The Extraction And Purification And Antioxidant Activity Analysis Of Metallothionein From Scapharca Subcrenata

Metallothionein（MT）is a class of low molecular weight multi-functional induced protein,which is rich in Cys,has metal binding properties and widely distributes in animals,plants and microorganisms.At present,MT has become a research hotspot in environmental science,bio-toxicology and medicine.Domestic and foreign scholars have studied various extraction and purification methods of metallothionein and corresponding analytical techniques,but there is no international standard method for the separation and quantitative analysis of marine animal MT.In order to provide a theoretical basis for the quality control and functional evaluation of marine active substances.In this study,the marine source Scapharca subcrenata was selected as the research object.The high-efficiency extraction and purification technology of the active component metallothionein in the Scapharca subcrenata and its antioxidant activity was studied.The main research contents are as follows:（1）Raw material of extracting metallothionein:Scapharca subcrenata was selected as the research object.The Scapharca subcrenata is a filter-feeding bivalve shellfish,which is easily enriched in heavy metals in the body.It is ideal raw material for extracting metallothionein and widely distributed in China.In this study,Zn²⁺was used to induce the production of metallothionein in the Scapharca subcrenata body,and the extraction method of metallothionein in the Scapharca subcrenata was optimized.The content of Zn²⁺in the Scapharca subcrenata was increased from 21.84mg/g to 72.40mg/g.The MT content increased from 0.39 mg/g to 0.65 mg/g,and the induction effect was significant.（2）Study on the extraction process of metallothionein from Scapharca subcrenata:Tris-HCl buffer with stable properties and good compatibility with physiological fluids was used as extractant,and MT was extracted by centrifugation.According to the results of single factor experiment,and Box-Behnken experimental principle,the response surface methodology was performed in this experiment:when the extraction buffer pH is 8.45,the buffer concentration is 0.18 mol/L,the extraction temperature is 35°C,the material ratio is1:6,the extraction time is 30 min,the optimum extraction effect of MT in the Scapharca subcrenata tissue is obtained,and the average extraction content is 0.69 mg/g.（3）Optimization of separation and purification technology of metallothionein in Scapharca subcrenata:Firstly,the optimal UV-detection wavelength of Zn-MT in Scapharca subcrenata was determined by protein purifier.The MT were separated from the hybrid proteins by Enrich SEC 70 high-precision molecular sieve pre-packed column,and eluted with 0.01 mmol/L Tris-HCI buffer solution at a flow rate of 1.00 mL/min.The preliminary purification target MTs were obtained.The extraction rate was 37.96%and the purity is 92.43%;The MTs obtained by the above method were subjected to DEAE anion exchange chromatography,and eluted with a gradient of 0.50 mol/L NaC1 buffer solution at a flow rate of 1.00 mL/min to separate the MT-Ⅰ and MT-Ⅱ components in the Scapharca subcrenata;The purified MT-Ⅰ and MT-Ⅱ components were desalted and the final product was collected.The extraction rate of MT-Ⅰ was 43.66%,the purity was 95.12%,the extraction rate of MT-Ⅱ was 45.83%,and the purity was 95.56%.Quantitative analysis of proteins based on the characteristics of metallothionein-binding metal elements and an accurate and efficient double detection method for metallothionein were established.ICP-MS was used to detect the signal value of zinc ion in metallothionein and UV detection was used to distinguish different MT in different purification stages.（4）Determination of the molecular weight of metallothioneins in the Scapharca subcrenata.The metallothionein in the Scapharca subcrenata is relatively close to the molecular weight of the metallothionein in the rabbit liver,which was determined by Tris-Tricine-SDS-PAGE gel electrophoresis.The relative molecular weight of the preliminary purified product MTs was 17.79 kDa,and the relative molecular weights of MT-Ⅰ and MT-Ⅱ isolated by anion exchange chromatography were 17.63 kDa and 17.16kDa,respectively.The relative molecular weight of rabbit liver Zn-MTs was 18.12 kDa.（5）Antioxidant activity of different stages of purification of metallothioneins:The experimental results show that the metallothioneins obtained in different purification stages have good antioxidant capacity（T-AOC）compared with Vitamin C（Vc）.Metallothionein has a strong ability to scavenge hydroxyl radicals and DPPH radicals.The total antioxidant capacity of the MTs obtained by preliminary purification was 4.25 U/mg,the total antioxidant capacity of MT-Ⅰ was 3.30 U/mg,and the total antioxidant capacity of MT-Ⅱ was 3.21 U/mg.The order of hydroxyl radical scavenging rate of 50 ug/mL protein solution was:MTs（70%）>Vc（62%）>MT-Ⅰ（57%）>MT-Ⅱ（51%）.The order of DPPH·clearance rate of 60 ug/mL sample solution was:MTs（89%）>Vc（78%）>MT-Ⅰ（56%）>MT-Ⅱ（50%）.