Purification And Biochemical Characterisation Of A Novel Fibrinolytic Protein,PSLTro02,from A Medicinal Animal Porcellio Scaber Latreille

?Compendium of Materia Medica?records insect drugs mostly with blood circulation,pass through effect of shuluo.Modem research shows that play such pharmacological effects of substances,mostly proteins or polypeptides,Fibrinolytic proteins such as turtle,scorpion enzyme anticoagulant polypeptides,single unicinctus plasmin.In the composition of animal drugs,the proportion of very large proteins,such as venom protein content within 90%,centipede body protein content of 60%to 70%,deer blood protein content of 70%to 80%,while the protein content of ants also have 42%to 67%.Scorpion Scorpion hydrolysis anticoagulant polypeptide from the extract obtained in vivo anticonvulsant,antiepileptic and analgesic sedative effect,Modem research shows that acceptable for the treatment of cancer and other malignant diseases;extracted from the earthworm in the PPA has fibrinolytic effect.Isolated and purified from natural medicine having a high fibrin-specific plasminogen has become one of the hot current study.Our group has been successfully isolated from traditional Chinese medicine Armadillidium Armadillidium vulgare Latreille creampie Porcellio plasmin,its relative molecular mass of 3.849 ×10⁴,preliminary study found that its enzymatic properties,the optimum temperature for plasmin Porcellio 40?,optimum reaction pH of 7.0;p-toluenesulfonyl chloride-L-lysine methyl ketone?TLCK?,ethylenediaminetetraacetic acid?EDTA?,ethylene glycol diethyl ether diamine tetraacetic acid?EGTA?,benzene methanesulfonam phthalocyanine fluorine?PMSF?inhibition is relatively weak,and the remaining plasmin activity were higher than 75%,while aprotinin,pepstain A murine women plasmin fibrinolytic activity is completely inhibited;And its orally active.To make it play a wider range of uses,this experiment simulated gastrointestinal environment Porcellio enzymatic activity of crude protein was obtained after separation of the smaller molecular weight,high potency protein or polypeptide.Pepsin and trypsin enzymatic extraction is commonly used two kinds of proteases.Pepsin?pepsin?belong to aspartic acid proteolytic enzymes,proteolytic enzyme digestion in the stomach,relative molecular mass of3.1 ×10⁴?3.6 × 10⁴,which is a precursor pepsinogen.Trypsin?trypsin?is a protease,the relative molecular mass of less than 2.4 × 10⁴,is extracted from cattle,sheep,pig pancreas a serine protease.Proteomics analysis,trypsin is the most widely used protease,since many proteins rich in lysine and arginine,thus after trypsin fragments obtained by amino acid arrangement is suitable for mass spectrometry analysis of proteins.In this paper,Porcellio for the study,the first attempt to Armadillidium fibrinolytic crude protein digestion in simulated gastrointestinal environment,and hydrolysates having fibrinolytic activity was isolated and purified.Consists of four parts,the first part of the second chapter,the impact of pepsin and trypsin factors and fibrinolytic activity of fibrinolytic activity Deactivation;the second part is the third chapter,Armadillidium fibrinolytic activity in crude protein analog the third part is divided into chapters IV and V,separation and purification procedure introduces Porcellio plasmin PSLTro02 and enzymatic properties of PSLTro02;enzymatic gastrointestinal environment Chapter ? fourth part is about the Porcellio plasmin PSLTro02 part vivo pharmacodynamics.Specific conclusions are as follows:1.Studied pH,temperature,metal ions,inhibitors and surfactants four factors in simulated gastrointestinal environment pepsin and trypsin fibrinolytic activity to fibrin plate for the detection of targets,the more successful best of enzyme inactivation process.In order to make better enzyme inactivation process can be more widely applied,further studies preferred enzyme inactivation process on Armadilhdium currently on the market and commonly used Lumbrokinase urokinase?and Our previous separation and purification such as fiber obtained fibrinolytic activity of plasmin PSLTro01 and optimal inactivation process,That is the premise of maintaining maximum stability of urokinase plasminogen under PPA and Porcellio make fibrinolytic activity of pepsin and trypsin effectively inactivate the optimal process:pepsin optimal solution for inactivation of pH 6.0?8.0;trypsin inactivation optimal solution for the mass concentration of 25 mg/mL TLCK and concentration of 1 mmol/L EDTA mixed preparation.2.Study Porcellio fibrinolysis crude protein in simulated gastrointestinal digestion process environment.First,compare before and after enzymatic hydrolysis in vivo pharmacodynamic part,fibrinolytic activity of enzymatic hydrolysates after slightly stronger than the crude protein digestion before.the main role in pre-digested crude protein coagulation process intrinsic coagulation ways and common coagulation pathway,delayed clotting time.and with the active protein concentration increases,the anticoagulant effect of the stronger.After enzymatic hydrolysates were acting on coagulation process intrinsic,extrinsic and common coagulation pathway,and the results are better than the crude protein group.And to explore the environment in a simulated gastrointestinal enzymatic activity on the process conditions effective parts,respectively,from the time of pepsin,trypsin hydrolysis time,hydrolysis temperature.enzyme-substrate ratio of four factors orthogonal experiment design,hydrolysis obtained optimum process:enzyme-substrate ratio of 1:1,pepsin time 4h,trypsin time 4h,reaction temperature 40 C.Finally.under the optimal hydrolysis conditions were compared before and after digestion SDS-PAGE gel electrophoresis band changes,the discovery of new substances in the molecular weight of the segments are.3.Mainly for crude protein Armadillidium fibrinolytic activity through artificial simulated gastrointestinal environment enzymatic hydrolysates resulting PSLTro02 separation and purification,primarily through HiTrap Capto Q?100mL?ion exchange chromatography,Sephacryl S-200 HR,purification methods Superdex 75 pg gel filtration chromatography purification step,etc.,and finally through Amicon???ultra-15?3K,5K and 10K?and concentrated by ultrafiltration centrifugal tube,and purifying the resulting hydrolyzate?PSLTro02?molecular characteristics?including purity,molecular weight,amino acid sequence?were expressed.HPLC purity test the purity of 93.86%.4.Characterization studies showed that the optimal reaction pH PSLTro02 7.0,stability in neutral and alkaline environment of higher stability in acidic environments;optinum temperature PSLTro02 is 40?,20? environment,can be stored for long periods,after higher than 50?,PSLTro02 can not be stored for long periods;Na⁺,K⁺,Mg²⁺has no effect on PSLTro02 fibrinolytic activity,Cu²⁺,Mn²⁺,Fe²⁺,Ca²⁺,Zn²⁺,Al³⁺,Fe³⁺are different the degree of inhibition of fibrinolytic activity,especially Fe²⁺,Fe³⁺ inhibited its activity;PepstainA,KTI,SBTI,Aprotinin at each concentration of fibrinolytic activity PSLTro02 large,the concentration of PMSF,TLCK,TPCK,EDTA,EGTA,SDS the basic PSLTro02 fibrinolytic activity was not affected;PSLTro02 specific hydrolysis of the substrate is S-2251,has the characteristics of the tissue-type plasminogen;pepsin and trypsin inactivation optimal conditions also apply to PSLTro02 fibrinolysis enzymes.5.Vivo pharmacodynamic experiments and found PSLTro02 fibrinolytic activity and anticoagulant activity are slightly stronger than PSLTro01.