| The heavy metal pollution and control in the marine environment have attracted more and more attentions from the people for a long time.However, the scientific problems involving contents, distribution pattern and migration and transformation of heavy metals and the function of influencing the chemical factors in the marine environment are the focus of attention by the domestic and overseas scientific researchers, and also how to effectively govern the heavy metal in the marine environment currently becomes one of the urgent researches. At present, the heavy metal governance of the ocean water by the macroalgae is a technology with low investment and high benefits, which has the definite detoxification function on the ingested heavy metals after combining with the sulfydryl protein. The metallothionein-like protein (MTL) and polypeptide compound in the frond is a kind of low molecular weight protein rich in cysteine generated under the metal ion induction, which possesses very strong heavy metal combining capacity and plays an important role in the aspects of detoxification and metabolism of heavy metal. This paper provides the scientific basis and technical support for the purpose of researching the interaction between the metallothionein-like protein and polypeptide in the frond and the heavy metal as well as researching the biological effect, exploring the changes of the enrichment capacity of heavy metals and toxicity resistance due to the changes of the protein and polypeptide in the frond after the metal induction, further exploring indicating the heavy metal pollution status in the marine environment by the peptide and protein in the seaweed as the marker, and developing the potential of the seaweed for the recovery effect on the heavy metal in the marine environment. The main research work was made as follows.Firstly, the microwave digestion pretreatment technology and the quantitative method of measuring the trace elements in the frond by the inductively coupled plasma mass spectrometry were established. The method had higher accuracy, good linearity, and correlation coefficient of more than 0.999. In addition, the heavy metal contents in multiple kelp respectively cultivated with the heavy metal in the laboratory and fostered in Weihai aquiculture area were measured. The result indicated that the kelp showed up the enrichment capacities for Cu, Zn and Cd elements in varying degrees, and was relatively obvious with the changes of time and concentrations. Different species of kelp such as Benniu, Pingbancai, Sanhai, Shengchanzhong, Rongfu, Gracilaria and asparagus had difference in the enrichment contents of the same heavy metal, and different varieties of kelp had larger differences on the relative enrichment contents of different heavy metals.Secondly, the metallothionein-like protein extraction and purification technology in the frond was established, so as to separate and purify the metallothionein-like protein by using the sephadex column. The online collection of low concentration protein components was achieved in virtue of the protein binding metal signal. The range of metallothionein-like protein molecular weight was preliminarily confirmed in the SDS-PAGE method, with the molecular range of 6.5kDa～7.0kDa. The metallothionein-like protein molecular weight was accurately measured in the high sensitivity mass spectrometry to gain the metallothionein-like protein molecule of 6.570kDa. After optimizing the chromatographic conditions, the online technology of high performance liquid chromatography-inductively coupled plasma mass-spectrometry was established, which realized the quantitative analysis of metallothionein-like protein by the double detection, and explored the correlation between the protein ultraviolet absorption signal and the binding metal. The result revealed that it could realize the low concentration quantitative analysis of protein based the metallic element signal, and the combination of metalloprotein and metal ion was relatively stable.Thirdly, the chemical infiltration method was applied to extract the glutathione (GSH) compound from the seaweeds, and a simple and fast method of measuring the GSH content in the frond was established by utilizing the fluorescent derivatives from the reaction of fluorescent reagent o-phthalaldehyde and GSH.This method was convenient and swift with preferable accuracy. The enrichment capacity of kelp by Cd2+ stress cultivation and the variation trend of GSH were investigated, and the correlation between the metal Cd concentration and GSH variation was studied. The result manifested that the glutathione and Cd in the frond had the positive correlation, and the contents of glutathione in the frond increased progressively with the addition of the induced concentration, but the amplitude of variation was relatively small.Fourthly, the enzymatic reaction with glutathione (GSH) as the substrate composes the polypeptide compounds with the metal inducibility. In the research, the method of marking the polypeptide compounds by the single fluorescent reagent (Monobromobimane) was established. The derivative was marked as preferable stability, and the fluorescence signal was hardly changed within a week, with the relative standard deviation of less than 5%. The molecular weight of polypeptide compounds before and after being marked were measured by utilizing the mass spectrometry, and the final marking ratio of monobromobimane and polypeptide was 1:1. At the same time, it took the lead in building the simultaneous quantitative analysis method of GSH and six compounds of PC2-PC6. Six compounds could be completely separated within 14 min-23 min. Through detecting the series standard solutions, the result showed that the linearity was good, and the detection limit of each compound was lower. Meanwhile, the contents of polypeptide compounds in different varieties of kelp were measured. The polypeptide concentration in the frond were up to mg/g level, which laied a method foundation for researching the induced response of polypeptide for the heavy metals.Fifthly, under the same condition, the single heavy metal was utilized to culture the kelp, the background values of metallothionein-like protein and polypeptide were compared, and the expression tendency and difference of the metallothionein-like protein and the polypeptide induced by different heavy metals were studied. The result indicated that the metallothionein-like protein in the kelp showed the decline trend under the single element induction of Cu and Cd, while it showed ascending trend after induction of Zn. Under the induction of multi-elements, it decreased progressively. Upon the element induction of Zn and Cd, the concentration variation range of polypeptide compounds was relatively small, and the contents of PC2, PC3 and PC4 increased. From the concentration variation trend, the variation of PC3 and PC4 laged slightly. After two elements with the same concentration inducing the seaweeds, the concentration variation trend of polypeptide compounds in the seaweeds was relatively complex. This research provides some theoretical supports for deeply researching the induced response of peptides and protein in the frond for the heavy metals in the marine environment. |
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