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The metallothionein (metallothioneins, MTs) are a class of low molecular weight, cysteine-rich metal-binding proteins. Certain degree of water between the environmental concentrations of heavy metals in aquatic organisms MTs can be metal-induced and induced, may indirectly reflect the level of heavy metal pollution in the water environment, marine animals MTs has been considered seawater biomarkers of heavy metal pollution and sediment. Content of marine animals MTs was Ocean metal exposed to the risk assessment and health assessment tool. In the past two decades, the MTs used in biological monitoring of marine pollution, the domestic and foreign scholars have studied a variety of analytical techniques and extraction methods. Marine animals the MTs of separation and quantitative analysis is no international standard method, this study exposure to cadmium seawater Philippines clams (Ruditapes philippinarum) as test material, mercury saturation method in the literature and spectrophotometry basis, respectively, of the two methods of extraction and determination conditions for optimization. Optimization of mercury saturation method, followed by orthogonal design and single factor design to optimize the extraction conditions of internal organs and gills MT (sulfhydryl content in crude extracts). Visceral best homogenate obtained conditions were as follows: Tris-HCl buffer solution concentration of 0.02mol / L, pH = 7.8, of offal and buffer volume ratio by mass (m / v) to 1/5, wherein the buffer solution concentration on the extraction effect. The optimum impurity conditions: the homogenate first at 4 ° C, 20000g centrifuged 30min, and then heated in a water bath of 80 ℃ 10min, then centrifuged at 4 ℃ 15000g 10min. Obtained gill preferred homogenizer conditions of: Tris-HCl buffer solution concentration of 0.02mol / L, a pH value of 7.8, a visceral buffer volume ratio by mass (m / v) to 1/4, wherein the buffer solution pH on the extraction effect. Best impurity: homogenate First at 4 ° C, 25000g centrifugation 20min at 80 ℃ 10min, and then centrifuged at 4 ℃ 15000g 10min. Optimum Method for Determination Ruditapes philippinarum gill tissue as test material, and one by one the determination of the method to extract liquid mercury content of gill tissue Cd-MT, resulting mercury saturation determination bivalve MT appropriate The conditions were as follows: Cd-MT extract diluted four times with Hg2 concentration of 50mg / L of 10% trichloroacetic acid on an equal volume ratio after mixing, incubation reaction 5min, further added 1.5-fold volume of 2% bovine heme solution, quickly centrifuged at 20000g 20min. Mercury saturation method optimized the relative standard deviation of the clams Cd-MT 4.95%, the recovery rate was 71.08% -78.86%, the detection limit for 0.24μgHg / L, corresponding to 4.47μgMT / g tissue. Single factor design For spectrophotometry optimization, optimize impurity removal process and determination of conditions of the internal organs and gills MT (sulfhydryl content in crude extracts). The visceral impurity optimum conditions were as follows: take lml supernatant was added 1ml of absolute ethanol (-20 ℃ precooling) and 80μl chloroform, centrifugal supernatant with 3 times the volume of ethanol -20 ° C precipitated 1H, 6000g centrifugation The supernatant was discarded after 10min, take the precipitate was washed precipitate was added 1.74 ml of anhydrous ethanol and 260μ1 Tris-HCl (containing sucrose) oscillation, centrifuged precipitate, N2 drying MT dry powder. Best measuring conditions: Ellman's chromogenic reagent NaCl concentration in 0.5 mol / L,. Spectrophotometric determination of an optimized spectral visceral MT relative standard deviation of 3.20%, the recoveries in the range of 90.09-96.85% detection limit 9.98μgMT / g tissue. Gills the MT impurity phase optimum conditions: take lml supernatant lml ethanol (-20 ℃ pre-cooling) and 40μl chloroform, centrifuged supernatant added three times the volume of ethanol -20 ° C precipitation 1h 6000g centrifuge for 10min the supernatant was discarded, N2 drying MT dry powder. Best measurement conditions: Ellman chromogenic reagent contain NaCl. The optimized spectral photometric method for determination of gill MT relative standard deviation was 2.92%, spiked recoveries in the range of 91.88-101.29% detection limit 6.61μgMT / g tissue. In summary, with respect to mercury saturation method, spectrophotometry is simple, reproducible, high accuracy, low cost, and test equipment is easy to get, more suitable for general laboratory selection, and can guarantee the authenticity and reliability of the results can be detected reliably as Ruditapes etc. bivalves MT analysis techniques.
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