Chemical Modification Of The Earthworm Fibrinolytic Enzymes

Earthworm fibrinolytic enzymes (EFE)is a kind of serine type protease, which can hydrolyze fibrin.Because of its strong ability to hydrolyze fibrin,EFE can be used as a medicine to treat thrombus desease. In the study,EFE was separated and purified from Eisenia faetida.Then EFE was chemically modified with big molecule,orEFEwas hydrolyzed restrictedly. Furthermore,serverl conditions of modifying reaction arid the character of the modified EFE were also studied .Through autolyzation,ethanol fraction precipitation,DEAE-cellulose ion exchange chromstography and ultrafiltration,EFE was purified by 6.67 folds with a specific of 2.29 104U/mg and a yield of 17.0%.The reactive conditions for EFE modified were optimized.The optimum conditions for EFE modified with PEG1 are as follows: EFE (1.0mg) was dissolve in 2.0ml of 0.1M sodium tetraborate, pH9.2. the solution was brought to 37 and activated PEG(25.5mg) was added .afterlh,the activity of modified EFEis 56.6% of free EFE,and the modification rate of amino residues is63.2%.Theoptimum conditions for EFE modified with Dextran are as follows: EFE(l.Omg) was dissolved in 2.0ml of 0.1M sodium tetraborate,pH8.4 . the solution was brought to 25 and activated Dextran (152.7mg) was added .after 18h,the activity of modified EFE is 27.4% of free EFE,and the modification rate of amino residues is 58.5%. The optimum conditions for EFE hydrolyzed by immobiled trypsin are as follows: EFE (l.Omg)was dissolved in 2.0ml of 0.1M'phosphate,pH7.4 . the solution was brought to 45 and immobiled trypsin 0.3 5g was added .after lh,the activity of modified EFE is 63.6% of free EFE, and the modification rate of amino residues is 72.1%.In addition ,the characters are compared between the modified EFE and the native EFE. And the results indicate that the affinity of PEG-EFE to casein is the higher than that of native EFE .Further more ,the ability is improved in counteracting hydrolyzation of trypsin to EFE and the thermal stability of PEG-EFE is enhanced.the Dextran-EFE has the similar changes as the PEG-EFE. The binding ability of PEG-EFE with antiserum retains 25.0% of that of native EFE and DEX-EFE is 32.5%. whereas the affinity of EFE to casein reduces when EFE was hydrolyzed by immobiled trypsin. and the thermal stability of hydrolyzed EFE is lowered. The binding ability of hydrolyzed EFE with antiserum retains 42.0% of that of native EFE.