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Electrochemical studies of Ferritin adsorbed at 3-Mercaptopropionate modified gold electrode

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Title:
Electrochemical studies of Ferritin adsorbed at 3-Mercaptopropionate modified gold electrode
Creator:
Pham, Mai Bach Thi
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Language:
English
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xiv, 80 leaves : illustrations ; 28 cm

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Degree Divisions:
Department of Chemistry, CU Denver
Degree Disciplines:
Chemistry

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Subjects / Keywords:
Ferritin ( lcsh )
Iron proteins ( lcsh )
Biological transport ( lcsh )
Biological transport ( fast )
Ferritin ( fast )
Iron proteins ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 74-80).
Thesis:
Department of Chemistry
Statement of Responsibility:
by Mai Bach Thi Pham.

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University of Colorado Denver
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Auraria Library
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ocm42613803
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LD1190.L46 1999m .P43 ( lcc )

Full Text
ELECTROCHEMICAL STUDIES OF FERRITIN ADSORBED AT
3-MERCAPTOPROPIONATE MODIFIED GOLD ELECTRODE
by
Mai Bach Thi Pham
B.S., University of Colorado at Denver, 1997
M.S., University of Colorado at Denver, 1999
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry
1999


This thesis for the Master of Science
degree by
Mai Bach Thi Pham
has been approved
by
Date
Xiaotai Wang


Pham, Mai Bach Thi (M.S., Chemistry)
Electrochemical Behavior of Ferritin Adsorbed at
3-Mercaptopropionate Modified Gold Electrodes
Thesis directed by Assistant Professor Donald C. Zapien
ABSTRACT
Ferritin is a protein whose principal functions are to sequester excess
iron, and to store and supply iron when it is needed. The mechanisms
of uptake and release are known to involve electron transfer steps. In
this work, the direct electron transfer behavior of adsorbed horse
spleen ferritin is studied at polycrystalline gold electrodes modified
with 3-mercaptopropionic acid. No electroactivity is observed when
the electrode is exposed to ferritin at 0.00 V, but is observed when
exposure is carried out at -0.45 V. The results from voltammetry
show that ferritin reacts similarly on both bare gold and modified gold
electrodes. The packing density is affected by certain conditions such
as dissolved ferritin concentration, adsorption time and adsorption
potential. Scan rate dependence studies reveal that the observed


electroactivity is due to adsorbed ferritin. In this study, potential step
chronoamperometry is used to measure the unimolecular electron
transfer rate constant. A value of 1.1 0.09 sec-1 is reported.
The voltammetry of horse spleen ferritin is also investigated using a
pyrolytic graphite electrode. Ferrocyanide reacts at the graphite
electrode similar to its behavior on gold or platinum electrodes.
Unfortunately, the electron transfer of ferritin iron on the modified
graphite carbon electron is not observed.
This abstract accurately represents the content of the candidates
thesis, i recommend its publication.
Signed
Donald C. Zapie


DEDICATION
I would like to dedicate this thesis and all of my work to my
brothers, sisters, and especially to my mother and in
remembrance of my father who is deceased. Because, of their
love, understanding, support and encouragement, I have been
able to pursue my educational goal. They have always been
by my side and I want to thank them for leading me to a
meaningful life.


ACKNOWLEDGMENT
Special thanks to Assistant Professor Donald C. Zapien for his
support, patience and guidance throughout this work. I also
wish to express my gratitude to Professor John A. Lanning
and Assistant Professor Xiaotai Wang for helping and advising
me. Individual thanks to Moon-Son Pyon for her contributions
into this work, to Ofentse Molefe for his support and
encouragement and to Tiffany Hays and Jim Halsey for
helping me.


CONTENTS
Figures.....................................................xi
Tables......................................................xiv
Chapter
1. Introduction............................................1
1.1 Cellular Ferritin Structure.............................1
1.2 Cellular Ferritin Function..............................3
1.3 Electrochemical Studies of Transfer Reactions in
Uptake and Release Iron in Cellular Ferritin and
Surface Modification....................................6
1.4 Electrochemical Methods: Tools for Studying
Mechanisms of Uptake and Release of Proteins............8
1.4.1 Indirect Methods Using Mediators........................9
1.4.2 Direct Methods Cyclic Voltammetry......................11
1.5 Cyclic Voltammetry and Potential Step
ChronoamperometryTools of Studying of
Mechanism of Electron Transfer of Proteins.............13
1.5.1 The Nernst Equation....................................13
1.5.2 Cyclic Voltammetry of Proteins.........................14
VII


1.5.3 Potential Step Chronoamperometry.......................20
1.6 Adsorption of Organized Monolayers on
Gold Electrode Surface.................................22
1.7 Research Objectives....................................25
2. Experimental Section (I)...............................26
2.1 Chemicals and Solutions................................26
2.2 Preparation of the Gold Electrode......................27
2.3 Purification of FerritinSize-Exclusion
Chromatography.........................................30
2.4 Equipment..............................................30
2.4.1 H-Cell.................................................30
2.4.2 Teflon Cell............................................32
2.4.3 Column Gravity Liquid Chromatography...................35
2.4.4 Other Equipment........................................35
2.5 Procedures...............................................36
2.5.1 Determine Concentration Ferritin (Ft)..................36
2.5.2 Experimental Cyclic Voltammetric Process...............36
2.5.3 Rate Constant Study Potential step chronoamperometry....39
3. Adsorption of Ferritin on Pyrolytic Graphite
Carbon Electrode Modified with
Hexamminechromium (111) Nitrate........................41


3.1 Preparation of Graphite Carbon Electrode................41
3+
3.2 Preparation and Characterization of Cr(NH3;6
Hexamminechromium (III) Nitrate.........................41
3.3 Voltammetric Experimental Configuration of Pyrolytic
Graphite Carbon Electrode...............................45
4. Results and Discussions.................................47
4.1 Cyclic Voltammogram (CV) of Clean Gold Electrode........47
4.2 CV of Modified Gold Electrode with
MPA Modifier............................................49
4.3 Voltammetry of Au/MPA/Ft at 0.00 V......................49
4.4 Voltammetry of Au/MPA/Ft at -0.45 V.....................52
4.5 Scan Rate Dependence....................................54
4.6 Potential Dependence Study..............................56
4.7 Concentration Dependence Study..........................58
4.8 Adsorption Time Dependence..............................58
4.9 Rate Constant...........................................61
4.10 Comparison of Voltammetry of Au/Ft vs. Au/MPA/Ft........63
4.11 Conclusions (I).........................................67
5. Results and Discussion: Adsorption of Ferritin
On Pyrolytic Graphite Carbon Electrode (II).............68
5.1 CV of Fe(CN)g on Carbon Electrode........................68
IX


5.2 CV of Ferritin on Carbon Electrode........................70
5.3 CV of Cr(NH3)g on Carbon Electrode........................70
5.4 Conclusions (II)..........................................73
Bibliography....................................................74
x


FIGURES
Figure
1.1 Ribbon Diagram of the Alpha Carbon Backbone
of a Horse Spleen Apoferritin Subunit..................2
1.2 Schematic Representation of the Horse Spleen Ferritin
Molecule. From the Schematic, a Molecular Four-Fold Axis
is Illustrating 4:3:2 Symmetry. A Sausage-Shaped Building
Brick Represents Each Subunit. The N-terminal Region of
the Polypeptide Chain Lies Close to the End of the N-
Terminal. E is the E helix Residues Lie Close to the End
Labeled E............................................4
1.3 Reduction of Ferritin Using
Methyl Viologen as a Mediator.......................10
1.4 Surface Modification.................................12
1.5 Cyclic Voltammogram..................................15
1.6 Excitation Wave Form.................................17
1.7 Current Response for a Reversible
Couple Obtained in CV...............................18
1.8 Chronoamperometry....................................21
1.9 An Ideal Organized Monolayer Alkanethiols on
a Gold Electrode Surface. All Molecules are
Closely Packed Together.............................23
2.1 Pyrolytically Water Distiller........................28
XI


2.2 Glass Sheathed Electrode............................29
2.3 H-cell Diagram......................................31
2.4 Diagram of Connecting from H-cell to
Potentiostat and X-Y Recorder.......................33
2.5 Teflon Cell Configuration...........................34
3.1 Graphite Carbon Electrode Configuration.............42
3.2 Glassware Apparatus in Collecting NH3 Liquid........43
3.3 Voltammetric Experimental Configuration
of Pyrolytic Graphite Carbon Electrode..............45
4.1 CV of Clean Gold Electrode in 1M H2S04. The
Voltammogram is Recorded form -0.35 to 1.50 V.
Scan Rate is lOOmV/sec..............................48
4.2 CV of Modified Gold Electrode with
MPA Promoter........................................50
4.3 Voltammetry of Au/MPA/Ft at 0.00V...................51
4.4 CV of Ferritin at -0.45V. Concentration of
Ferritin = 6.75 mg/mL. Scan rate = 100 mV/s.........53
4.5 Plot of Anodic Current Peak vs. Scan Rates..........55
4.6 Plot of Absolute Packing Density vs.
Absorption Potential................................57
4.7 Plot of Abs. Packing Density vs. Concentrations.....59
4.8 Plot of Packing Density against Adsorption Time.....60
4.9 Chronoamperomogram..................................62
xii


4.10 Plot of Ini vs. t...................................64
4.11 Cyclic Voltammetry: a), of Ferritin on Bare
Gold Electrode (Au/Ft) and b). of Ferritin
on Modified Gold (Au/MPA/Ft)........................65
5.1 CV of Fe(CN)*~on Carbon Electrode
in 0.05M of KCI.....................................69
5.2 CV of Ferritin on Carbon Electrode in
hepes solution......................................71
5.3 CV of Ferritin on Carbon Electrode in Hepes
Buffer Containing C|tNH3) ..........................72
xiii


TABLES
Table
3.1
Rate Constant Values from Potential
Step Chronoampermetry Study.........................63
XIV


1. Introduction
1.1 Cellular Ferritin Structure
Ferritin is a large protein, which can be found in most cell types of
humans and other vertebrates, invertebrate, higher plants, funguses
and bacteria [1-2]. Cellular ferritin is responsible for the storage of
iron in cells of biological systems. Cellular ferritin is about 12 nm in
diameter and 1.0 nm in shell thickness [1].
The molecular weight of ferritin is approximately 450,000 g/mol [6].
Ferritin contains 24 individual subunits (figure 1.1), which are
composed of varying ratios of L and H-type subunits [1] depending on
the organism and tissue type. Each subunit is folded into a four a-
helix bundle conformation. The whole ferritin molecule is a roughly
spherical globular protein (fig. 1.2) of 4:3:2 symmetry containing an
inorganic iron core whose formula is (Fe00H)8.Fe0.0P03H [29] (fig.
1.2). The numerous interactions between subunits, where the
subunits come together, both hydrophilic and hydrophobic channels
are formed which allow access of small molecules or ions into the
cavity [1, 30].
1


Interior
Exterior
Figure 1.1 Ribbon diagram of the alpha carbon backbone of a horse
spleen apoferritin subunit [4]
2


On the other hand, iron passes in and out of the core most likely
through the hydrophobic channels [1, 3].
The core, which is variable in size, can consist of as many as 4,500
iron atoms along with variable amounts of phosphate [1]. Under
certain conditions, in vitro experiments have discovered that iron can
exit the protein [5]. Although ferritin is not a redox protein, it has
been suggested that electron transfer steps are involved in the loading
and unloading of iron [7]. Studying ferritin electron transfer
properties is key to understanding the mechanisms of iron uptake and
release.
1.2 Cellular Ferritin Function
Cellular ferritin is considered as a sink for iron, an essential element
for virtually all-living organisms. In a large amount, iron is stored in a
soluble, nontoxic and available form [4]. The general reaction that
occurs inside and outside a ferritin is the reduction oxidation reaction
of iron where Fe3+ is reduced to Fe2+ and Fe2+is oxidized to Fe3+.
Fe3+ + e Fe:
Fe2+ Fe3+ +
3


8 Hydrophilic Channels
6 Hydrophobic Channels
Figure 1.2 Schematic representation of the horse spleen ferritin
molecule. From the schematic, a molecular four-fold axis is
illustrating 4:3:2 symmetry. A sausage-shaped building brick
represents each subunit. The N-terminal region of the polypeptide
chain lies close to the end of the N-terminal. E is the E helix residues
lie close to the end labeled E.
4


The mechanisms of iron storage, in vivo experiments, are uncertain,
but in vitro iron-core formation in ferritin involves the oxidation of
Fe(ll) and hydrolytic polymerization of Fe(lll) [1,4, 26-28], it is known
that the hydrolyzed Fe(lll) are stored inside their protein shells [21-
25]. Some studies have shown that the threefold axes, which are
hydrophilic channels and passageways of metal ions, are also known
to bind metal ions including Cd2+, Zn2+, Ca2+ and Tb3+ [4, 23, 29, 36].
The funnel-shaped threefold channels open toward the outside of the
molecule and narrows toward the cavity. Metal ions may be guided
inwards through the funnel opening [29, 31-33, 36]. It has also been
suggested that Fe(ll) is chelated by carboxyl groups residing in the
channels, and that the channels are also sites for oxidation and the
build-up of polynuclear Fe(lll) species [4, 34, 35, 37].
Release of Fe(lll) from the shell is very slow. In general, after
conversion of Fe(lll) to Fe(ll), iron release is faster and more complete
in the presence of a complexing agent [9-11]. Reductants that are
effective in vitro include reduced flavins, sulfhydryl compounds
(thioglycolic acid, dithionite, dihydrolipoic acid) [9, 12, 13], Cu(ll) plus
5


ascorbate [14] free radicals of oxygen (superoxide) and methylviologen
[15-17].
Some reports have pointed out that the rates of uptake and release
depend on the age of the protein coat or iron core [19], the disorder of
the core [20] and a variety of environmental conditions such as pH,
buffer ions [18], anions, metal cations, chelators and subcellular
distribution.
1.3 Electrochemical Studies of Transfer Reactions
in Uptake and Release Iron in Cellular Ferritin
and Surface Modification
Numerous research groups have studied the interfacial properties of
electron transfer proteins by applying direct electron transfer at
various electrode surfaces. These investigations aim to discover the in
vivo behavior of such cellular proteins and also a basis for their
application to practical bioelectrocatalytic systems.
By using viologen derivatives as electrochemical mediators, ferritin
was found to be reduced electrochemically in micro-coulometric
studies [46-50]. However, it is known that rapid electron transfer
between electrodes and ferritin is difficult to obtain. Various suitable
6


promoters [38,39,51-55] and surface modification [56] including
surface-confined mediators [57,58] and foreign adatoms [59] are used
for enhancing the rate of electron transfer [55]. Allen et al. [60] have
reported various surface modifiers for cytochrome c at gold electrodes
and presumed a structure for the surface-modifiers at the electrode
surface. A functional group X of the surface modifiers should contain
nitrogen, phosphorous or sulfur to absorb or bind to the gold electrode
surface. The other functional group Y of surface modifiers should be
anionic or weakly basic, which interacts favorably with the positively
charged electron transfer domain (lysine residues) of cytochrome c.
Besides 4,4-bipyridyl and bis(4-pyridyl) disulfide which are effective
surface modifiers, a carboxylic acid terminated alkanethiol is also
used broadly and successfully for the study of the direct
electrochemistry of adsorbed cytochrome c at self-assembled
monolayers on gold electrode [61].
The heterogeneous electron transfer reactions between electrodes and
c-type cytochromes in the bulk solution have been extensively studied
by many researchers during the last decade [42-45]. At various
electrodes such as mercury, platinum, silver, gold and illuminated p-
7


type silicon, the electrode reactions of cytochrome c at these
electrodes have been reported to be irreversible [42-45].
Hill and his colleagues found that horse heart cytochrome c undergoes
a rapid electron transfer reaction at the gold electrode in the presence
of various surface modifiers which are electron transfer promoters
[38,39]. In the presence of surface modifiers by using a UV-vis
electroreflectance spectroscopic technique coupled with voltammetry
the redox properties of the adsorbed cytochrome c on gold electrode
was studied by Hinnen and Niki [40], To investigate the adsorption
behavior of cytochrome c and electron transfer promoters on a gold
electrode, infrared reflection-absorption spectroscopy (IRRAS) was
applied and the IRRAS spectra showed the existence of cytochrome c
and 4,4-bithiodipyridine on the surface of the gold electrode. The
molecules of this layer adsorbed strongly on the electrode [41].
1.4 Electrochemical Methods: Tools for Studying
Mechanisms of Uptake and Release of Proteins
Numerous voltammetric methods have previously been applied to
studying the electron transfer reactions mechanisms of uptake and
release of proteins. The most widely used methods have been linear
8


sweep and cyclic voltammetry [62-65], impedance methods such as
ac voltammetry and impedance spectroscopy [62,66,67], differential
pulse and normal pulse voltammetry [62,68], and chronoamperometry
[69,70]. But first, electrochemical methods can be classified into
those, which involve direct electron transfer, and those, which use
electrochemical mediators.
1.4.1 Indirect Methods Using Mediators
Fig 1.3 illustrates the reduction of a protein in the presence of a
mediator. Historically, methyl viologen has been used extensively as a
mediator for electron transfer of proteins. After applying a potential to
reduce methyl viologen, the same in turn, reduces the protein in the
solution. The oxidized mediator returns to the electrode surface to be
reduced again. The cycle of reduction and oxidation continues until all
the protein is electrolyzed. Unfortunately, this method is not useful
for studying the mechanisms or kinetics of protein electron transfer,
however, the value of n can be defined as shown below:
(96485 Coul/mole)
Charge passed in exhaustive
electrolysis measured in
n =
Number moles of Protein
9


MV+
Ferritin (Oxidized)
Ferritin (Reduced)
Figure 1.3 Reduction of Ferritin using methyl viologen as a mediator.
10


1.4.2 Direct Methods Cyclic Voltammetry
Although electrochemical techniques are useful tools for studying the
redox properties of electroactive species, there are some problems
when these species are proteins. Because the redox centers reside
(sometimes rather deeply) in the globular matrix, the redox centers
sometimes cannot be directly oriented in a favorable position to the
electrode surface. As a result, the probability for electron transfer is
low. Proteins adsorb irreversibly to a metal electrode, forming an
insulating layer of denatured protein, thus poisoning the electrode
surface, precluding electron transfer with the electrode [71].
To address these problems, an adsorbed layer of organic molecules,
called promoters, is used to modify the surface of the electrode and
enhance the kinetics of a redox reaction (fig 1.4). A promoter can
cause the species (redox center) in the solution to approach the
electrode in a suitable orientation for induce electron transfer [71].
11


i
Electrode /
I
! N-S-S-@N
Figure 1.4 Surface modification
12


1.5 Cyclic Voltammetry and Potential Step
Chronoamperometry Tools of Studying
Electron Transfer Properties of Ferritin
1.5.1 The Nemtst Equation
To a half-cell, experimentally, the potential is affected mainly by
temperature, concentration of the electroactive species and the
number of electrons transferred per molecule. Mathematically, the
relationship between the potential of a half-cell consisting of a metal in
contact with its ions and the variables involved in the redox reaction is
given by the Nernst equation:
E = E RI ln[redl (1)
nF [ox]
where: E = potential (emf) of the half- cell
E0 = potential of half-cell under standard conditions
R = constant (80314J/C)
T = absolute temperature (298K)
n = number of electrons transferred during reactions
F = Faraday number (96,495 Coulomb)
[ox] = concentration of oxidized form
13


[red] = concentration of reduced form
Equation (1) shows the relationship between E, the emf of the half-cell,
and the concentration of the oxidized and reduced forms of the
components of the solution.
1.5.2 Cyclic Voltammetry of Proteins
Cyclic voltammetry is a powerful electrochemical method for
diagnosing reaction mechanisms. The current is measured as a
potential ramp is applied. Fig 1.5 illustrates a typical cyclic
voltammogram for ferrocyanide reacting at a platinum electrode.
From left to right, the oxidation of ferrocyanide takes place:
Fe(CN)f Fe(CN)36 + e"
6
After the potential scan is reversed, ferricyanide is formed based on
the reduction reaction at the electrode surface below:
Fe(CN)f + e"-> Fe(CN)f
6 6
A working electrode is immersed in a quiescent solution, a potential
ramp is applied over a given range and then reversed so that a
descending ramp returns, usually to the original potential. The ramp
is reversed at some time after the electroactive species reacts,
14


Figure 1.5 Cyclic voltammogram [81]
15


inducing the reoxidation of products generated by the forward sweep,
as shown in fig 1.6. This triangular potential excitation signal of the
working electrode is switched between two designated values which
are called the switching potentials. Usually, the voltage on the time
axis is tracked by an X-Y recorder to report a current response as a
function of applied potential. The scan rates in the forward and
reverse directions are normally the same. Cyclic voltammetry can be
used to provide single-cycle or multicycle scans; however, the
multicycle scans do not give identical currents because of changes in
concentrations at the electrode surface.
Cyclic voltammogram can give information on the anodic and cathodic
peak potentials (Ep)a and (Ep)c, the anodic and cathodic peak currents
(ip)a and (ip)c, and the half-peak potentials (Ep/2)c at which the cathodic
and anodic currents reach half of their values. The independent
variables are the voltage scan rate and the range of potential over
which the scan is made (fig 1.7). The peak current for an oxidized
species is given as:
ip = (2.69 x 105) n3/2 A Dox1/2 vI/2Cox
where A = area of the electrode surface in cm2,
16


(to
Figure 1.6 Excitation wave form
(a) Forward sweep
(b) Reverse sweep
17


Figure 1.7 Current response for a reversible couple obtained in CV
[74]
18


D = diffusion coefficient in cm2/sec,
C = concentration in mol/cm3
v = scan rate in V/sec.
For a reversible reaction, the number of electrons transferred in the
electrode reaction can be determined from the separation between the
peak potentials:
(EP)a-(Ep)c = 0057
n
this equation is valid when the switching potential is at least
lOOn/mV past the cathodic peak potential [72-75].
Summarily, cyclic voltammetry is especially well-suited to the
study of electron transfer mechanisms of electroactive species at
about the millimolar level [75]. Cyclic voltammetry can provide
valuable information on peak potential separation and scan rate
dependencies information useful in determining whether the electron
transfer step is fast compared with mass transport to the electrode
surface or if chemical reactions are couple to the electron transfer
step [8].
19


1.5.3 Potential Step Chronoampermetry
Chronoamperometry is the electroanalytical method used to measure
the current that flows through the working electrode as a function of
time. The excitation signal, usually a voltage, is either constant or
some simple function of time. In unstirred solution, the current-time
curve is recorded. The mass transfer to such an electrode will occur by
diffusion, and the resulting current-time curve will be an exponential
decrease of current with time.
In chronoamperometry (figure 1.8), the initial potential EQ is normally
set sufficiently anodic (or cathodic) where the reduction (or oxidation)
reactions being observed does not occur to any measurable extent. In
potential-step chronoamperometry, the potential of the electrode is
then stepped from the equilibrium potential, where no electrolysis
occurs, to some other final potential at which a reaction takes place.
In the chronoamperogram the rapid i-time curve the relationship of
the current-time curve and rate constant is given in below equation:
i = kapp Q exp(-kappt)
20


(a)
E
E final
E initial (Ej)
(b)
Time
Figure 1.8 Chronoamperometry
(a) Excitation potential step
(b) Chronoamperogram
21


where kapp is the rate constant apparent. When increasing of rate
constant kapp can cause the value of current i decreased and the i-time
curve becomes stiffer.
Chronoamperometry is a useful method for studying rates of electrode
processes, diffusion coefficients, adsorption parameters, and rates of
chemical reactions coupled to electron transfer reactions [72, 74].
1.6 Absorption of Organized Monolayers on
Gold Electrode Surface
Figure 1.9 shows an ideal organized monolayer of alkanethiols on a
gold electrode surface. Organized monolayer can be defined as single
molecular layers in which all constituents share a common orientation.
Monolayers have to contain identical head groups, which attached
onto the substrate surface, and identical extended tail groups, which
are often alkane chains. Monolayers can be deposited on the
electrode surface by self-assembly (SA) or the Langmuir-Blodgett (LB)
method.
From a homogenous solution, the self-assembled monolayer (SAM) is
formed by adsorption of the head groups on the electrode
surface. The tail, groups are closely packed and extended from the
22



^ue^ofeB

ft0
>\A
oNS
<;^>e'
. A0S
e\e
23


substrate. A covalent or coordinate covalent bond is formed between
the head groups and the surface.
Self assembled monolayers whose head groups are composed of
thiols, disulfides and sulfides are known to have strong adsorption
on the electrode surface, i.e. gold, platinum and mercury. The
covalent bonds between gold atoms and sulfurs are very stable,
allowing the monolayer to survive the electrochemical experiment
while maintaining both coverage and orientation.
Especially in aqueous electrolytes, self-assembled monolayers based
on thiols have a high packing density, being highly impermeable
toward both solvent and electrolyte ions. With these remarkable
qualities, SAM can be used to form a spacing layer between the
electrode surface and a redox couple that is freely diffusing from the
electrolyte or anchored on the substrate.
Gold is one of the most widely used electrode materials substrate to
be deposited because gold does not form an oxide layer when in
contact with air. Using electrochemical cycling in concentrated acid,
clean hydrophilic gold surface is achieved. Cleaning treatments
24


should be done before depositing an adsorbed layer because the bare
gold surface can be contaminated with various adsorbed species. A
clean cyclic voltammogram should be obtained to check surface
cleanliness. All residues of organic contaminants should be removed
before immersing the substrate into thiol solution.
1.7 Research Objectives
This study investigates whether ferritin adsorbs onto Au/MPA in an
electroactive form. The surface of a gold electrode will be modified
with 3-mercaptopropionic acid (MPA), then exposed to dissolved
ferritin under a variety of conditions. The packing density dependence
on adsorption potential, adsorption time and dissolved ferritin
concentration will be investigated using cyclic voltammetry.
Comparisons of Au/MPA/ferritin voltammetry to that of ferritin on
bare gold will be used to investigate the effect of surface groups on
adsorbate mobility. Estimates of the electron transfer rate constant
will be determined using potential step chronoamperometry.
25


2. Experimental Section (I)
2.1 Chemical and Solutions
All of the following chemicals were purchased from Sigma Chemical
Company (St. Louis, MO): 3-mercaptopronanoic acid (3-MPA) (99.3%),
horse spleen ferritin (>85%), Bovine albumin (fraction 5 powder),
disodium hydrogen phosphate (Reagent Grade), phosphoric acid
(Reagent Grade), and sodium dichromate (98%). Phenylmethyl
Sulfonyl fluoride (PMSF) (>99%), were purchased from Aldrich
Chemical Company (Milwaukee, Wl).
Baxter Scientific Co., Inc. (McGraw Park, IL) was the commercial
supplier of sodium chloride (99.2%) and sodium azide (>99%). Bio-
Rad Protein Assay Dye was purchased from Bio-Rad Laboratories
(Hercules, CA). Alfa_Johnson Matthey (Danvers, MA) was the source
of polycrystalline gold foil (99.99%), and fused 0.125 mm diameter
gold wire.
Pyrolytically distilled water (PDW) was purified by distilling de-ionized
water through a heated platinum catalyst in the presence of oxygen.
26


Distilled water was used in preparation of solutions and in cleaning
procedures in this experiment. The water still is illustrated in figure
2.1.
Chromic acid cleaning solution, which was used to for cleaning
purpose of gold surface electrode, Teflon cells, and glassware, was
prepared by dissolving 92 g of sodium dichromate (Na2Cr204.H20) into
458 mL of water. Then, 800 mL of sulfuric acid was added while
stirring.
2.2 Preparation of The Gold Electrode
One end of a 10-cm length of 0.25 mm gold wire was threaded
through holes at the top of a 0.5 cm x 1.0 cm gold foil, then the foil
portion was shaped into a 3-mm diameter cylinder (fig 2.2). The
electrode was annealed in a natural gas/air flame and soaked in
chromic acid for cleaning. The electrode was inserted into an 8.00
mm O.D. glass sheath connected to a nitrogen source (fig 2.2). The
gold electrode and glassware were soaked in chromic acid cleaning
solution for at least four hours and rinsed with pyrolitically distilled
water (PDW).
27


[\)
00
Figure 2.1 Pyrolytically water distiller
Cr
Nichrome
coil tube
furnace
(outside)
Pt gauze
catallyst
(inside)

\
water in


Nitrogen in (from top)
Thermometr Adapter

Gold Wire
Glass Sheath
Gold foil
Figure 2.2 Glass sheathed electrode
29


2.3 Purification of Ferritin Size-Exclusion
Chromatography
Prior to any analysis commercially available horse spleen ferritin was
purified by size exclusion chromatography. G-200 Sephadex gel was
prepared by soaking 4 g of G-200 Sephadex gel in 300 ml_ storage
phosphate buffer (0.05% NaN3, 0.0002 M PMSF, 0.9% NaCI and
0.02 M NaH2PP4) in at least 24 hours. Hydrated G-200 was poured
slowly down the column to avoid trapping air bubbles. A large volume
of phosphate buffer was applied to the column and flushed through
the column under nitrogen pressure. The column was charged with 3
mL of Ft (102 mg/mL) and was eluted through the column. Fractions
of 1 mL were collected, and those containing ferritin were combined.
2.4 Equipment
2.4.1 H-Cell
Fig 2.3 shows the diagram of an electrochemical H-cell. The H-cell
contained 2 chambers: (1) a side chamber that contained the
reference electrode (silver/silver chloride) and auxiliary (platinum)
electrodes, and (2) the main chamber which contained the
30


Main Chamber
(Working Electrode Chamber)
Fritted Bridge
Small Chamber (Reference Chamber)
Figure 2.3 H-cell diagram
31


electrochemical solution and gold electrode under study. From the
cell, the reference and working electrodes were connected to the
potentiostat as illustrated in fig 2.4.
These two chambers were separated by a 1-cm fritted glass disc that
allowed ions to flow between the compartments and kept
contaminants from the side compartment from entering the main
chamber. Nitrogen was led in the nitrogen outlet from the bottom of
the main chamber and then allowed to flow out of the cell at an
exhaust port at a level above the solution surface
2.4.2 Teflon cell
A cavity was drilled into a 1.5" diameter Teflon rod to accommodate a
solution volume of 3.5 mL. Nitrogen (for deaeration of the solutions),
auxiliary and reference electrodes were led into the cell through ports
around the cell, while the gold working electrode was directly inserted
into the sample solution through the top of the rod, (fig 2.5). Ferritin
adsorption took place in this Teflon cell.
32


n2
in
Figure 2.4 Diagram of connecting from H-cell to potentiostat and X-Y
recorder
33


Figure 2.5 Teflon Cell Configuration
34


2.4.3 Column Gravity Liquid Chromatography
The column specifications: 9.1 mL swollen Sephadex G-25 media, bed
height-5 cm, diameter-1.8 cm, initial equilibration volume-25 mL,
sample volume-2.5 mL, eluant volume-3.5 mL. G-200 Sephadex was
purchased from Pharmacia (Alameda, CA), and column gravity liquid
chromatography column purchased from Kontes Glass Co. (Vineland,
NJ).
2.4.4 Other Equipment
Voltammetric scans were performed with a Cypress Model Omni 90
Potentiostat (Lawrence, KS) and a BioAnalytical Systems Model RXY
recorder (West Lafayette, IN). The concentration of ferritin following
size-exclusion chromatography was determined by using a Perkin-
Elmer (Norwalk, CT) Model 552 UV-Visible Spectrophotometer.
Flame atomic absorption spectroscopy, AA-575 Atomic Absorption
Spectrophotometer (Varian Analytical Instruments, Palo Alto CA), was
used to determine the average number of iron atoms per ferritin
molecule.
35


2.5 Procedures
2.5.1 Determine Concentration Ferritin (Ft)
The concentration of ferritin was determined using UV-Visible
spectroscopy (wavelength = 595 nm). A Bio-Rad Protein Assay was
used as a guide to determine concentration of ferritin. A series of
standards of bovine albumin from 0.020-0.080 mg/ml_ was prepared.
5 mL of. dye diluted in ratio 1:5 with water was added into each
standard. The absorbance of a ferritin sample similarly prepared was
projected onto the concentration axis of the calibration curve.
2.5.2 Experimental Cyclic Voltammetric Process
There are six steps performed in each experiment: (1) cleaning the
gold electrode, (2) absorption of MPA on clean gold, (3) rinsing the
electrode, (4) absorption of ferritin onto the modified gold electrode,
(5) rinsing the electrode and (6) scanning the i-E curve. Except for
step (5) which was done in the Teflon cell, all others steps were
performed in H-cells. The described following steps were applied for
all studies in this experiment.
36


Step 1: Cleaning Electrode
The gold electrode, which was soaked in chromic acid cleaning
solution, was inserted into an 8.0 mm O.D. glass sheath connected to
a nitrogen source (fig 1.1). The electrode was immersed into 1 M
H2S04 in the main chamber of H-cell for cyclic cleaning purpose.
Subsequently, the electrode was cleaned by cycling the potential
between 1.5 and -0.35 Volts, which are potential limits for gold
electrode in 1M H2S04, at one-minute intervals. Usually after ten
cycles, a clean electrode surface was confirmed by scanning its cyclic
voltammogram to ascertain that all contaminants were removed from
the electrode surface (see fig 4.1 for a clean electrode voltammogram
in the next section). The gold electrode was completely rinsed free off
excess sulfuric acid and any dissolved adsorbate material with
distilled water.
Step 2: Absorption of MPA on Clean Gold (Au/MPA)
After the gold electrode was cleaned and rinsed, the reference
electrode was transferred into another cell which contained 1M
sulfuric acid solution in the small chamber; while the working
electrode was immersed into 7.54 x 10'4 M of 3-mercaptopropionic
37


acid solution in 1M H2S04 in the main chamber of the same cell. With
nitrogen flowing into the cell from the side and potential at 0.00 Volts,
the gold electrode was immersed in this cell in 3 minutes for MPA
absorption which was determined by a reference previous study.
Step 3: Rinsing Electrode
This cell was used for rinsing purpose. In pH 7 phosphate buffer
solution, the electrode was flushed several times, so that all excessive
MPA was rinsed off from the electrode surface.
Step 4: Absorption of Ferritin on Au/MPA
A stream of nitrogen blanketed the electrode while it was transferred
from the rinsing cell into the Teflon cell. The nitrogen was turned off
before the electrode was immersed in the ferritin solution to avoid
denaturing ferritin. The Au/MPA electrode was then immersed into
the ferritin sample. For the purpose of reducing ferritin, an adsorption
potential of -0.50 V was applied for 3 minutes. The electrode was
then rinsed free (under nitrogen) of excess ferritin before it was
transferred in the second rinse cell. Note: ferritin is in the oxidized
form from 0.1 V to 0.45V.
38


Step 5: Rinsing Electrode
With nitrogen, the electrode was flushed several times in clean
phosphate buffer at -0.50 V to rinse the electrode free of dissolved
ferritin.
Step 6: Scanning Final Cyclic Votammogram
The Au/MPA/Ferritin electrode was transferred into pure buffer
solution where the potential was scanned between -0.45 V to +0.10 V.
Step 7: Cut and Weigh Method
At the end of the study, the voltammograms were cut out then
weighed. Three similar scans were obtained for each trial, and an
average anodic peak area was determined.
2.5.3 Rate Constant Study Potential Step
Chronoamperometry
Instead of setting potential at -0.45 V in step 2, potentials were
gradually final set at potentials of -0.120, -0.175, -0.225, -0.275 and
-0.325 V, and -0.50 V for the initial potential. After the electrode was
thoroughly rinsed free of MPA and transferred in the Teflon cell, the
potential was stepped to -0.50 V at which ferritin was allowed to
adsorb on the electrode. The chronoamperogram was obtained by
39


recording the current time curve following a potential step to 0.20 V.
From the chronoamperomograms, current values and times were
extracted. A plot of ln(i) vs. time was constructed for each trial; and
the rate constant extracted from the slope.
40


3. Adsorption of Ferritin on Pyrolytic Graphite
Carbon Electrode Modified with
Hexamminechromium (III) Nitrate
3.1 Preparation of Graphite Carbon Electrode
A block of pyrolytic graphite carbon with dimension 3.5 x 3.5 x 10 mm
was prepared. The block of pyrolytic graphite was inserted into a PVC
tube containing epoxy resin. After drying, both ends of the tube were
ground flat, exposing a clean surface of graphite. The pyrolytic
graphite electrode configuration is illustrated in figure 3.1.
1 cm length of heat shrink tubing was shrunk around one end of the
tube; mercury was then added through the top of the heat shrink
tubing. Platinum wire was inserted into the mercury, and the heat
shrink tubing sealed with Teflon tape.
3.2 Preparation and Characterization of Cr(NH3) ]+
Hexamminechromium (III) Nitrate
The glassware was set up as in figure 3.2. Liquid ammonia was
condensed by passing gaseous ammonia across a jacketed beaker
filled with dry ice/acetone, and collected in the 3-neck flask.
41


Platinum Wire--------
m
Mercury
(b)
_PVC_
Heat Shrink
Tubing
(a>-
Edge-Oriented Pyrolyic Graphite Carbon
Epoxy
(a) Bottom of electrode
(b) Top of electrode
Figure 3.1 Graphite Carbon Electrode Configuration
42


Dry ice / Acetone
NH3 (g) in
Dry ice/Acetone
Figure 3.2 Glassware apparatus in collecting NH3 liquid


Approximately 100 mL of NH3 liquid was collected and transferred to a
250-mL beaker that was inserted in 400-mL beaker.
Hexamminechromium (III) Nitrate was synthesized based on the
reaction below:
CrCI3 + 6 NH3 ^ [Cr(NH3)6]CI3
[Cr(NH3)6]CI3 + 3 HN03 [Cr(NH3)6](N03)3 + 3 HCI
The synthesis was done according to a procedure outlined by
reference from Oppegard and Bailar [82] Approximately 625 mg of
clean sodium metal and 25 mg of iron (II) ammonium sulfate were
added to the beaker containing liquid ammonia. After the
disappearance of the blue color, 6.25 g of anhydrous chromium (III)
chloride was added in 0.25 g portions while the solution was
constantly stirred.
After the addition was complete, the brown precipitate was allowed to
settle. The slightly colored supernatant was decanted from the
beaker, and the residue was transferred to an evaporating dish and
dried. Approximately 20 mL of 2 M HCI was used to dissolve the
residue. The mixture was filtered and the filtrate was treated with 50
mL of concentrated nitric acid in order to precipitate
44


hexamminechromium (III) nitrate. The bright and yellow precipitate
was collected in a Buchner funnel, washed with cold distilled water
containing a little nitric acid, washed with alcohol (ethanol), then
ether.
The product was stored in a brown bottle, which protects the powder
from strong light. The pure powder-like hexamminechromium (III)
nitrate was dried in a vacuum oven.
3.3 Voltammetry Using the Pyrolytic Graphite
Electrode
A voltammetric experiment using the pyrolytic graphite carbon
electrode was set up as shown in fig 3.3. In this study, H-cell,
potentiostat and X-Y recorder were used in the same manner as the
experiments in which gold electrodes were used. The surface of the
electrode was gently polished on a nylon polishing disk, then rinsed
with distilled water. In general, the electrode was immersed into the
solution of interest and the potential scanned negatively from 0.10 V
at 100 mV/sec.
45


Figure 3.3 Voltammetric experimental configuration of pyrolytic
graphite carbon electrode
46


4. Results and Discussions
4.1 Cyclic Voltammogram (CV) of Clean Gold Electrode
Shown in fig 4.1 is the current potential curve of clean gold electrode
in 1 M H2S04. The features of this voltammogram are denoted as
peaks A, B, C, D, and E. In the negative potential region, peak A
represents the bulk evolution of hydrogen:
2H+ + 2e~ H2
Peak B indicates the re-oxidation of the hydrogen near the electrode to
H+. Though peak D represents the bulk evolution of oxygen, the gold
2 H20 + 4e~ 02 + 4H+
surface acts as a catalyst for oxidizing water to form a surface oxide at
potentials less positive than that of process D. It is still unclear which
oxide species in formed on the surface (-OH, -O-, -02, etc). The two
small peaks shown at position C are probably due to oxides forming at
two different crystal planes of polycrystalline gold. The peaks
represent the process known as underpotential deposition, or UPD.
The cathodic peak E potential near 0.90 V represents the cathodic
stripping of the surface oxide.
47


Figure 4.1 CV of clean gold electrode in 1M H2SO4. The voltammogram is
recorded from -0.35 V to 1.50 V. Scan rate is 10OmV/second.
48


4.1 CV of Modified Gold Electrode with MPA Modifier
When the potential of the Au/MPA electrode is cycled between 0.00
and -0.45 V, the current-potential curve shown in figure 4.2 is
obtained. In the negative direction, a peak appears at around -0.30 V,
indicating the presence of an adsorbed layer. It is not clear which
process is represented by this peak, perhaps the reductive cleavage of
the Au-sulfur bond.
4.3 Voltammetry of Au/MPA/Ft at 0.00V
It can be inferred from the result discussed above that MPA can be
adsorbed on gold at a controlled potential of 0.00 V. As a first
approximation, it would seem that ferritin can be adsorbed at the
Au/MPA electrode under the same conditions as those for MPA on
gold. After the surface was modified with MPA, the electrode was
transferred to the ferritin solution. The Au/MPA electrode was
exposed to feritin at 0.00 V in three minutes. The current-potential
curve is shown in figure 4.3. No cathodic or anodic peaks are
observed indicating that either ferritin doesnt adsorb on the modified
49


Figure 4.2 CV of Modified Gold Electrode with MPA Promoter
50


Figure 4.3 Voltammetry of Au/MPA/Ft at 0.00V
51


surface, or ferritin adsorbs but is not electroactive. Conceivably,
ferritin might absorb on the electrode surface at a different potential.
In the interest of discovering the suitable adsorption potential for
ferritin, a wide range of potential was applied.
4.4 Voltammetry of Au/MPA/Ft at -0.45V
Figure 4.4 is the CV of ferritin adsorbed at -0.45 V. The scan,
recorded in the range of-0.45 to 0.10 V yielded fairly large currents,
an anodic peak at -0.20, and a cathodic peak at -0.35 V. As
discussed above, at 0.00 V no faradaic currents were observed, but at
-0.45 V both cathodic and anodic are shown. Evidently, -0.45 V is
conducive to the formation of an electroactive layer, while 0.00 V is
not. Said in another way, the currents are only observed when a very
negative potential is applied. Since ferritin is reduced at -0.45 V, it is
reasonable to conclude that ferritin adsorbs as an electroactive
species when it is reduced. At 0.00 V, perhaps ferritin adsorbs on the
electrode surface, but not in an electroactive form. Future work will
address this question.
52


Figure 4.4 Cyclic voltammogram of ferritin at -0.45 Volts.
Concentration of ferritin = 6.75 mg/mL. Scan rate = 100 mV/s
53


4.5 Scan Rate Dependence
Fairly well-defined current potential curves are obtained after the MPA
modified electrode is immersed in ferritin solution at -0.45 V, scanned
in fresh pH7 phosphate buffer solution, and recorded at scan rates
100, 200, 300 and 400 mV/sec. The anodic current and cathode
peaks are observed at -0.17 and -0.33 V, respectively. The higher
scan rates provide higher peak currents as predicted by theory.
ip = k S n(3/2) D0(1/2) d(1/2) C
where ip = current peak potential; k = proportionality constant; S =
surface area of electrode in cm2; n = number of electrons transferred;
D0 = diffusion constant in cm2/sec; u = scan rate in V/sec; C =
concentration in moles/L .
The anodic peak currents of 40.75, 69.40, 93.75 and 123.33 are
calculated for 100, 200, 300 and 400 mV/sec. This data are
illustrated in plot of anodic current peak vs. scan rate, shown in fig
4.5. A linear equation is obtained from the plot with a correlation
coefficient of 0.999, which indicates that the peak current has a linear
54


Peak Current
Plot of Anodic Peak Current vs. Scan Rate
Figure 4.5 Plot of anodic current peak vs. scan rates.
55


dependence on scan rate. This behavior is indicative of an
electroactive-adsorbed species.
4.6 Potential Dependence Study
In this experiment, the effect of adsorption potential on relative
packing density was studied. In the range of -0.50 V to 0.10 V, the
anodic and cathodic peaks are observed at around -0.20 V and -0.35
V, respectively. The AEPof the potential peaks is about 0.15V. At
potential -0.45 V, the CV is seen with the highest peak current. At
-0.1 V, -0.2V, 0.0V and 0.1V, no current was observed. The inference
can be made that at -0.45 V more ferritin is reduced, hence more
electroactive ferritin adsorbs. The redox reaction of ferritin can take
place at a potential of-0.30 V, however the result is not as
pronounced as when -0.45 V is used because though ferritin is
reduced, the amount reduced is less than that at-0.45 V. This result
is also demonstrated in fig 4.6, the plot of absolute packing density vs.
potential showing the calculated packing densities for ferritin
adsorbed at various potentials. The plot indicates that the more
negative potential, the more ferritin becomes electroacitve.
56


Abs. Packing Density
Abs. Packing Density vs. Absorption Potentials
Figure 4.6 Plot of absolute packing density vs. absorption potential
57


4.7 Concentration Dependence Study
From the CV, in this study the lower dissolved ferritin concentration
yields a smaller peak current than those with higher concentration.
The current-potential curves are obtained in the potential range of-
0.45 to 0.10 V, the anodic and cathodic current peaks appearing at -
0.15 and -0.33 V, respectively. Figure 4.7 demonstrates the plot of
absolute packing density vs. concentration. It can be seen that as the
dissolved ferritin concentration increases, the packing density
increases. The observed adsorption isotherm is typical for globular
proteins.
4.8 Adsorption Time Dependence
After the gold electrode was modified with MPA, the electrode was
exposed to ferritin solution at -0.45 V at varying duration, namely 30,
60, 90, 90, 120 and 180 seconds. Again, the current-potential curves
were scanned from 0.45 to 0.10 V. A plot of absolute packing
density versus adsorption time is shown in figure 4.8. The scan shows
that the more time ferritin is allowed to adsorb the higher the packing
density.
58


Abs. Packing Density vs. Cocentration
Figure 4.7 Plot of absolute packing density vs. concentrations
59


Abs. Packing Density
Abs. Packing Density vs. Adsorption Time
Time (s)
Figure 4.8 Plot of absolute packing density versus adsorption time
60


The data show a limiting packing density after about 120 s adsorption
time, suggesting that the adsorption kinetics are principally governed
by diffusion of ferritin to the Au/MPA surface or surface attachment
rather than the changes in structure or orientation of ferritin following
initial attachment. The observed time dependent adsorption is in
sharp contrast to that observed with ferritin adsorbed at tin-doped
indium oxide (ITO) electrodes. In the latter case, the limiting packing
density is reached only after hours of exposure time. The ITO surface
is highly ionized at neutral pH; leading to the more significant
coulombic forces between the ITO and the protein. The lesser
electrostatic repulsion between protein and carboxylate requires less
time for the protein to find its most stable orientation on the surface.
4.9 Rate Constant
A typical chronoamperomogram for an adsorbed layer of ferritin is
shown in figure 4.9, which was performed from -0.50 to -0.325 V. At
t = 0, the working electrode potential is stepped from the initial value
of -0.50 V to a value of -0.325 V. After a very high initial current, the
current decays sharply as expected. Values of current at various
61


Figure 4.9 Chronoamperomogram
62


times are extracted from the chronoamperogram and the In i is plotted
against time as shown in figure 4.10. The unimolecular rate constant
can be extracted from the slope. A mean value of 1.1 0.09 s1 is
calculated for the rate constant. Rate constant values which are
obtained from each trial are shown in table 3.3 below.
Table 3.3 Rate Constant Values from Potential Step
Chronoamperometry Study
Potential Range (Volts) Rate Constant (sec1)
-0.50 V to -0.325V 1.0534
-0.50V to-0.275V 0.9888
-0.50V to -0.225V 1.1094
-0.50V to -0.175V 1.1902
-0.50V to -0.125V 1.0585
4.10 Comparison of Voltammetry of Au/Ft vs.
Au/MPA/Ft
The cyclic voltammetry of ferritin on bare gold electrode (Au/Ft) is
obtained in figure 4.11. The cycle is scanned from -0.50 V to 0.10 V.
Besides the familiar anodic and cathodic peaks seen on the
voltammogram, there is an anodic shoulder at a potential more
63


In(i) vs. t (-0.5 to -0.325 volts)
Figure 4.10 Plot of Ini vs. t
64


CD
CD
a). Ft/Au
b). Ft/MPA/Au
-0.5 -0.3 -0.1 0.1
POTENTIAL (VOLTS vs Ag/AgCI)
Figure 4.11 Cyclic voltammetry: a), of ferritin on bare gold electrode (Au/Ft) and b). of
ferritin on modified gold (Au/MPA/Ft)


positive than that of the principal adsorbed species [79-80]. The
i
differences between voltammograms of Ft/bare gold and Ft/MPA/Au
is shown in figure 4.11. Szucs and Novak have reported somewhat
similar behavior with adsorbed cyt c on bare gold electrodes,
attributing the extra feature to adsorbed, oligomerized cyt c. Although
the cause of the shoulder is not known, a similar inference can be
made to try to explain the result. When ferritin proteins are
oligomerized or aggregated, the structure of the globular network can
change, resulting in a change in the energy level of the redox center.
As a result, redox potential of the couple of the aggregate will change.
However, the participation of surface groups can prevent
oligomerization. Surface groups such as carboxylate groups can
prevent lateral movements of ferritin and hence the aggregation of
adsorbed ferritin molecules. This reasoning might be used to explain
the current-potential behavior observed on Au/MPA electrodes. The
anodic shoulder is not observed when the electrode is modified with
MPA. It can be reasoned then that the presence of MPA surface
groups on the electrode can prohibit the lateral migration of ferritin
molecules, thus preventing aggregation. Although the extra
66


peak is not pronounced, this observation suggests that ferritin may be
mobile on bare gold, but not on Au/MPA.
4.11 Conclusion (I)
Through this study, adsorption of ferritin on modified gold electrode
with 3-mercaptopropionic acid is observed. At 0.00 V potential,
neither cathodic nor anodic peaks are observed. But at -0.45 V,
ferritin is reduced and becomes electroactive; experimentally, its
redox reaction on the modified gold surface is observed by cyclic
voltammetry in the potential range of-0.45 V to 0.1V. The packing
density increases with concentration and adsorption time. Similar
behavior is observed for ferritin adsorbed on bare gold electrodes. In
this study, a limiting packing density is shown after 120-second
adsorption time. The peak current is proportional to scan rate
indicating that the electroactive species is adsorbed ferritin. From
potential-step chronoamperometry, a rate constant of 1.1 0.09 sec'1
is estimated for the redox reaction of ferritin indicating quasi-
reversible electron-transfer kinetics are involved in the electron
transfer of ferritin at the modified gold electrode surface.
67


5. Results and Discussion Adsorption of Ferritin
On Pyrolytic Graphite Carbon Electrode
5.1 CV of Fe(CN)g" on Carbon Electrode
The cyclic voltammetry of Fe(CN)g~ in 0.05M of KCI on pyrolytic
graphite (PG) carbon electrode is shown in figure 4.13. The purpose
of this scan is to test the working condition of the graphite carbon
electrode. The scan is obtained in the range of-0.40 to 1.00 V. Both
anodic and cathodic current peaks are seen around 0.20 and 0.05V in
the scan, respectively. Ferrocyanide is used because it has been
thoroughly studied using gold and platinum. The principal peaks are
similar to those observed using gold or platinum electrodes. From this
result, it is concluded that the constructed graphite electrode works
well enough to be used in the investigation of ferritin electrochemistry.
68


-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
Figure 5.1 CV of Ferrocyanide on Carbon Electrode in 0.05M of KCI
69


5.2 CV of Ferritin on Carbon Electrode
Dissolved oxygen is removed by bubbling nitrogen through the ferritin
solution for 10 minutes. Using the graphite electrode, the cyclic
voltammetry of ferritin is shown in figure 5.2. The scan is observed in
the scale from -1.20 to 0.00 V. No anodic current is seen, however, a
small cathodic peak is barely visible at -0.85 V potential. A peak
current of roughly 5^A is measured, a current which is too small for a
diffusion controlled reaction (section 1.5.2). This result could mean
that ferritin is adsorbed on the PG electrode.
5.3 CVofCrfNHs)^ on Carbon Electrode
To see if chromium (III) can catalyze the redox reaction of ferritin at
the electrode surface, a small amount of Cr(NH3)6+ was added to the
solution of ferritin in hepes buffer. The scan is shown in figure 5.3.
The CV is scanned from -1.20 to 0.00 V. There is no anodic peak, but
a small cathodic peak is observed at around -0.75 V. Another CV is
scanned from -1.20 V to a very positive potential at 0.4 V in order to
70


Figure 5.2 CV of ferritin on carbon electrode in hepes solution
71


Figure 5.3 CV of ferritin on carbon lectrode in Hepes buffer
containing Cr(NH3)g+
72


incorporate Cr(NH3)|+ into the carbon surface. However, when this
electrode was immersed into ferritin and the potential scanned, no
currents attributable to ferritin were observed.
5.4 Conclusions (II)
The results of this study indicate that dissolved probably doesnt react
at the surface. The scan of ferrocyanide indicates that the prepared
PG electrode is suitable for voltammetry. That chromium (III) did not
modify the electrode might suggest that the compound synthesized
may not be the right compound, or that hepes is not a suitable buffer
in the modification of the electrode. These issues will be investigated
in future work.
73


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