| Metallothionein(MT)is a low molecular weight , cysteine–rich and heavy metal-binding protein. Its biosynthesis is induced by metals, hormone, bad stimuli, etc. It widely distributes in human, animal, higher plants and microorganisms. MT plays a role in storing and carrying trace element, energy metabolism, detoxifying of heavy metals, radioresistance, scavenging of free radicals, growing, delaying aging, preventing cancer and so on. It has been found the following characteristics: (a)Low-molecular-weight (usually<10,000Da); (b)heavy metal-binding (zinc, copper, and/or cadmium); (c)High content of cysteine ( Rich in Cys-X-Cys sequence domain ) ; (d)Non-aromatic amino acid, histidine content is extremely low. There is an acetylated methionine at the end of MT peptide chain. The number of cysteine, the cysteine location and the basic amino acid residues are conservative. These features provide more theoretical basis for the MT extraction and purification.At present, there are almost two kinds of methods to extract MT crude: the heat-based method and the classical purification method. The former, according to the physical and chemical characteristics of MT: solid conformation, heat-resistant, not easy to be enzymatic hydrolysis, etc. The heating method depends on heating to remove miscellaneous macromolecular proteins in the process of purification, while the classical method is based mainly on the size of miscellaneous protein molecules and fat composition to complete separation. After extracting the crude, the next is to achieve the further purification, the most commonly used method is ultrafiltration chromatography and ion exchange technology. Under normal circumstances, animal tissues contain only small amounts of MT. There are many types of protein in the organization. And it is difficult to purify. A kind of mouse/ human metallothionein was constructed and expressed successfully with genetic engineering methods, but the cost was more expensive relatively. At present, most research has fully affirmed its important physiological function. This study was based on the classic protein extraction theory and technology to explore better ways of separation and purification.[Objective](a)To purify the MT from the livers of mature rats by typical procedures and make some improvements and separate the two forms through DEAE chromatography.(b)To prove the improved purification technology can be used to get MT from other organizations or other species.[Methods](a) Preliminary treatment of samples: the samples (rat liver) are homogenate including the following methods: high-speed broken homogenate way, glass homogenate devices, ultrasonic treatment repeated freeze-thaw method, chemical treatment method and so on. According to the number of sample volume and other limitations, this study used the more common treatment—high-speed broken method.(b)Optimization of purification: Even though the time of heating method has been significantly reduced, the crude needed to get rid of the darker by repeated high-speed centrifuge. In addition the non-uniform temperature control in the short heating time could make the crude remain more substantial residual impurities. Certainly, the chromatography method was effective at removing impurities, but the uncertain impurity components were not conducive to the use of duplicate column material. Heating method temperature was generally greater than 80℃, which was easy to generate disulfide bonds.This process might change the structure and function of MT and make it difficult to obtain near physiological conditions of crude for the further corresponding scientific research.In contrast, the classical method under mild conditions were more conductive to carry out some structural and functional analysis. Some steps could be improved. We could make use of the physical and chemical properties of MT to conduct its preliminary identification.[Results](a) The hollow-fiber ultrafiltration (8h) could reduce the purification cycle significantly instead of the concentration of ammonia dialysis (72h);(b)The extraction of crude protein didn't have the general characteristics of the 280 nm peak and the absorption shoulder disappeared(200—300nm)when added acid .(c)The electrophoresis results showed that its molecular weight was around 6.5 K Dalton.(d) The two forms were separated through DEAE chromatography. The best elution conditions include the following aspects: the elution concentration gradient with 0.02-0.3 mol·L-1 Tris-HCL, elution volume was 600 ml, speed of flow was 0.8 ml·min-1.(e) After initial identification, the atomic absorption showed that the protein extracts contained zinc, copper, cadmium: Zn (0.176 +0.035μg·g-1), Cu(0.093+0.026μg·g-1), Cd(0.012+0.001μg·g-1). Zinc was the most significant metal element.[Conclusion]The improved method is more suitable for laboratory preparation. This reflects some specific significance:(a) The application of improved technology for MT production enterprises.(b) Enable the study of molecular biology more in-depth and explore the pathogenesis of related diseases.(c) To analyze the structure, type (subtype) under the natural state of MT in order to prepare the corresponding antibodies.(d) It is very significant to extract MT from different tissues (target organ) in vivo animal model. Take MT as one of important target indicators will be more effective for disease prevention and clinical treatment... |
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