Structural Characterization And Antioxidant And Antimicrobial Studies Of Metallothionein-like Proteins Induced In C. Vugularis Exposed To Heavy Metals

Metallothioneins （MTs） are a class of low-molecular-weight proteins which are rich in thiolgroups and heavy metal ions. MTs have many biological functions, such as antioxidant,anti-tumor, antidote and immune regulation, so scientists are interested in studying of MTs.Many researches have reported that metallothionein like （MT-like） proteins exist in microalgaecell and their contents induced by heavy metals can be markedly promoted. Based on this, themetal binding metallothionein like （matal-MT-like） proteins induced in C. vugularis exposed toheavy metals were extracted and purified, and the proteinic structural and bio-activities includingantioxidant and antimicrobial were investigated in this dissertation.This dissertation consists of six chapters. The chapter one details the structures of MTs, thephysiological function and analytical methods, and introduces the biological activity ofC.vugularis. The biological activities of C. vugularis ars also summarized.Based on the optimization of culture conditions of C.vugularis, chapter two investigatesthe influences of different concentrations of heavy metals on the algal biomass and the amountof induced metal-binding proteins. Through the orthogonal experiments, the culture conditionof C.vugularis was optimized as followings,1.0g L^-1NaHCO3,0.8g L^-1KNO3,0.02g L^-1K2HPO4,0.6mg L^-1VB1and2μg L^-1VB12in seawater. The4×107cfu mL^-1of algal biomasscould be achieved under the optimized medium. For the tests of agla growth inhibition and theinduction of metal-binding proteins, the alga were exposed to Zn²⁺, d²⁺and Cu2+（in the formsof CdCl₂, ZnCl₂, CuS0₄·5H₂O） with six concentrations of5,10,20,40,60and80μmol L^-1,respectively, for each heavy matal. And the tests were named as cadmium groups, zinc groupsand copper groups according to the exposed metal ions. Three replicates for each concentrationwere set. The results showed that both of the agal biamass and metal-binding proteins increasedwith the increase of metal concentrantions under the lower concentration ranges, the mostamouts of biamass could be obtained when the stress concentrations of Zn²⁺, d²⁺and Cu2+were20μmol L^-1,40μmol L^-1and5μmol L^-1, respectively. When the stress concentrations ofwere60μmol L^-1for Zn²⁺,60μmol L^-1for d²⁺,and40μmol L^-1for Cu2+, the most amounts ofmetal-binding proteins were induced in the algal cells. Both of biomass and induction weredecreased when the concentrations of these metals were continually increased. In chapter three, C. vugularis exposed to60μmol L^-1of Zn²⁺was cultivated for five days.After being harvested by centrifugation, the washed algal cells were homogenized by asupersonic cell disintegrator. After centrifugation, the supernatant, a crude protein-containingextract, was separated with a Sephadex G-75gel filtration column and purified with a desaltingG-25gel filtration column. The pureness were62.4%and99.5%for the crude and pure protein,respectively. The characteristic UV spectra of the proteins were analyted and found to be similarto that of the standard MTs from rabbit liver. Therefore, the Zn-binding proteins were referred asmetallothionein-like （Zn-MT-like） proteins. In addition, the molecular weight of the proteinsmeasured with Tricine-SDS-PAGE was about8.2kDa.0.2%（w/w） of purified Zn-MT-likeproteins could be obtained from the fresh alga.In chapter four, the structure of Zn-MT-like proteins was characterized with differenthyphenated analytical methods. Two of the isoforms of proteins were seperated with a DEAESephadex A-25anion exchange column detected with AFS, and the isoforms at the secondchromatograpic peak was found to be similar to that of Zn-MTs-2from rabbit liver. Three mainsub-isoforms could be separated on a reversed-phase C18column （2.1i.d.×250mm,5μm）detected with ESI-MS/MS. And the molecular weights of the three sub-isoforms were about6222.3Da,6162.5Da and6132.2Da, respectively. The analysis of amino acid compositionshowed that the content of Cys amino acid residues was15.4%for all78amino acid, andaromatic amino acid and His amino acid were found in the proteins. In additon, about two znicions were observed in one molecule of Zn-MT-like proteins detected with ICP-MS.In chapter five, the Zn-binding proteins were studied on the anti-oxidation and antimicrobialactivities. The results showed the concentrations of50%elimination rates of Zn-MT-likeproteins were13.12mg/L,2.23mg/L and48.59mg/L for hydroxyl radical, DPPH radical andsuperoxide anion radical, respectively. The scavenging effects on three radical were much higherthan those of GSH and Zn-MTs. Base on the agar hole pervasion method, the activities ofanti-bacterial and anti-fungal of the Zn-MT-like proteins were tested and compared with those ofthe standard Zn-MTs from rabbit liver. Results showed that the Zn-MT-like proteins stronglyexhibited the anti-microbial activities against gram-positive bacteria （including Staphylococcusaureus and Bacillus subtilus）, Penicillium chrysogenum and Aspergillus niger, but wasinsensitive to Escherichia coli and brewer’s yeast. The diameters of inhibition zone for1mg/mLof Zn-MT-like proteins were13.2mm,17.4mm,7.2mm and9.8mm for Staphylococcus aureus,Bacillus subtilus, Penicillium chysogenum and Aspergillus niger, respectively. However, no anyanti-microbial activity was found for the standard Zn-MTs from rabbit liver. In addition, thebound znic in the Zn-MT-like proteins colud be removed at low pH （2.0） to form theapo-MT-like proteins. However, the apo-MT-like proteins could easily bind copper or selenium in neutral medium to form Cu-MT-like or Se-MT-like proteins.