**2. Various scientific developments in metallothionein research**

The scientific research history of MTs begins in 1950s almost seven decades back. The developments in MTs research includes the contributions from scientists in fields of environmental hygiene/medicine, biochemistry, medicine, pathology, nutrition and toxicology and are best illustrated in number of articles that are been published. During this time, in protein research methods, there is a considerable development in isolation and analytical methods for identification. In beginning the interest in MTs was focused on two main topics. The major interest were in the protein chemistry and in the toxicological, *i.e*. the metabolic pathways for the metal toxicology specifically in relation to the cadmium kinetics. In the recent years, the key contributions are brought in the molecular biology. Consensus and the conclusions reached at various scientific meetings on the metallothionein are based up on the published data as depicted in the **Table 1**.

In 1957, the data related to cadmium binding protein in the equine tissue were published. However, in the beginning this study was first initiated through a report that was in a form of abstract that deals with cadmium in the human organs. In most of the animal species, cadmium is present in small amounts in tissues and the


#### **Table 1.**

*Various scientific events held on the metallothionein.*

body fluids. For explaining this unexpected finding some of the hypotheses were postulated. Either cadmium can be present as bound to macromolecule or so had some natural function in the biological system or the cadmium also could be just a contaminant. The first report in 1960 was published on metallothionein [15]. For the first time that a cadmium containing protein that are isolated from the equine renal tissue, was termed as 'metallothionein' based on the higher content of sulfur (4.1%) followed by Cadmium (2.9%) and Zinc (0.6%). Later researchers estimated that the molecular weight was found as 10000 ± 260 Da.

The absorption at specific wavelength of 250 nm revealed the cadmium mercaptide bonds. The MTs lack an aromatic amino acid due to the non absorption at precise wavelength of 280 nm. These were further examined through amino acid analyses. Higher sulfur concentration of about 4.1% was explained through the amino acid analyses that revealed high cysteine content. The eactive protein mercapto groups at this time were examined through titration with the p-chloro mercuri benzoate (CMB), N-ethylmaleimide (NEM) and silver ions. The Amino acids were determined using both ion exchange chromatography and two-dimensional paper chromatography. The cysteine residues were found as cysteic acid after metallothionein oxidation with the performic acid and as N-ethylmaleide derivatives. The Sedimentation constant were determined by Schlieren diagram with the use of sedimentation through ultracentrifugation to 1.75 (S020w). Partial specific volume, diffusion constant, and friction ratio were obtained. The estimated protein molecular weight was still varying between 9790 and 10500 Da. These were partly explained by several artifacts during the preparation. Metal analyses for Cd and Zn reported 5.9% of MTs weight and 5.2 g atoms mol−1 for cadmium and for zinc as 2.2% by weight and 3.3 g atoms mol−1. Some exchange between Cd and Zn was taking place obviously. It was suggested that the bonding with 3 SH-groups together with an atom of either Cd and Zn was formed.

In the years between 1970s and early 1980s, there were only few research groups performed researches that are related to MTs. A workshop was arranged with nearly 25 invited participants who had submitted their background manuscripts and the tentative report [16] was prepared and were distributed in advance to each of the participant. A consensus report was agreed during the meeting held in the year of 1978.

During this Ist international meeting about MTs, held in the Zurich consensus was reached about, e.g. nomenclature and methods for protein preparing. This first meeting had been followed by another meeting held at Zurich in 1985 that were more open but still with a workshop consensus. In between, a meeting was arranged at Aberdeen in1981.

#### **3. Structure and occurrence of metallothionein**

The structure of MTs was first reported by Kagi and Schafer [15]. MTs have 2 domains consists of a cluster with three and other with 4 metal atoms. The gene are located on the chromosome 16. This protein contains a number of isoforms that are coded with several alleles. The ratio of the mRNA for MT-I and MT-II genes remains constant during the induction with metals, e.g. Cd, Cu and Zn. The MTs amino acid sequences from mammalian sources reveals that all of them contain nearly 61 amino acids that are more similar in their composition. All contain 20 cysteine residues which remain invariant to the amino acid sequence. Every cysteines do participate in the coordination of the seven moles of Zinc or cadmium per mole of the MT [17]. The MTs are the cytoplasmic proteins; but, their occurrence during fetal development in nucleus are given by immuno histochemistry. The Plant MTs were first recognized in roots of the copper-resistant strain of *Agrotis gigantean* and also in roots of the tomato

plants that are been exposed to higher concentrations of Cadmium chloride in medium are found in numerous other plants like Cd-treated maize, tobacco, wheat, rice and cabbage and also added found in some cell suspension cultures of plants [18, 19].

Metallothioneins from mammals contain 20 cysteins residues and bind seven cadmium or zinc ions in two discrete clusters. The thiolate side chains of these proteins act as bridging and terminal ligands to form Cd3SCy9 and Cd4 SCy11 clusters.
