENTS Acknowledgements ................................................ ................................................ ........ .. .......... iii Abstract ................................................................................ .. .................................. .. ............... iv Table of Contents ................................................... ................................................................... vi List ofAbbreviations ............................................................. .................................................... xi List of Figures ......................................................................................................................... xiv List of Tables .................................................................... ....................................................... xvi List ofSchemes .............................................................................. ......................................... xvi CHAPTER ONE ...................................................................................................................................... 1 OVERALL INTRODUCTION ............................................................................................................... ! CHAPTER TW0 ..................................................................................................................................... 5 ELECTROCHEMICAL PRINCIPLES AND TECHNIQUES .................................... .......................... 5 2.1 BASIC PRINCIPLES OF ELECTROCHEMISTRY................................ ........................................ 5 2. 1.1 Electrode Solution lnterphase ...................................... ........................................................... 5 2.1. 2 Transport to the Electrode .......................... ............................................................................ 7 2.1.3 Definition ofElectroanaly tical techniques .............................................................................. 8 2.1.4 Electrode Reaction ......................... ................................... ..................................................... 9 2.1. 5 The Three Electrode, Electrochemical Cell ................................ ........................................... 12 2.2 CYCLIC VOLTAMNIETRY ........................................................................................................ 15 2.2.1 Reversibility.................... .. ........................................................................................... ......... J6 2.2.2

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