Biophysical characterization of a recombinant metallothionein and its possible use as a biosensor (Rekombinant metallotioninler’in biyofiziksel karakterizasyonu ve biyosensor olarak kullanılabilirliği)

Heavy metals are one of the most toxic contaminants for the environment and affect not only the ecological balance but represent a serious threat to human health. Detection and quantification of heavy metals at trace level are important for environmental and health protection [1]. A common pollutant is cadmium (Cd), which leaks into the environment from several sources including fertilizers, industrial plants and sewage [2]. Several methods, such as spectroscopic or electrochemical analyses are routinely used for detection of heavy metals. Recently attempts to develop sensors which can be used locally allowing determination of trace quantities of heavy metals led, among others, to the use of biomolecules as the recognition element in metal sensors. Metallothioneins (MT) belong to a group of low molecular weight, cysteine-rich and aromatic amino acid-lacking proteins that bind heavy metals such as Cd, arsenic (As) and mercury (Hg). MTs consist of two metal binding domains (α and β) that are assembled from cysteine clusters. Cysteine sulfhydryl groups participate in the coordination of heavy metals. Due to their high metal binding capacity for different metals, MTs are highly valuable for detoxification, remediation and recycling in agricultural areas. Their potential use for development of metal biosensors for environmental and therapeutic purposes is also recognized. We identified an mt gene in Cd resistant durum wheat and the recombinant protein (dMT) was overexpressed in E. coli as a GST-fusion protein (GSTdMT). GSTdMT was purified with Cd as a dimer in monodisperse solutions [3]. Structure of GSTdMT was investigated by small angle X-ray scattering (SAXS), circular dicroism (CD) and UV-vis spectrophotometric measurements. Inductively coupled plasma optical emission spectroscopy (ICP-OES) and EXAFS measurements showed that GSTdMT binds about 4 Cd2+/protein in a tetrahedral arrangement. The aim of this study was to investigate the utilization of dMT as the recognition element in a Cd biosensor. GSTdMT was immobilized onto epoxy- and thiol-modified surfaces and changes upon Cd-binding were detected by surface plasmon resonance (SPR). Our results demonstrate the possibility of using GSTdMT as a biosensor for Cd2+ detection for different applications.