Studies of Potential-Dependent Metallothionein Adsorptions Using a Low-Volume Electrochemical Quartz Crystal Microbalance Flow Cell 


Alejandro L. Briseno, Fayi Song, Alfred J. Baca, and Feimeng Zhou* 
Department of Chemistry and Biochemistry, California State 
University, Los Angeles, Los Angeles, CA 90032  

     An intracellular protein that is known to be involved in both regulation of essential metals and
detoxification of nonessential metals is metallothionein (MT).[1-5] MTs are cysteine-rich,
low-molecular-weight proteins or polypeptides of high metal content. MTs are purported to play central roles in the intracellular fixation of the essential trace metals such as Zn and Cu and regulation of their flow to the cellular destinations, in the detoxification of heavy metals such as Hg, Cd, and Ag, and in the protection of adverse conditions such as oxidative stress.[1-3, 6] Although MTs are small molecules in size, their diverse biological functions and complex properties have intrigued scientists since their discovery in the late 1950s.[7] Part of the complexity stems from the complete absence of any chromophores (e.g., aromatic amino acids) that can be examined as handles by spectroscopic techniques.[4, 5] Some of the metals involved (e.g., Zn) are also optically silent, making structural identification and functional elucidation challenging. 
     In the past few years, electrochemical methods have been demonstrated as viable means to study certain properties of MTs because the cysteine residues and most of the metal ions associated with MTs (Cd, Hg, Ag, and Cu) are electroactive.[8-10] A number of electrochemical studies, focusing primarily on the measurement of the redox potentials of the different isoforms and subisoforms of MTs and their binding affinity to various metals,[10-19] have been published. We recently observed two reversible redox peaks (Ep = -0.63 and -0.91 V, respectively) at thin mercury films (TMFs) deposited onto glassy carbon disk electrodes and assign the redox reactions to the oxidation peaks of the MT adsorbates.[20] It is generally believed, due in part to the strongest affinity of MTs to mercury (the hierarchical order for MT to bind different metals is Hg2+ > Ag+ > Cu2+ > Cd2+ > Zn2+)[2, 6], that labile metals originally present in MTs can be replaced by Hg.[8-10] Certain metals in solution (e.g., Cd2+ added to MT solutions) have also been shown to be complexed by the cysteine residues in MTs accumulated at the dropping mercury electrodes.[8-10] 
     In assessing the surface properties of the MT adsorbates at an electrode or the heterogeneous
electron transfer of MTs at the electrode/solution interface, two aspects that have been difficult to
determine are the surface coverage of the MT adsorbate and the average number of electrons (n)
involved per MT redox reaction. Knowledge about the n value is essential since the number of sulfhydryl groups per MT molecule participating in the electrode reaction(s) can be evaluated. Such information is vital to a detailed interpretation of the intra- and inter-molecular metal transfers and exchange processes between MTs and the substrates.[2, 4, 5, 8-10, 21] While the n value can be estimated indirectly using the slopes of the MT redox potential-pH curves, [10] the treatment is complicated by the uncertainty in resolving the overlapping oxidation peaks between MT-metal complexes at, and the free metals in the mercury electrodes, as well as by the possible protein structural change between two very different pH values (e.g., from pH 6 to pH 11 in Ref [22]). Another ambiguous aspect concerning the MT adsorbates is that the amount of MT adsorption might be affected by the potentials imposed to the mercury electrodes. Electrochemical studies of MTs inevitably involve the use of an externally applied potential which may or may not alter the MT adsorption and the structure of the final MT adsorbate. While the interaction between the MT molecules and the mercury surface is predominately via the formation of the cysteine-mercury thiolates,[8-10, 20] the likelihood of electrostatic interaction between MTs and the electrode surface cannot be excluded. For example, at neutral pH, rabbit liver MTs are positively charged due to the greater number of positively charged amino acids (7 lysines in MT-I and 8 in MT-II).[2] When a potential is applied, the electrode surface will become charged and the net positive or negative charges at the electrode surface can repel or attract the MT molecules. Conceivably, in addition to the chemisorption through the interaction between the cysteine residues of MTs and the mercury surface, electrostatic forces can also be operative for the MT accumulation onto the electrode surface. 
     To date, owing to the incompatibility or limited amenability of mercury surface to many surface
analysis techniques (e.g., scanning probe microscopy and optical spectroscopic techniques such as infrared and UV-vis reflectance spectrometry), amounts of MT adsorption onto mercury surfaces at different potentials have not been quantified. Relating the MT adsorption to the observed electrode processes at certain electrode potentials is also not straightforward because the applied potential can be sufficiently negative or positive to trigger the metal transfer and/or to cause redox reactions of the metals released by the MT adsorbates. 
     We report here our study of the potential-dependent MT adsorption processes using two techniques that can be used in-situ or on-line with a TMF electrode housed in an electrochemical flow cell, viz., flow-injection electrochemical quartz crystal microbalance (FI-EQCM [23-25]) and electrochemical inductively coupled plasma-atomic emission spectrometry (EC/ICP-AES [26]). The former technique is based on the combination of a homemade, low-volume EQCM flow cell with a simple flow-injection device. With this design, the adsorption of an analyte while it enters the flow cell can be monitored in real-time at a preset electrode potential. The amount of MT adsorption at a TMF electrode was found to be dependent on the relative position of the applied potential with respect to the point of zero charge (PZC) of the TMF surface.[27] The magnitude of the MT oxidation peak current was correlated to the MT surface coverage. 
       The use of the latter technique, EC/ICP-AES, on the other hand, allowed us to quantify the amount of Cd2+ released during the MT oxidation [8, 10] and or that accumulated into the TMF at a rather negative potential. Since the oxidation (stripping) potential of Cd at a dropping mercury electrode coincides with the major MT oxidation peak, it has been difficult for conventional voltammetric techniques to resolve these two separate faradaic processes. Quantification of the amount of Cd that might be accumulated into the mercury film was accomplished by subtracting from the total mass change monitored by the FI-EQCM experiment, the amount of Cd2+, measured in a separate EC/ICP-AES experiment. The average number of sulfhydryl groups per MT involved in the MT oxidation reaction was also deduced.Copper is an essential trace element that is crucial for the biological activity of numerous enzymes and metalloproteins.  Although the requirement for dietary copper is well known, the mechanisms by which this trace metal is transported intracellularly and inserted in apometalloproteins is poorly understood.   The properties that underlie the biological function of copper are also responsible for the toxic properties it exhibits when free in solution.¹  It is therefore necessary for organisms to limit the concentration of excess free copper to a minimum, which raises the question of how copper is transported within an organism and how the metal is made available for incorporation into apometalloproteins.