Electroactivity of proteins
and its use in biomedicine
PROTEINS (history)
The word protein comes from the Greek  ("prota"), meaning
"of primary importance" and these molecules were first described
and named by JJ Berzelius in 1838. However, proteins central role
in living organisms was not fully appreciated until 1926, when J B
Sumner showed that the enzyme urease was a protein.
The first protein to be sequenced was insulin, by F Sanger, who
won the Nobel Prize in 1958. The first protein structures to be
solved included hemoglobin and myoglobin, by Max Perutz and Sir
John C Kendrew, respectively, in 1958. Both proteins threedimensional
structures were first determined by x-ray diffraction
analysis; the structures of myoglobin and hemoglobin won the 1962
Nobel Prize in Chemistry for their discoverers.
Proteins are an important class of biological macromolecules present in all biological organisms, made
up of such elements as carbon, hydrogen, nitrogen, oxygen, and sulfur. The polymers, also known as
polypeptides consist of a sequence of 20 different L--amino acids, also referred to as residues. For
chains under 40 residues the term peptide is frequently used instead of protein. To be able to perform
their biological function, proteins fold into one, or more, specific spatial conformations, driven by a
number of noncovalent interactions such as hydrogen bonding, ionic interactions, Van der Waals' forces
and hydrophobic packing. In order to understand the functions of proteins at a molecular level, it is
often necessary to determine the three dimensional structure of proteins. This is the topic of the
scientific field of structural biology, that employs techniques such as X-ray crystallography and/or NMR
spectroscopy, to determine the structure of proteins.
A certain number of residues is necessary to perform a particular biochemical function, and around
40-50 residues appears to be the lower limit for a functional domain size. Protein sizes range from this
lower limit to several thousand residues in multi-functional or structural proteins. However, the current
estimate for the average protein length is around 300 residues. Very large aggregates can be formed
from protein subunits, for example actin - collagen and -synuclein in Parkinson's disease
4
Electrochemistry showed its usefullness in
the past and present analysis of DNA.
Can electrochemical analysis be equally
useful in the present protein research?
Electrochemical analysis of proteins
was succesfully applied in biochemistry, pharmacy,
medicine and particularly in clinical oncology research for
several decades in the middle of the 20th century.
Later the attention of electrochemists turned to
direct electrochemistry of a limited number of redoxactive
center-containing proteins and the potentialities
of the electrochemical methods as tools for protein
analysis in molecular biology and biomedicine were
neglected.
On CARBON and MERCURY electrodes
some amino acid residues can yield
analytically useful
ELECTROCHEMICAL SIGNALS
Electrochemical responses of proteins are dependent
on the orientation of the protein molecule at the electrode
surface
GOLD electrodes are preferable used
but at these electrodes amino acid
residues produce NO REDOX
RESPONSES
GOLD electrode
ET e-
protein
electrode
tunnelling
distance no electron transfer
redox active site
redox active
site
Small conjugated proteins (mostly metalloproteins)
undergo fast reversible processes at electrodes
Phase-sensitive a.c. voltammetry
or a.c. impendance measurements
can provide information about PROTEIN ADSORPTION/DESORPTION
behavior in dependence on the electrode potential and/or a.c. frequency
LIQUID MERCURY
ELECTRODE provides
very SMOOTH
SURFACE and best
REPRODUCIBILITY
reorientation
desorption
adsorption
Tyr and Trp oxidation at carbon electrodes
Constant current chronopotentiometry or
square wave voltammetry
1996
d.c. polarography
These techniques contain
sophisticated base line correction
(compensating the high background
currents at carbon electrodes)
ELECTROACTIVITY OF AMINO ACIDS IN PROTEINS
Tyrosine and tryptophan residues are oxidized at carbon electrodes
Chronopotentimetric peak H
which is due to the ability of proteins to catalyze hydrogen evolution,
is obtained at mercury and solid amalgam electrodes
Peak H ...... because of Heyrovsky J, Hydrogen evolution, High sensitivity
Present proteomics requires sensitive methods for the analysis of all
proteins. We wish to show that electroactivity of amino acid residues
in proteins can be utilized in the analysis of practically all proteins,
including those important in biomedicine.
Electrochemistry has shown its usefulness in the analysis of DNA and in studies
of some conjugated proteins.
Can electrochemical analysis be equally useful in the
research of all proteins?
10
According to F A Armstrong (Encyclop. Electrochem. Vol. 9, 2002) a
problem with commonly used metal electrodes, such as gold, mercury,
platinum and silver, is that they lead to denaturation and irreversible
adsorption of the resulting inactive protein.
In my talk I wish to show that proteins can remain native at the bare
mercury electrodes and combined with CPS peak H these electrodes can be
useful in studies of changes in protein structures, incl. denaturation
11
Peak H differs from the previously described polarographic
and voltammetric electrocatalytic signals of proteins
(i) by its ability to detect peptides and proteins down to
nanomolar and subnanomolar concentrations and
(ii) by its remarkable sensitivity to local and global changes
in protein structures. Denatured
native denatured
Weaker adsorption at Hg surface
hydrophobic groups (and some thiol
and disulfide groups) burried.
Electroactive groups less accessible
Stronger adsorption at Hg surface
hydrophobic groups, thiol, disulfide and
other groups better accessible
Greatly increased electroactivity
Ostatná V., et al.. (2006): Native and denatured bovine serum
albumin. D.c. polarography, stripping voltammetry and constant
current chronopotentiometry. J. Electroanal. Chem. 593,
172-178.
Native
Ostatná V., Palecek E. (2008): Native, denatured and reduced
BSA. Enhancement of chronopotentiometric peak H by
guanidinium chloride. Electrochim. Acta, 53, 4014-4021
Ostatna V. et al. (2008) Constant current chronopotentiometry
and voltammetry of native and denatured serum albumin at
mercury and carbon electrodes, Electroanalysis 20, 1406-1413
12
Previously shown measurements were performed at alkaline pH's (around pH 9.3)
Qualitatively similar results were obtained also with other proteins such as human serum albumin,
-globulin, -globulin, concanavalin, -crystallin, myoglobin, avidin, etc.
I will show that also at neutral pH large differences between peak H heights of native and denatured
BSA and other proteins can be observed. On the other hand under certain conditions denaturation of
BSA at the electrode surface may take place.
50 mM sodium phosphate, pH 7.0
urea-denatured BSA
Denatured proteins produce much higher peak H than their native forms
(both in alkaline and neutral media)
native
denatured
Such a large difference
between peaks of native and
denatured BSA can be
observed only at very fast
potential changes as in CPS
at relatively high stripping
currents. Much smaller
differences were found with
CV at 4 V/s scan rate.
100 nM native and urea-denatured BSA in sodium phosphate (pH 7) in presence 56 mM urea, Istr -30 A,, tA 60 s, EA -0.1 V.
H2
H1
native BSA denatured BSA
0.2 M phosphate
Ionic strength-dependent BSA transition at the electrode surface
80 mM
Such transition is NOT observed in solution as indicated by
the protein CD and fluorescence spectra
50 mM phosphate
100 nM BSA was adsorbed at HMDE from 50mM/200 mM sodium phosphate pH7 (for tA 60 s at EA -0.1 V) in
presence of 56 mM urea and then transferred either to 50 or 200 mM sodium phosphate, pH7, where the
chronopotentiogram was recorded (ex situ)
Is BSA denatured on HMDE in 0.2 M sodium phosphate, pH 7 at -0.1 V (against SCE) ?
Ex situ experiments indicated
that adsorption from
0.2 M sodium phosphate, pH 7
does not result in BSA surface
denaturation. Such denaturation
does take place, however, at more
negative potentials.
At neutral pH BSA is not denatured around pzc at the Hg electrode surface.
BSA surface denaturation can take place at more negative potentials and higher ionic strengths
15
Ex situ peak H of native (red) and denatured BSA (black) adsorbed at HMDE from 50 mM sodium acetate pH 4.5 in presence of urea 56 mM.
BSA-modified electrode was transferred to A. 50 mM sodium phosphate buffer, pH 7 B. 50 mM sodium acetate, pH 4.5, where the
chronopotentiogram was recorded with stripping current Istr -40 A at 18.5 °C. Other details as Fig.1 C.
50 mM Na-acetate
pH 4.5
50 mM Na-acetate
pH 4.5
50 mM Na-phosphate
pH 7
Native BSA
Denatured BSA
BSA in acid media is not denatured when adsorbed at -0.1 V but it undergoes
surface denaturation at more negative potentials
Palecek E., Ostatna V. (2009): Potential-dependent surface denaturation of BSA in acid media.
Analyst 134, No. 10, 2076-2080
Surface denaturation of proteins in acid media
16
100 nM native (red) and urea-denatured (black) BSA was adsorbed at HMDE for tA 60 s at different accumulation potentials, EA A. -0.1 V, B. -0.5 V, C. -1.1 V, D. -1.4 V
and at E. -1.6 V from 50 mM sodium acetate pH 4.5 in presence of urea 56 mM followed by transfer of BSA modified HMDE to 50 mM sodium phosphate, pH 7. F.
Dependence of peak H height on accumulation potential. Other details as Fig. 2B.
Dependence of peak H of native and denatured BSA on
accumulation potential, EA at pH 4.5 (ex situ as in the previous slide)
Native BSA
Denatured BSA
EA -0.1 -0.5 -1.1 -1.4 -1.6 V
Between EA +0.1 and ~ -0.5 V surface denaturation of BSA can be neglected.
Around EA -1.1 V BSA is almost fully denatured. Compared to neutral and alkaline pH's
at pH 4.5 BSA surface denaturation is much faster
17
At neutral pH large changes in peak H are taking place at higher ionic
strengths with BSA immobilized at the bare mercury electrode
charged to highly negative potentials. We tentatively explain these
changes by strong electric field acting on the protein molecule tightly
bound to the mercury surface.
At acid pH's (e.g. pH 4.5) native and denatured BSA produce peak H
of almost the same heights. BSA surface denaturation takes place at
potentials negative to pzc but not at the open current potential and
potentials positive to pzc.
Denaturation of proteins at the electrode surface
Peak H recognizes peptide and protein redox states and
specific binding of low m.w. compounds to protein molecules
Interaction of riboflavin with Riboflavin-Binding Protein
(RBP, a carrier of riboflavin, RB) plays an essential role in the embryo development.
RBP produced electrocatalytic peak H, capable to
discriminate between apoprotein and holoprotein
forms of RBP nanomolar concentrations.
SWV at CPE required
higher concentrations
of RBP and displayed
almost identical
oxidation peaks of
apoprotein and
holoprotein.
Bartosik M., Ostatna V. and Palecek E.
(2009): Electrochemistry of riboflavinbinding
protein and its interaction with
riboflavin. Bioelectrochemistry 76, 70-75
19
Peptides
Angiotensin I (1296.5)
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu
Angiotensin II (1046.2)
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
Angiotensin III (931.11)
Arg-Val-Tyr-Ile-His-Pro-Phe
[Val4]-Angiotensin III (917.08)
Arg-Val-Tyr-Val-His-Pro-Phe
Angiotensin II anti peptide (899.01)
Glu-Gly-Val-Tyr-Val-His-Pro-Val
Angiotensin IV (774.92)
Val-Tyr-Ile-His-Pro-Phe
Bradykinin (1060.2)
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
[Met(O)5]-Enkephalin (589.7)
Tyr-Gly-Gly-Phe-Met(O)
1-Mating Factor (1684.0)
Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr
Bombesin (1619.87)
Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2
SH38 (1154.2)
Asn-Arg-Cys-Ser-Gln-Gly-Ser-Cys-Trp-Asn
SS38 (1152.2)
Asn-Arg-Cys-Ser-Gln-Gly-Ser-Cys-Trp-Asn [­S-S­ 3-8]
[Lys8]-Vasopressin (1056.2)
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2 [­S-S­ 1-6]
Proteins
bovine serum albumin
human serum albumin
-globulin
-crystalline
myoglobin
-globulin
concanavalin A
aldolase A
lysozyme
micrococcal nuclease
RNA-ase A
azurin
cytochrom C
-synuclein and its mutants
-synuclein
protein p53 and its mutants
p53 core domain and its mutants
p53 C-terminal domain
insulin
thioredoxin
MutS
metallothionein
histones
avidin
streptavidin
Peptides and proteins
producing peak H
all tested peptides and proteins yielded
peak H
in contrast to BCR for peak H
presence of cysteine in peptides or proteins is not
necessary
peptides without cysteine produced
peak H only at neutral and acidic pH
but not at alkaline pH
amino acid residues with labile protons
such as lysine, arginine, cysteine are responsible
for peak H
2 PH(surf) +2e -- 2 P(surf)
+ H2(g)
P(surf)
+ BH(aq) -- PH(surf) + B(aq)
Recently we found that sulfated polysaccharides
such as carrageenans and dextran sulfate produce
peak HPS
S. Strmecki et al. Electrochem.Commun. (2OO9) in press
Palecek E., Ostatná V. (2007): Electroactivity of non-conjugated proteins and peptides.
Towards electroanalysis of all proteins. Electroanalysis, 19/23, 2383-2403.
20
All tested peptides and proteins produced CPS peak H. Differences in peak
heights and potentials were observed in different peptides and proteins. Peak H
responds sensitively to changes in protein structures. Very fast potential
changes in CPS play an important role in this analysis. BSA is NOT denatured at
potentials close to zero charge but surface denaturation may occur at negative
potentials.
Ionic strength-dependent surface denaturation of BSA at neutral pH and
potential-dependent denaturation at acid pH can be observed.
Redox states in peptides and proteins (such as thioredoxin or protein p53) can be
easily determined by means of peak H. The observed differences in this peak
result probably from different adsorption modes of reduced and oxidized forms
at the positively charged mercury surface. Similarly recognition of holo- from apo
RBP forms required protein adsorption at positive potentials.
Concluding remarks
21
In addition to various peptides and proteins we are currently studying two
proteins important in biomedicine by electrochemical methods:
Tumor suppressor protein p53
declared ,,The Molecule of the Year" by Science magazine in 1993 perhaps the
most important protein in the development of cancer. DNA-p53 protein
interactions are very important in performing the p53 function. Electrochemical
signals of DNA and p53 protein can be utilized in studies of these interactions
-synuclein (Asyn)
a major component of Lewy bodies associated with Parkinson disease. It is natively
unfolded but undergoes aggregation leading to fibrillar structures, in which the
protein adopts a -sheet secondary structure
Understanding the mechanism of aggregation and the factors that modulate it, is
important for devising therapeutic strategies.
The number of methods for studying the process of aggregation is limited and
electrochemistry appears to be suitable for this purpose.
Great changes in peak H not only during the AS aggregation and formation of
mature fibrils but particularly during the early stages of AS incubation in vitro.
CONSTANT CURRENT CHRONOPOTENTIOMETRY of Asyn (natively unfolded protein)
unfolding
oligomerization
aggregation
Palecek, E.,et al. (2008): Changes in interfacial properties of -synuclein preceding its aggregation. Analyst 133, 76-84.
NO Cys, NO Trp
in the molecule
23
MEEPQSDPSV EPPLSQETFS DLWKLLPENN VLSPLPSQAM DDLMLSPDDI EQWFTEDPGP
70 80 90 100 110 120
| | | | | |
DEAPRMPEAA PPVAPAPAAP TPAAPAPAPS WPLSSSVPSQ KTYQGSYGFR LGFLHSGTAK
130 140 150 160 170 180
| | | | | |
SVTCTYSPAL NKMFCQLAKT CPVQLWVDST PPPGTRVRAM AIYKQSQHMT EVVRRCPHHE
190 200 210 220 230 240
| | | | | |
RCSDSDGLAP PQHLIRVEGN LRVEYLDDRN TFRHSVVVPY EPPEVGSDCT TIHYNYMCNS
250 260 270 280 290 300
| | | | | |
SCMGGMNRRP ILTIITLEDS SGNLLGRNSF EVRVCACPGR DRRTEEENLR KKGEPHHELP
310 320 330 340 350 360
| | | | | |
PGSTKRALPN NTSSSPQPKK KPLDGEYFTL QIRGRERFEM FRELNEALEL KDAQAGKEPG
370 380 390
| | |
GSRAHSSHLK SKKGQSTSRH KKLMFKTEGP DSD
Amino acids which can contribute to the p53 protein electroactivity are
shown as larger letters. C, cysteine; W, tryptophan; Y, tyrosine; R, arginine;
K, lysine. Core domain is underlined.
N-terminus is acidic, C-terminus is strongly basic
The N-terminal domain (aa 1- ~100)
contains a transactivation region (aa
1- 42) and a proline-rich region; this
domain contains two Trp's (W), one
of them in the transactivation
region, but no Tyr and Cys (C)
residues.
The central (core) domain (CD, aa
~100- ~300) is involved in sequencespecific
binding to DNA
contains all Cys in p53 molecule
C-terminal domain is involved in nonspecific
binding to DNA and
contains no Trp and Cys and only
one Tyr
Amino acid sequence of human tumor suppressor p53 protein
Superstable p53 T-p53C core domain
The low thermodynamic stability of wt p53 protein makes the
protein difficult to study by biophysical and structural
methods.
More suitable for many experimental purposes were
stabilized p53 variants. Stabilization of p53 means an
increase in the thermodynamic stability of the protein,
derived from its folding -unfolding equilibrium.
The most stable substitutions forms superstable quadruple
mutant (T-p53C) M133L/V203A/N239Y/N268D.
The T-p53C provides a more rigid and stable structural
framework, while maintaining the overall structural
characteristics of the wild type protein.
T-p53C was received from Prof. Alan R Fersht from MRC, Cambridge UK
p53 core domain Prof. Z. Shakked from Weizmann Institute, Rehovot, Israel
We applied the electrochemical method to study p53 protein and
showed that
(i) redox states of core domain p53 can be determined and
(ii) wild type and mutant p53 produce different electrochemical
signals depending of 3D structure at different temperatures
(iii) The responses of wt differed from tested mutants in a wide
temperature range in a different extent. Responses of F270L
and V143A around 20 °C were almost the same.
(iv) We followed the time dependence of the EDTA effect on CPSA
peak H1 and H2 of superstable Tp53C (MRC), p53 CD (WIS).
Tp53C displayed much slower kinetics as compared to p53 CD.
Treatment of R175H with EDTA had no effect on the mutant
responses.
Label-free assay of DNA-protein interactions
using double-surface technique.
Suitable for almost all proteins
Determination of point mutations by MutS protein
Paleček, E. et al. (2004). "Sensitive electrochemical determination of unlabeled MutS protein and detection of point mutation in DNA."
Anal. Chem. 76(19): 5930-5936.
Label-free assay of DNA-protein interactions
using double-surface technique.
MAGNETIC BEADS
Suitable for almost all proteins
Determination of point mutations by MutS protein
Paleček, E. et al. (2004). "Sensitive electrochemical determination of unlabeled MutS protein and detection of point mutation in DNA."
Anal. Chem. 76(19): 5930-5936.
Label-free assay of DNA-protein interactions
using double-surface technique.
MAGNETIC BEADS
Suitable for almost all proteins
Determination of point mutations by MutS protein
Paleček, E. et al. (2004). "Sensitive electrochemical determination of unlabeled MutS protein and detection of point mutation in DNA."
Anal. Chem. 76(19): 5930-5936.
Label-free assay of DNA-protein interactions
using double-surface technique.
MAGNET
MAGNETIC BEADS
Suitable for almost all proteins
Determination of point mutations by MutS protein
Paleček, E. et al. (2004). "Sensitive electrochemical determination of unlabeled MutS protein and detection of point mutation in DNA."
Anal. Chem. 76(19): 5930-5936.
Protein dissociation and
electrochemical determination at
carbon or solid amalgam electrodes
Carbon or solid amalgam electrode
Label-free assay of DNA-protein interactions
using double-surface technique.
MAGNET
MAGNETIC BEADS
Suitable for almost all proteins
Determination of point mutations by MutS protein
Paleček, E. et al. (2004). "Sensitive electrochemical determination of unlabeled MutS protein and detection of point mutation in DNA."
Anal. Chem. 76(19): 5930-5936.
ELECTROCHEMISTRY OF BIOMACROMOLECULES
First polarographic measurements of proteins were done in J. Heyrovský
laboratory about 80 years ago. About 30 years later electrochemistry
entered the field of nucleic acid research, followed by application of
electrochemical analysis of lipids and membranes. Until very recently
electrochemical analysis of polysaccharides was limited to a few papers
dealing with adsorption phenomena. Recent labeling of polysaccharides
with Os(VI)L complexes and finding the ability of (unlabeled) sulfated
polysaccharides to catalyze hydrogen evolution and produce peak HPS
open the door for wider use of electrochemistry in the polysaccharide
research. Thus at present electrochemistry can be applied in the
research of four main classes of biomacromolecules, i.e. PROTEINS,
NUCLEIC ACIDS, LIPIDS AND POLYSACCHARIDES.
MPI BPC, Göttingen
T.M. Jovin
C. Bertoncini
synuclein
Ankara University
B. Uslu, S. Ozkan
B.Dogan
(BSA electrochem.)
Weizmann Inst.,Rehovot
Z. Shakked
wt and mutant p53CD
MRC, Cambridge
A. Fersht, A. Jörger
Dept. Biophys. Chem. Mol. Oncology
- predominantly DNA
Protein Group
Hana Cernocka
Mojmir Trefulka
Petra Mittnerova
Emil Palecek
Lida Rimankova
Martin Bartosik
Veronika Ostatna
Collaboration
IPC JH, Praha
M. Heyrovsky
L. Novotny
B. Yosipchuk
amalgam electrodes,
electrocatalysis
Merry Christmas and
a Happy New Year
2010!
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