Quantification of Zn2+ and Cd2+
Released from MT Adsorbates.
The metals released by the MT adsorbates in the
various electrode reactions are quantified with EC/ICP-AES
and the results are listed in Table 1. We also used
1.6 ?M MT solution to form the MT adsorbates, but
the amount of metal release for Zn2+ was below the
detection level of our EC/ICP-AES. A detailed
examination of the tabulated values revealed four
trends: (1) the smaller the concentration of MT in
the solution for the controlled-potential MT
adsorption, the less the amount of Cd2+ or Zn2+
measured; (2) The extent of overall Cd2+ replacement
(release) is greater than that of Zn2+; and (3) The
percentage of metal replacement was found to be
quite high at the lower concentration (e.g., 5 ?M
MT) but decrease to a more-or-less constant value
(within the experimental uncertainties) at a MT
concentration greater than 16 ?M.
The first trend is conceivable since
a low MT concentration in the solution will result
in a smaller extent of MT adsorption, which in turn,
will release a lower amount of metals upon redox
reactions. The second trend, however, is somewhat
unexpected. As mentioned in the Introduction about
the hierarchical order of MT for metal binding, Cd2+
should possess a higher affinity than Zn2+. This
unusual trend appears to be consistent with the
observation that the addition of Cd2+ into a MT
solution does not remove the zinc initially bound to
the MT.[18] This may imply that the removal of Zn2+
might be kinetically controlled. It is possible
that, as far as the heterogeneous electron transfer
(ET) process is concerned, certain Zn-binding sites
are more inaccessible than the Cd-binding sites. Our
postulation is based on the possibility that the ET
reaction of redox proteins is highly dependent on
the relative distance(s) of the redox center(s) from
the electrode and the interaction of the protein
molecules with the electrode surface (e.g., via
covalent bonding or electrostatic
interaction).[38-41] In their study about the
monitoring of the zinc ion flux in reactions of
Zn-reconstituted metallothioneins (Zn7MT) with
electrospray mass spectrometry, Zaia and coworkers
found that it was difficult to remove the last Zn
ion from a MT molecule.[6] In a separate report by
Ju and Leech,[42] it was suggested that the
cysteines in the MT ? domain might be in contact
with the electrode surface, leaving the ? domain
exposed to the solution. Thus, it appears that the
varied accessibility of various thiolates located at
different distances away from the electrode surface
to the ET reaction is a plausible argument for
reasoning the unexpected order of metal
release/replacement. To interpret the third trend,
we suggest that the surface coverage and packing of
the MT molecules might be different when the
concentrations of MT solutions used for the
controlled-potential MT adsorption are varied.[42]
At low concentrations, most of the MT molecules can
adsorb onto the electrode surface. The metals in the
adsorbates will be released from upon ET reactions.
As the MT concentration increases, the Hg surface is
fully saturated with the MT molecules and
consequently most MT molecules will remain in the
solution. Since the metal release most likely
originated from the redox reactions of the MT
adsorbates, the ratio between the metal release with
respect to the total amount of MTs introduced into
the cell will decrease. Such a suggestion is not
entirely unsubstantiated. In our previous
mechanistic study about the redox reactions of
cysteines and cysteine-metal thiolates in MT
adsorbates,[27] we have found that the adsorbate
film, formed under an open-circuit condition,
constitutes almost a monolayer. We later used flow
injection EQCM to quantify the amount of MT
adsorbate at a potential more negative than the PZC
of the Hg surface and found that the electrostatic
interaction between the Hg and the MT molecules in
the solution would produce an adsorbate film
slightly larger than a monolayer.[28] Thus, the data
in Table 1 seem to suggest that, at 5 ?M or less,
the MT surface coverage is at or!less than a
! monolayer and consequently most of the pristine
! metals in the MT adsorbates can be released via
! redox reactions.
As shown in Table 1, the
quantitative aspect of!the EC/ICP-AES approach is
quite satisfactory. This is evidenced from the
comparison of ICP-AES results from the Cd2+/Zn2+
standards to that from the Zn2+-spiked Cd2+/Zn2+
! solution. As the Zn2+ concentration increased by 1.8
times upon the addition of the Zn2+ standard (i.e.,
from 7.7 ?M to 7.7 ?M + 6.2 ?M = 13.9 ?M), the total
Zn3+ quantified (by combining Zn2+measured under
Peak I and Peak II) was up by 1.9 times (i.e., from
9.4 ? 0.4 to 18 ? 0.8 in Table 1). It is interesting
to note that the increase in the peak currents
between the two samples in the DPVs (the solid and
the dotted line curves in Figure 4a) do not show
such a proportional increment (Peak 1 increased by
almost 3.5 times). This suggests that the EC/ICP-AES
measurements are much more reliable. Another
interesting point worth mentioning is that the Zn2+
" augmentation from vhe 5 ?M MT solution to the
Zn2+-spiked 5 ?M MT sample does not quantitatively
reflect"the amount of the additional Zn2+.
" Specifically, in a 5 ?M MT solution, thg
" concentravion of cll the initial Zn2+ is about 2.8
?M. Therefore, an increase of about 3.2 times is
expected if 6.2 ?M of Zn2+ was spiked ((2.8 ?M + 6.2
?M)/2.8 ?M)and all the Zn2+ ions are in the free
form. The actual increase in Zn2+ concentration
observed by ICP-AES is only 2.3 times (i.e., (8.1 ?
0.7)/(3.6 ? 0.2)). This discrepancy suggests that
(1) not all of the initial Zn2+ complexed by the MT
molecules have been released into the solution and
(2) it is probable that some of the added Zn2+ might
have been complexed by the MT molecules in the
sample solution and retained by those MT molecules
that are attached to the electrode surface. For the
! purpose of contrasting the quantitative features of
DPV and EC/ICP-AES, we summarized the peak currents
(under Peaks 1 and 2 from DPVs) for all the cases
examined. As can be seen in Table 1, no obvious
trends can be found from the DPV peak currents. As a
consequence, quantifying the total Zn2+and Cd2+ and
calculating the percentages of metals replaced by
voltammetry for each case is not possible. Thus, it
is clear that the data obtained with EC/ICP-AES are
more accurate and can provide additional information
about the MT metal transfer/release processes.
Figure 2.Time-resolved
ICP-AES responses to the elution of Cd2+ from the
flow cell (b) and to the Zn2+ elution (c) after the
first DPV scan. In acquiring (c), the DPV scan was
halted at –1.0 V for monitoring the first Zn2+
elution peak (Peak I) and resumed after Peak I had
completely evolved.
Table 1.
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