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Alfred J. Baca: Quantification of Metals Released
by Metallothionein Adsorbates at Mercury Film Electrodes by Differential
Pulse Voltammetry and Electrochemical ICP-Atomic Emission Spectrometry |
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Quantification of Metals Released by Metallothionein
Adsorbates at Mercury Film Electrodes by Differential Pulse Voltammetry
and Electrochemical ICP-Atomic Emission Spectrometry
Alfred J. Baca, Yajaira Garcia, Alejandro
L. Briseno, and Feimeng Zhou*
Department of
Chemistry and Biochemistry, California State
University, Los Angeles, Los Angeles, CA 90032
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Studies of the metal ion flux between metallothioneins
(MTs) and their biological partners have recently generated a great deal
of interest [1-8] MTs are a family of small metal-binding proteins that
have been purported to play an important role in heavy metal detoxification
and intracellular regulation of essential metals.[8-10] Most studies have
either focused on the understanding of the metal transfer process under
various redox conditions or dealt with the quantification of the metal
release associated with the MT redox reactions. [1-7] The two focal points
are related to the attempts in verifying the hypothesis that the metal
transfer between MTs and their substrates is influenced by the cellular
redox conditions. Recent research has indicated that the metals released
by MTs to a substrate or that received by apo-MTs from a metal-containing
species is facilitated by a redox-active species (e.g., glutathione (GSH))
and the directionality for the metal transfer is mediated by the status
of the redox-active species.[1, 2, 8] For example, the reduced form of
GSH favors the Zn transfer from enzymes to MTs, whereas the glutathione
disulfide (the oxidized form of GSH) enhances the reverse process of Zn
release from the protein.[1, 2, 8]
Various analytical techniques have been used to investigate
the metal transfer between MTs and different metalloenzymes or species
possessing metal-binding ligands.[6, 11-14] Among them, electrochemical
techniques offer certain unique advantages.[12-14] First, the redox potentials
of MTs at the electrode surface or the electrode/solution interface can
be accurately determined and the mechanism through which electroactive
groups undergo electron transfer reactions with the electrode can be probed.
Second, the precise control of the electrode potential provides an excellent
opportunity to trigger the metal release from the MT molecules or to examine
the metal uptake by the MT molecules. A prodigious amount of voltammetric
work has been performed to measure the redox potentials of various types
of MT isoforms or subisoforms.[12-27] Quite a few papers have also been
published on the use of differential pulse voltammetry (DPV) to study the
MT metal binding constants and the binding events.[Erk, 1998 #13; Erk,
1999 #1; Erk, 2000 #126;[18] We recently studied the redox behavior of
rabbit liver MT adsorbates formed onto thin mercury films (TMFs) under
both open-circuit [27] and controlled-potential conditions[28]. We found
that the voltammetric behavior of the MT adsorbates is analogous to that
at dropping mercury electrodes. The utilization of TMFs allows other non-electrochemical
techniques to be used in conjunction with voltammetry. For example, quartz
crystal microbalance (QCM) was employed to measure the amount of MT adsorption
in the presence or absence of an applied potential[28]. The determination
of the surface coverage enabled us to estimate the number of cysteine participated
in the MT complex oxidation reactions.[27, 28] Our studies and other groups’
reports have both shown that MTs exhibit interesting but complex redox
activities. Specifically, many peaks, given rise by the various electroactive
groups (e.g., cysteine, cystine, and cysteine-metal thiolates containing
different metal ions) appear in the voltammograms and a large number of
them could be overlapped. As a consequence, quantification of the metals
released by MT can be difficult sometimes, due to the limitation of many
conventional voltammetric techniques in resolving overlapping peaks and,
particularly, in quantifying metals which exist in several possible forms
(free, amalgamated or complexed).
The solution for accurately quantifying the metal transfer
induced by electrochemical reactions of MTs resides in the combination
of voltammetry with other analytical techniques that are more suitable
for trace metal analysis. One of such combination, electrochemical inductively
coupled plasma-atomic emission spectrometry (EC/ICP-AES)[29, 30] or mass
spectrometry (EC/ICP-MS)[29, 31, 32], has been shown as a powerful hybrid
technique for enhanced metal detection and for unraveling complicated electrode
reactions whose products contain metals. In the present work, we show that EC/ICP-AES can be used to quantify both Zn2+ and Cd2+ ions that are released/replaced
from the MT adsorbates involved in the various electrode reactions. The
comparison and correlation between the DPV results and the ICP-AES data
allowed a better understanding about the electrode processes. The cumulative
results from our previous voltammetric and QCM studies, together with the
present EC/ICP-AES measurements, help us provide a rather comprehensive
description about the various electrode reactions at mercury surfaces.
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