锌胁迫对小球藻抗氧化酶和类金属硫蛋白的影响

Activities of antioxidant enzymes and Zn-MT-like proteins induced in Chlorella vulgaris exposed to Zn^(2+)

通过对抗氧化酶活性和类金属硫蛋白的测定,考察在0、5、10、20、50和100μmol/L Zn2+(氯化锌)胁迫下锌对普通海洋小球藻的生物学影响。结果表明:不同浓度Zn2+均能抑制小球藻的生长,当Zn2+浓度大于10μmol/L时,小球藻生物量随培养时间延长而迅速下降;过氧化物歧化酶(SOD)活性随Zn2+胁迫浓度的增加而增加,当Zn2+浓度为50μmol/L时SOD活性达到最大,但继续增加Zn2+胁迫浓度反而导致SOD活性下降;而过氧化物酶(POD)活性则随着Zn2+胁迫浓度的增加而降低。同时,实验发现藻细胞内有两种主要的锌结合形态,其中Zn结合类金属硫蛋白(Zn-MT-like)与兔肝金属硫蛋白(MT)的分子量相近,且随着Zn2+胁迫浓度的增加而出现规律性地增多。因此,藻细胞内Zn-MT-like蛋白的诱导量可作为小球藻受Zn2+胁迫的响应指标。

Chlorella vulgaris（C.vulgaris）,a fast-growing unicellular micro-alga,is commonly used as a supplement of food for human and animal consumption.As C.vulgaris readily absorbs metals,this micro-algae has also been employed as an important indicator for monitoring the pollution and toxicity of metals in aquatic environments.Due to both natural geological sources as well as anthropogenic sources,the escalating contamination of aquatic environments by metals such as zinc（Zn） is becoming of increasing concern.A limited concentration of Zn is essential for growth of C.vulgaris,but limited knowledge exists concerning toxic impacts of Zn on the alga.The present study investigated various biochemical variables including biomass,the activities of antioxidant enzymes including superoxide dismutase（SOD） and peroxidase（POD）,the intracellular Zn assimilated,and the inducement of Zn-MT-like proteins in C.vulgaris when the alga was exposed to various Zn^2＋ concentrations.Results showed that the growth of C.vulgaris was inhibited by exposure to Zn^2＋ at concentrations ranging from 5—100 μmol l/L.The algal biomass rapidly decreased with increased Zn^2＋ exposure concentration.Algal biomass also decreased with increased exposure time to concentrations of Zn^2＋ 10 μmol/L.The results indicated that the growth of the alga was significantly inhibited by exposure to Zn^2＋,especially at higher exposure concentrations.The activities of SOD increased with the increase of Zn^2＋ exposure concentrations within the ranges of 0—50 μmol/L,but slight decreases in SOD activity occurred with exposure concentrations of Zn^2＋ 50 μmol/L.The highest activity of SOD was evident when the alga was exposed to 50 μmol/L of Zn^2＋,in which the value of SOD activity was two times more than that of the control group.However,the activities of POD within the alga significantly decreased（P 0.05） with the increase of Zn^2＋ exposure concentrations.For example,the activity of POD was 7.4 times less than that of the control group when C.vulgaris was exposed to 10 μmol/L of Zn^2＋.The results also showed that C.vulgaris has a strong ability to assimilate waterborne Zn^2＋.For example,the concentrations of intracellular Zn were about 17 and 34 times more than that of the control group when the alga were exposed to 5 μmol/L and 10 μmol/L of Zn^2＋,respectively.However,the concentrations of intracellular Zn did not increase when the exposure concentrations of Zn 10 μmol/L.A slight decrease of intracellular Zn was observed when the alga were exposed to 100 μmol/L of Zn^2＋.After being homogenized and centrifuged,the cell extracts of C.vulgaris after exposure to Zn^2＋ were separated with a size-exclusion high-performance liquid chromatography coupled with the detector of inductively coupled plasma mass spectrometry（ICP-MS）.Two Zn-binding species were observed within the cells of C.vulgaris,in which the compounds of the Zn-binding fraction at the retention time（tR） of 6.3 min were referred to as the Zn-MT-like proteins because the retention time（tR） was found to be similar to that（tR 6.1 min） of the standard Zn-MTs derived from rabbit liver.In addition,the amounts of Zn-MT-like proteins induced in the alga were found to increase with the increase of the Zn^2＋ exposure concentrations.The results indicate that the Zn-MT-like proteins induced in C.vulgaris can be used as the indicator to reflect the pollution of Zn^2＋in aquatic environments.