Inducement And Purification Of Metallothionein From Eisenia Foetida

The metal binding protein, with an induced character, can bind with metal exclusively and play an important role in adjusting metal metabolism inside organism. Therefore, it is significance to research the physical process of inducement, expression and metabolism. In this paper both inducement and purification of a group metal binding proteins, metallothioneins (MT), which perform an important founction on heavy metal inside the earthworm, are researched.According to the contrast research, a method is confirmed, which is to induce through half-immersing the earthworm in the 80mg/L CdCl₂ solution and 48h. Then research on the biology and change of the protein components for induced earthworms. The induced earthworm can survive within 48 hours, and besides, they react acutely and secrete a great bulk of yellow substance. The induced earthworm occurs a great change in a protein level, because coarse grind solution of the earthworm present 8 different protein components. Through comparing the induced group and thecontrol, relative content of the thioprotein in the former is four times higher than that in the latter. As UV-scanning illustrative plates shown, MT roughly extracted solution from induced earthworm is higher than that from the control. According to plumbago stove absorption spectroscopy, Cd content from induced protein sample is 21% higher than the control. The result indicates that it is effective to use 80mg/L CdCl₂ to induce the earthworm.The roughly extracted protein liquid is separated originally by the metal chelate affinity column combined with Cu²⁺ , and the MT is arranged in the polyacrylamid gel in term of the molecular weight. Then MT is purified by the Electro-Eluter and identified corresponding physical and chemical characters by determining molecular weight, UV-apex and metal cadmium concentration. The result indicates MT molecular weight is under 14.4kD. The absorption apex near the 400nm is slightly higher in the induced group than that in the compared group after the roughly extracted protein liquid reacts with thio- reagent. Through UV-scanning the purified sample under the acidic condition , there are two absorption apices at 190nm, which is possible to be two components of MT. The concentration from the purified and unpurified protein is generally consistent based on using the plumbago stove absorption spectroscopy to detect the metal cadmium content.