Extraction Of Zinc-binding Metallothionein-like Proteins Induced In C.Vugularis And Its Chelation To Cadmium

Metallothionein（MT）is a class of low-molecular weight non-enzymatic proteins capable of binding to metal ions.At present,the extraction yield of commercial metallothioneins from animal origin is low and the price is expensive.Therefore,it becomes a research focus to develop more economical raw materials to extract metallothioneins.Metallothionein-like proteins which have the similar structural features of metallothioneins can be produced from C.vugularis through the induction by zinc ions.Based on this,in this dissertation,the zinc-binding metallothionein-like proteins were extracted and separated from C.vugularis through Zn²⁺induction,and the chelating effects of the proteins to cadmium were conducted.The main research work is described as follows:In the first part,C.vugularis was cultivated under zinc stress,the effects of zinc stress on the synthesis of metallothionein-like proteins in the algal cells were investigated through the tests of different zinc salts（zinc chloride,zinc acetate and zinc gluconate）,different Zn²⁺concentrations(30-120μg mL^-1)and different stress time（0-6 d）.The results showed that the the highest induced amount of zinc-binding protein could be obtained when C.vugularis cells at mid-log phase were stressed by zinc chloride at a Zn²⁺concentration of 60μg mL^-1 for 3 days.The cell disruption conditions of C.vugularis were optimized for the protein extraction.Results showed that the break rate of algae cells up to 76.03%could be reached through three freeze-thaw cycles combined with ultrasonic cell-break.The crude protein extracts were separated and purified by Sephadex G-75 and G-25,respectively.And 31.1 mg of zinc-binding metallothionein-like proteins was obtained in 1 g of alga mud.After purified,the protein purity was 98.02%and the mercapto content was 1,228.76μmol mg^-1.The proteins present the typical characterization of metallothionein by infrared spectrum and circular dichroism.Each protein molecule combined with 7 zinc atom and consists of 80 amino acids,with 12 cysteines accounting for 15%of the total amino acids.In the second part,the chelating effects of C.vugularis zinc-binding metallothionein-like proteins to Cd²⁺were investigated by means of ultraviolet absorption spectroscopy combined with high performance liquid chromatography-inductively coupled plasma mass spectrometry（SEC-ICP-MS）.The binding zinc could be dissociated from the C.vugularis metallothionein-like proteins to form apo-metallothioneins under acidic media,and the apo-metallothioneins could be re-bound to zinc forming zinc-binding metallothionein-like proteins when the medium pH was re-adjusted to neutral and weakly alkaline condition.At the same time,the binding effect of Cd²⁺to C.vugularis metallothionein-like proteins was studied based on the SEC-ICP-MS method.It was found that cadmium could replace zinc-binding metallothionein-like proteins to form cadmium-binding metallothionein,and the chelation effect of Cd²⁺increased with the increases of the concentration of Cd²⁺.In the third part,the chelation of C.vugularis metallothionein-like proteins to Cd²⁺in a simulated digestive tract environment was investigated based on the dialysis method.In the environment of simulating gastric and intestinal digestive juice,C.vugularis metallothionein-like proteins were placed in the dialysis bag,and the chelation of metallothionein-like proteins on Cd²⁺with different concentrations in the gastrointestinal fluids outside the dialysis bag was investigated.Meanwhile,the effect of different protein concentrations and chelating reaction time on the chelating rate was also investigated.The results showed that the chelation of C.vugularis metallothionein-like proteins to Cd²⁺in the simulated intestinal fluid environment was stronger than that in the gastric juice environment.The chelating rate was 38.62%for 10μg mL^-1 of C.vugularis metallothionein-like proteins to 50 ng L^-1 Cd²⁺.Therefore,the extracted C.vugularis metallothionein-like proteins have a strong chelation effect to Cd²⁺,which is expected to be a bio-detoxification reagent for the heavy metal cadmium.