Screening, Isolation And CDNA Cloning Of Fibrinolytic Enzyme From Trichosanthes Kirilowii And Some Chinese Herbal Medicines

Modern pharmacology had proved that Chinese herbal medicines which had theeffect of activating blood and removing stasis could improve hemodynamics,hemorheology and microcirculation of blood. Fibrinolytic enzyme which can beuseful in treating some forms of thrombosis, has been isolated and purified fromanimals, microorganism and several Chinese herbal medicines, and most of thembelong to serine protease. This dissertation was focused on the studies on screeningand isolation some Chinese herbal medicines with fibrinolytic activity, and thencDNA clone of fibrinolytic enzyme from Trichosanthes kirilowii.Proteins were extracted by ammonia sulfate precipitation from 42 kinds of drugswhich were used to activate blood circulation and to remove blood stasis, and wereobserved on fibrin plate. Specific activity of Chinese herbal medicines screened wascalculated according to the standard curve of urokinase and concentration of proteins.It showed that proteins from 8 drugs had strong fibrinolytic activity on fibrin plate.Eight drugs were Trichosanthes kirilowii, Siegesbeckia pubescens, Cassia occientalis,Corydalis yanhusuo, Leonurus japonicus, Panax pseudo-ginseng, Achyranthesbidentata and Rhizoma alismatis in high to low order.The proteins of Achyranthes bidentata and Siegesbeckia pubescens wereextracted by ammonia sulfate precipitation, DEAE-Sepharose exchange chromato-graphy. There were five fractions with a potent specific activity which wereAchyranthes bidentata 40%-4, Siegesbeckia pubescens 60%-1, 60%-2, 60%-3, and80%-2.The protein extracted from Leonurus japonicus was inhibited with 8 proteaseinhibitors. The fibrinolytic activity was strongly inhibited with phenylmethylsulfonylfluoride (PMSF), soybean trypsin inhibitor (SBTI), chymostatin and benzamidine, butto a less degree with EDTA, leupeptin, aprotinin and pepstatin, indicating it may be aserine protease. The fibrinolytic protease was purified by affinity chromatography inthe diffrent concentration benzamidine which was immobilized on sepharose 4Bbeads. It showed that target proteins were effectively bound to the affinity mediumwhen the benzamidine was 380 mmol/L and fibrinolytic proteases with molecularweight of 60 and 33 kD were purified from motherwort. The crude protein from Trichosanthes kirilowii was proteolytic enzymes with apotent fibrinolytic activity, so it was called Trichosanthes kirilowii fibrinolyticenzyme extracts(TkPE). The fibrinolytic activity was inhibited by SBTI, benzamidine,and chiken ovomucoid, from high to low degree, indicating it may be a serineprotease. When the TkPE was incubated with fibrinogen and fibrin, they decreased asthe incubation time progressed. However, the TkPE did not act on bovine serumalbumin (BSA), hemoglobin, and human immunoglobulin G(IgG). It indicated that itwas relatively specific to fibrin or fibrinogen as a protein substrate. Onplasminogen-fibrin plate and plasminogen-free fibrin plate (heated at 85℃for 60 minto eliminate plasminogen), the TkPE showed different fibrinolytic activity. The resultmight indicate that its activity of plasminogen activator was stronger than itsfibrinolytic enzyme. So, it was a tissue type plasminogen activator fromTrichosanthes kirilowii.The fibrinolytic enzyme of fresh Trichosanthes kirilowii was purified by affinitychromatography on the SBTI which was immobilized on sepharose 4B beads. Theenzyme with molecular weight of 67 and 55 kD were purified from fresh Trichosan-thes kirilowii.The fibrinolytic enzyme from Trichosanthes kirilowii were separated on reversedphase chromatography, ion exchange chromatography, Sephaceral S-100 gel filtrationchromatography, and preparative electrophoresis. Furthermore, a protease labeledTkP-1 with molecular weight 62.5 kD was eluted with the gradient of NaCl from 0 to1 mol/L in 0.1 mol/L Tris-HCl (pH 7.5) and eluted with a buffer of NaCl 0.05 mol/Lin 10 mmol/L NH₄OAC (pH 5.0). The enzyme specific activity was 109.81 U/mg,which was purified 3.58-fold.The deduced amino acid sequences of several plant serine proteases have beenknown. So, the serine proteases conserved sequences were found using multiplesequence alignment of plant serine protease. The primers were framed according tothe conservative sequences. And the cDNA of Trichosanthes kirilowii was cloned byRT-PCR. A fragment of gene was obtained from Trichosanthes kirilowii.