Study On Separation&Purification&Determination Of Metallothionein And The Function Of Monitoring Heavy Metals

Metallothioneins (MTs) is a kind of metal binding protein, which described asphysiologically multifunctional because they are involved in the transport, storage anddetoxification of metals. A large number of studies have shown that MTs can be used as abiomarker of heavy metal pollution. At present, MTs has become a research hotspot inenvironmental science, bio-toxicology and medicine. The main contents of this article are asfollows：1: A purification method of MTs in earthworm was established. The factors influencingthe purification of MTs was studied by orthogonal experiment and response surface analysis.The optimum condition of sepration and purification of MTs by SephadexTMG-75column andDEAE SephadexTMA-25column was studied. Under optimized conditions, the purity of theextraction MTs as high as90%. The factors influencing the results were quantitied and abetter reproducibility was obtained. It was the basis of the determination of MTs, and it wasbetter for the comparison of MTs from different of biological samples.2: A new fluorescent method for the quantification of metallothionein was establishedusing lomefloxacin-europium (Ⅲ) complexas fluorescent probe. The linear rang was0.08-20mg/L with the detection limit of0.022mg/L, and the recovery was91.9-104.4%. Setting theenzyme-linked immunoassay as a standard, when compared with the differential pulsepolarography, this method was more accuary. This method has been successfully applied tothe determination of metallothionein in Eisenia andrei induced by Cd.3:A contact method of filter paper was used to investigate Sb inducing MTs in Eiseniafoetida. Spectrophotometrical and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gelelectropheresis) analyses were used to determine the nature of the induced MTs samples.Rabbit liver metallothioneins was used as a standard. The results showed that the molecularweight of induced protein was approximately6-7kDa. A characteristic absorption peak at268nm was observed, which were consistent with the standard. The amounts of Sb-MTsquantified by cadmium hemoglobin saturation method showed a significant positiverelationship with increasing Sb exposure.The experiments prove that Sb can induce Sb-MTsin Eisenia foetida as a biomarker of antimony pollution.4: Earthworms MTs was investigated whether it can be treated as biomarkers in heavy metals (Zn, Cu, Cd, Sb, and Pb) pollution soil. ICP-MS was used to determine the content ofmetal and cadmium hemoglobin saturation method was used to determine the amount of MTs.The experimental results show that the amount of MTs was related to the logarithmicconcentration of heavy metals in soil, and the two are in line with Lorentz fitting model. Theresponse concentration of MTs to different metal were lower than the background value. Theresponse concentration order was CZn≈CCu<CCd<CPb<CSb. Besides, MT-1, MT-2isoformexpressions were related to the metal. We prove that earthworms MTs were biomarkermonitoring heavy metals (Zn, Cu, Cd, Sb, and Pb) in soil.