Probing the characteristics of metal-binding proteins using high-performance liquid chromatography–atomic absorption spectroscopy and inductively coupled plasma mass spectrometry

Abstract

The primary structure of metallothionein (MT) and MT-like proteins (MLP) contains a high proportion of cysteine residues which are capable of complexing a variety of metals. Due to the unique metal-binding properties of MT and MLP, metal saturation assays have been used as an indirect method of quantifying the concentration of these proteins in various samples. In adapting Cd-saturation methodologies of MT from mammalian sources for MLP from freshwater mussels, the redox state of the MLP cysteine residues was indirectly monitored by adding Cd in the presence, or absence, of a reducing agent followed by high-performance liquid chromatography (HPLC)-microatomization (MA)-atomic absorption spectroscopy (AAS) and/or HPLC-inductively-coupled plasma-mass spectrometry (ICP-MS). The presence of 2-mercaptoethanol(2-MCE) in freshwater mussel extract results in a higher concentration of Cd present in the MLP fraction, indicating that these proteins may be partially oxidized in vivo and/or during isolation under anoxic conditions. Standard preparations of rabbit MT obtained by induced synthesis due to controlled exposure to Cd are fully reduced and saturated with metal (Cd and Zn) and, therefore, did not respond similarly to the reduction treatment used in these studies. Thus, as conventional metal saturation assays exclude a reduction step, they may be unsuitable for the quantitative analysis of MLP from molluscan tissues without further modification.

Evaluation of an Ultrasonic Nebulizer Interface for Capillary Electrophoresis and Inductively Coupled Plasma Mass Spectrometry

“…Metallothioneins are a family of relatively low-molecular-weight, heavy metal-binding proteins that have been characterized and separated by CE with photodiode-array spectrophotometric detection (36). The MT concentration in animal tissues has also been employed as a metal pollution biomarker in aquatic environments (37). Capillary electrophoresis ICP-MS should provide a technique to explore the functional significance of individual isoforms.…”

Development of a Stable Isotope Approach for the Inductively Coupled Plasma–Mass Spectrometry Determination of Oxidized Metallothionein in Biological Materials

“…Techniques for the determination of MTs include metal-saturation assays (4,5), polarography (6), and immunological assays (7). Electrochemical techniques based on the quantification of MT fraction via sulfhydryl groups may suffer interferences by other redox systems present in the sample.…”

Identification, characterization and determination of metal-binding proteins by liquid chromatography. A review

“…The first common approach to solve the problem consisted in the separation of the metalbinding proteins and further analysis of the metals by electrothermal atomic absorption spectrometry (ETAAS). The mentioned procedure has been used for the separation and detection of cadmium-containing proteins [24] and for the determination of selenium bound to serum proteins [23]. ETAAS has also been applied to the determination of lead-binding proteins present in red cells [44].…”

“…From the analytical point of view, high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are the most widely used methods to separate proteins. A comprehensive review on determination of metal-binding proteins by CE has been recently delivered by Stutz et al [22] The detection of the metals has been carried out with electrothermal atomic absorption spectrometry (ETAAS) [23,24], inductively coupled plasma-atomic emission spectrometry (ICP-AES) [25], and inductively coupled plasma-mass spectrometry (ICP-MS) that has been applied in the speciation of metals in human serum [26]. A complete review has been published on bio-inorganic speciation analysis by hyphenated techniques [27].…”