Biomedical Engineering Reference
In-Depth Information
As depicted in Fig. 6.1, the asymmetric peak currents (
I
a
/
I
c
0.76, at 100 mVs
1
)
150 mV, at 100 mVs
1
) essentially
indicate that the electrochemical process of Cu, Zn-SOD is quasi-reversible at the
SAM-modifi ed electrode. The formal potential [
E
0
E
a
E
c
and the large peak separation (
∆
E
p
E
c
)/2, was
65 mV vs Ag/AgCl (262 mV vs NHE). This value is slightly smaller than those recently
obtained by other groups by using cyclic voltammetry; 0.30 V vs NHE obtained at the
cysteine-modifi ed Au wire electrode in phosphate buffer (pH 7.0) [123] and 0.32 V
vs NHE obtained at the Au electrode in 0.10 M NaClO
4
solution (pH 7.2) containing
2
], estimated as (
E
a
10
4
M 1,2-bis(4-pyridyl)-ethene [121]. The
E
0
values have been also determined
by using coulometric titrations [129], visible spectroelectrochemical techniques [130,
131] and EPR-monitored redox titrations [132]. The differences in the
E
0
obtained
thus far (ca. 0.12
0.403 V vs NHE at pH 7.0) are probably due to the differences in
the enzyme preparation, the experimental conditions used, or others.
A study of the relationship between the peak currents obtained in Fig. 6.1 and
potential scan rate (
v
) indicates that the peak currents are proportional to
v
(not
v
1/2
)
in the examined range of 10 to 1000 mVs
1
, demonstrating that the observed volt-
ammetric response corresponds to the electrode reaction of Cu, Zn-SOD confi ned on
the SAM of cysteine. This is very different from the redox reaction of Cyt.
c
in solution
phase at the same cysteine-modifi ed Au electrode, in which the peak current increased
linearly with
v
1/2
and thus the redox process is expected to be a diffusion-controlled
electrode reaction of solution-phase species [133]. In the latter case, the cysteine pro-
moter can be considered to be able to “transiently” orient Cyt.
c
onto the electrode sur-
face without a deactivation in such a way that the prosthetic group is disposed towards
the electrode surface and, as a result, the direct electron transfer takes place. In addition,
the binding between the cysteine promoter and Cyt.
c
is reversible so that the exchange
with solution-phase Cyt.
c
can occur. Possibly in a different way, the SOD enzyme can
be regarded as being “permanently” confi ned via the SAM of cysteine to the electrode
surface in the present case, i.e. cysteine bridges between the SOD and the electrode.
The relevant kinetic parameters of the electrode reaction, i.e. the rate constant of the
electrochemical process (
k
s
) and anodic and cathodic transfer coeffi cients (
α
c
)
of the Cu, Zn-SOD were estimated according to the Laviron's equation [134] and calcu-
lated to be
k
s
α
a
and
0.2) s
1
,
0.02 [98].
It can be deduced from the demonstration mentioned above that the Cu, Zn-SOD
is properly oriented on the Au electrode via the cysteine monolayer so as to allow a
rapid electron transfer of the Cu
2
site to/from the electrode. Bovine Cu, Zn-SOD
has a net negative charge at pH 7.0 (p
I
(1.2
α
a
0.39
0.02, and
α
c
0.61
4.9) [135]. The p
K
a
values of
ˆ
COO
and
ˆ
NH
2
groups of cysteine are 1.71 and 10.78, respectively [136] and thus the cysteine
immobilized on the Au electrode via the formation of Au-S bonding can be expected
to behave as a zwitter ion at pH 7.0. Therefore, the orientation of Cu, Zn-SOD on the
Au electrode through the SAM of cysteine and the resulting facilitated electron trans-
fer may be considered to be not only simply due to an electrostatic interaction between
the SOD molecule and these functional groups, but also due to a unique interaction
on a molecular level. For example, the
ˆ
NH
2
and
ˆ
COO
groups of cysteine are
considered to cooperatively function to bind the SOD; the hydrogen bonding between
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