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Title: | STUDIES ON THE PURIFICATION AND METAL BINDING PROPERTY OF METALLOTHIONEINS FROM MARINE CRUSTACEANS | Authors: | CHONG PENG SIONG | Issue Date: | 1997 | Citation: | CHONG PENG SIONG (1997). STUDIES ON THE PURIFICATION AND METAL BINDING PROPERTY OF METALLOTHIONEINS FROM MARINE CRUSTACEANS. ScholarBank@NUS Repository. | Abstract: | Metallothioneins( MTs) are small, cysteine rich, metal-binding proteins that are known to be distributed widely in both vertebrates and invertebrates. Due to their suspected role in the Zn, Cu homeostasis and heavy metal detoxification, these proteins have, over the past forty years been a subject of great interest to many protein chemists. However, the purification of these proteins has been plagued by the vulnerability of these molecules towards oxidation due to their high cysteinyl content. The efforts made by many workers in solving this problem, have so far been inadequate. In this work, we have attempted to address this problem through the development of a new purification method employing the combined use of hydrophobic interaction chromatography (HIC) and chemical modification. Our revised protocol was tested against the conventional purification method that was commonly reported in earlier literatures utilising MTs from samples that were taken from hepatopancreas of the crab, Portunus pelagicus. Improvement in favour of our method can be seen in two main areas; firstly, there was a lower loss of protein to oxidation for every batch of raw material processed, and secondly, the time taken for the entire purification process was shortened significantly. Materials that were purified with the HIC were then subjected to chemical modification so as to protect these proteins in subsequent processing steps by blocking the reactive thiol groups (of the cysteinyl residues) in the MTs. Two chemical reagents were used for the blocking, they are 4-vinylpyridine and sodium sulphite. Of these two, blocking by the latter is reversible. Hence it is possible to regenerate apo-proteins from the sulphonated molecules and this is done so by the addition of dithiothreitol , a reducing agent. It is with the aim of reconstituting MTs from apo-proteins that were regenerated from the sulphonated MTs, that a series of metal saturation experiments was conducted. Two general procedures were followed for the metal saturation experiments. In one procedure, the apo-proteins were titrated with cadmium in alkaline medium. In the second method, the pH of the apo-protein solution was slowly increased in the presence of excess cadmium ions added straight from the start. From these experiments, we were able to propose a working model to describe the metal loading mechanism of these MTs, and also to use this model to correlate with some aspects of the observed behavior of MTs both in vivo and in vitro. Besides the crab, the new purification method was also used for the isolation of two MT isoforms from another species of crustacean, Thenus orientalis., a marine crayfish found in tropical waters. In this purification, we also incorporated the use of ion exchange chromatography and electrophoresis lo further clean up the proteins after chemical modification. Of these two techniques, only the ion exchange chromatography was scaled up for preparative application. Two protein isoforms were obtained separately from both the crayfish and the crab species and these molecules were subjected lo sequencing analysis. Partial sequences were elucidated for three of these proteins, with MTs from Portunus pelagicus being suspected to be N-terminally blocked. Despite the incomplete sequencing results, both the amino acid analysis results as well as the partial primary structure firmly established the proteins isolated in this work to be MTs. Furthermore, from this preliminary comparison between the primary structure of the proteins of the crayfish and those from the crab, Scylla serrata and the lobster, Homarus americanus, considerable degree of homology was noted. | URI: | https://scholarbank.nus.edu.sg/handle/10635/175568 |
Appears in Collections: | Master's Theses (Restricted) |
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