| Metallothioneins (MTs) are a family of low molecular weight metal-binding proteins, which contain a high proportion of cysteine. In many aquatic species metallothioneins can be inducted by metals, and the degree of MTs induction is correlated with metal concentrations, therefore the level of metals in aquatic environment can be reflected indirectly by the induction of MTs. At present, MTs in marine animals have been recognized as potential biomarker of heavy metals in seawater and sediment. MTs concentrations in marine organisms are also proposed as an important tool to evaluate the risk of metal exposure and assess the health of oceans. In the past two decades, many researchers both at home and abroad have applied MTs to marine pollution monitoring, and developed different analysis techniques and extraction methods. Until now, there is no standard method, neither isolation nor quantification in the studies of marine animals. In this study, Ruditapes philippinarum exposed to seawater containing cadmium was used as a material, the extraction and determination condition of the established mercury saturation method and spectrometric method were optimized.For the mercury saturation method, extraction conditions for maximum MT recovery from viscus and gills, expressed as content of sulfhydryl group (-SH) in the crude extract, were optimized by orthogonal design and single-factor design. The optimized homogenization process of viscus was as follows:the buffer concentration was 0.02 mol/L, pH7.8 and m/v was 1/5. MT yield was primarily affected by Tris-HCl buffer concentration. The optimal process obtained for removing impurity was that the homogenate was centrifuged at 20000g at 4℃for 30 min, and then the collected supernatant was heated at 80℃for 10 min, followed by a second centrifugation at 15000g at 4℃for 10 min.The optimized homogenization process of gills was as follows:the buffer concentration was 0.02 mol/L, pH7.8 and m/v was 1/4. MT yield was primarily affected by Tris-HCl buffer pH. The optimal process obtained for removing impurity were that the homogenate was centrifuged at 25000g at 4℃for 20 min, and then the collected supernatant was heated at 80℃for 10 min, followed by a second centrifugation at 15000g at 4℃for 10 min.When the determination condition was optimized, gills in R. philippinarum were used as material, the established mercury saturation method was optimized by single-factor design, expressed as the content of Hg in solution. The optimized determination condition was as follows:the 4-fold dilution of supernatant contained Cd-MT was mixed with HgCl2 solution at 50 mg Hg/L in 10% trichloroacetic acid in the same volume. After incubating for 5 min,1.5-fold of 2% bovine hemoglobin was added and rapidly centrifuged at 20000g for 20 min. The relative standard deviation of this optimized method was 4.95%, the recovery of standard addition was 71.08%-78.86%, and the detection limit was 0.24μgHg/L, equivalent to a tissue concentration of 4.47μgMT/g.For the spectrometric method, the conditions of removing impurity and determination condition for MT from viscus and gills, expressed as content of sulfhydryl group (-SH) in the crude extract, were optimized by single-factor design. The optimal process obtained for removing impurity for viscus was as follows:for per 1 ml of the supernatant, 1ml cold (-20℃) absolute ethanol and 80μl of chloroform were added. Subsequently, the samples were centrifuged and the supernatants were added to 3-volume of cold ethanol, stored at-20℃for 1h and centrifuged at 6000g for 10 min. Then, the pellets after centrifuged were washed with 1.74ml absolute ethanol and 260μl Tris-HCl homogenization buffer (containing sucrose), centrifuged again and the pellets were dried by N2(99.99%). The optimal determination condition was Ellman reagent containing 0.5mol/L NaCl. The relative standard deviation of this optimized method was 3.20%, the recovery of standard addition was 90.09%-96.85%, and the detection limit was 9.98μgMT/g.The optimal process obtained for removing impurity for gills was as follows:for per 1 ml of the supernatant, 1ml cold (-20℃) absolute ethanol and 40μl of chloroform were added. Subsequently, the samples were centrifuged and the supernatants were added to 3-volume of cold ethanol, stored at-20℃for 1h and centrifuged at 6000g for 10 min. Then, the pellets after centrifuged were dried by N2(99.99%). The optimal determination condition was that the NaCl was not contained in Ellman reagent. The relative standard deviation of this optimized method was 2.92%, the recovery of standard addition was 91.88%-101.29%, and the detection limit was 6.61μgMT/g.In short, compared with mercury saturation method, spectrometric method is more simple, repeatable, accurate, low-cost, and materials can be obtained easily, and can guarantee the facticity and dependability of the results. Therefore, spectrophotometric method can be used for the dependable determination of MTs in marine bivalve such as R. philippinarum.
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