Study On The Isolation And Purification Of Natrual Hirudin From Leech

Hirudin, as a novel anticoagulant and antithromotic agent, is a kind ofprotein that is obtained from the salivary gland secretion of one kindblood-sucking leech. Now the scientists all over the world have intensivelyinvestigated the isolation and purification of hirudin. But the leech was killed inorder to obtain hirudin in most methods of extracting hirudin from leech in thesepapers and patents. This will increase the cost. So hirudin's clinical useremained limit because this substance was not available in adequate amounts fortherapeutic purpose.The three methods of salting-out with (NH₄)₂SO₄ saturation, precipitatingwith organic impregnant and changing pH of protein in isolating and purifyinghirudin were studied firstly, and it is certain that the method of ammoniumsulfate salting-out to isolate hirudin is the primary step. The results of severaltimes centrifuge and two times centrifuge showed that the range of 35%～45%(NH₄)₂SO₄ saturation (W_a) was the suitable range for natural hirudin salting-out. And meanwhile, it confirmed that the optimum treatment to salting-out inorthogonal design: 40%(NH₄)₂SO₄, pH 5.5, the time of deposited is 20 rain, herethe.yield of protein(W_p)=34.37%, the yield of hirudin (W_h)=69.33%, purificationfold (R_hp)=2.02, special activity (S_p)=5.33 AT-U/mg.Then, separately followed by an anion exchange chromatography on DEAESephadex A-50 and DEAE Sepharose CL-6B, tow batches of clarifying hirudinsolution can be obtained. And through comparing the result, it can conclude thatthe anion exchange chromatography on DEAE Sepharose CL-6B is better thanthe anion exchange chromatography on DEAE Sephadex A-50 in isolation andpurification of native hirudin with special activity; yield, purification(fold). Inthe experiment of DEAE Sepharose CL-6B ion exchange chromatography, withthe conditong of 100mL(10mg) volumes of sample, the eluted velocity of30mL/h, 0.025mol/mL phosphate buffer and 0～1.0mol/L NaCl are separatelyconsidered, then the yield of protein(W_p)=5.07%, the yield of hirudin(W_h)=77.76%, purification fold(R_hp)=15.34, special activity (S_p)=191.69 AT-U/mg.Also, in the experiment of desalting by Sephadex G-25 Gel filtration, the elutedvelocity 30mL/h are considered. The protein concentration=0.295μg/mg, yieldof protein(W_p)=0.27%, the yield of hirudin(W_h)=47.88%, purification fold(R_hp)=8.73, special activity(S_p)=1017 AT-U/mg.At last, it is isolated and purified by reverse-phase HPLC, gathering thepart of activity apex that its purity is more than 90 percent. Otherwise, the finalproducts of hirudin were identified by SDS-PAGE, RP-HPLC. Natural hirudin which has high activity, steady configuration, uneasilylosing activity and low cost is an ideal material for anticoagulant drugs. In thefollowing study, it had been prepared for freezing, desiccating and identifyingfurther.