Purification, Characterization And Pharmacodynamic Activity Of Proteases And Its Isoenzyme From Nereis Virens

Nereis virens (N-V) protease, a kind of proteolytic enzyme in Nereis succinea, had been extracted successfully in our laboratory. N-V protease is able to hydrolyze fibrinogen and fibrin significantly both in vivo and in vitro which can be developed a de-fibrin & thrombolytic drug and can be utilized in clinic as a therapeutic approach for heart and brain infarction, for treating and preventing heart and brain thrombus diseases. We have claimed patent for this protease with the application number 02144828.0, and patent number 1500873. We have also submitted it to Swiss protein with the ID P83433. It was found in our research that N-V proteases are a group of isoenzymes which mean they share same function, similar properties of enzymology and cross-reacting antigen determinant but have structure difference. They possess the pharmacodynamic avtivities to lower the fibrin, to counteract platelet aggregation, to amendment hemorrheology and to dissolve thrombus which makes it possible to be developed into a new thrombolytic drug. The study is not only a rational research but also an application research; some advanced techniques and methods are applied in this study. We studied isolation and purification process of the N-V protease; its biochemistry characteristics and pharmacodynamic avtivities. The first part is the study on purification of N-V Protease. Ammonium sulfate salting-out, Sephacryl gel filtration chromatography (GFC) and hydrophobic chromatography of phenyl Sepharose techniques were used to separate and purify the N-V protease; optimized salting-out and chromatography experimental conditions were reported in this part. Polyacrylamide gel electrophoresis (PAGE) was adopted to purify the enzyme; dialysis and Sephadex G-25 gel filtration techniques were used to desalt; ultrafiltration and Sephacryl S-200 GFC were used to remove pyretogen; bacterial endotoxin detection and rabbit were used to test pyretogen. In order to search for a better freeze-dry technique, we compared two pre-freeze methods: quick freezing and slow freezing, and analysised the temperature of sublimation and the time of resolution to map freeze drying curve. Fibrin plate method was used to detect Neresis protease fibrinolytic activity in vitro, and ultraviolet absorption & Bradford methods were applied in order to definite the content of protein.The second part of this paper is the study on characterization of N-V protease and its isoenzymes. The purity of N-V protease was identified by PAGE, immobilized pH gradient isoelectric electrophoresis (IPGIEF) and gel filtration high performance liquid chromatography (HPLC) ; relative molecular weight was determined by using high-performance liquid chromatography - mass spectrometry (LC / MS) and structure was analysised by detecting the peptide mass fingerprint (PMF) with MALDI-TOF-MS mass spectrometer; Amino acid sequence was preliminarily definited by N-terminus analysis of amino acid sequence; polyclonal antibody was harvested from immunifaction rabbit, antibody titer and antigenicity was determined by antigen-antibody reaction; the optimum reaction temperature and pH were definited by fibrin plate, and four kinds of specific protease inhibitors were used in order to determine classification of N-V protease. Fibrin plate which contain agarose as supporting dielectric with and without plasminogen were used to analysis its fibrinoclase and kinase activities.The third part of this paper is the study on pharmacodynamic avtivity of N-V protease. Fibrin plate was adopted to assess valence and the fibrinolytic bioactivity of N-V protease in vitro. The fibrin plate method was used in order to assess N-V protease activity on hydrolyzing fibrin in vitro; the content of Fg and D-Dimer, ELT were assayed to study the effect of N-V protease on fibrinolysis in normal rabbits; PT, APTT were measured to observe the effect of N-V protease on blood coagulation function. Adenosine diphosphate (ADP), arachidonic acid (AA) and collagen (CG) were used to induce platelet aggregation in rats and the maximum ratio of platelet aggregation was detected.The hemorheological effect was observed by building the model of hyperviscosity blood syndrome in rats with high molecular dextran. The effects on antithrombus formation were observed by thrombosis experiment of rats and rabbits in vivo and in vitro.The experimental results are as follows:1. Purification of the N-V protease.By the methods we used to purify the N-V protease, the protein concentration and fibrinolytic activity in raw material of Nereis succinea, fractions from ammonium sulfate salting-out, Sephacryl gel filtration and phenyl chromatography showed that the recovery of N-V protease reached 75% and the specific activity was increase by 60-fold. Five different pre-installed chromatographic columns were used to choose hydrophobic medium. By analysising PAGE results and assaying content and activity, it showed that Phenyl Sepharose HS is the best purification media. By optimizing the elution conditions, it was showed that the optimal concentration of ammonium sulfate was 1.5mol·L-1.By comparing desalting methods, dialysis and Sephadex G-25 gel filtration chromatography , the latter was the final choice because it could deal with large samples and be monitored easily; By comparing the ultrafiltration and Sephacryl S-200 gel filtration chromatography to remove the pyretogen, ultrafiltration membrane with the molecular weight of 100kD was chose. pyrogen was detected by bacterial endotoxin test and poyrogen test, pyrogen test is a standard method, and it is generally considered that negative bacterial endotoxin test result was valid, positive bacterial endotoxin test result needs to be rechecked by pyrogen test, those with negative results in pyrogen test could be administrated clinically. Slow freezing method was optimal, sublimation temperature was–5℃and the time of resolution was 4 hours.2. characterization of the N-V protease and its Isoenzymes.Three strips were showed clearly on polyacrylamide gel. Among them, stripⅡwas found to be the major product (50%) , StripⅠandⅢwere minor band (30% and 20% respectively) by the analysis of the density scan. The isoelectric focus electrophoresis showed three strips, which exhibited the isoelectric point of the N-V protease point at 3.5 -4.5. No significant change of isoelectric point was observed among three components. HPLC analysis of the N-V protease revealed three major peaks. The total integrated area was 95.24%. Three fractions were collected and their activities were examined. The results showed that all of them possessed fibrinolytic activity. Purity of the N-V protease reached 95% by HPLC analysis, which was consistent to PAGE results. The molecular weight of three compositions was 29248.75D, 29007.66D, and 28954.17D, determined by mass spectrum (MS). The PMF confirmed that the three compositions were new proteins without having been known the sequences. The structure of two of them (componentSⅡ,Ⅲ) was resemble and had high homology, and the other was different in peptide finger printing map. ComponentsⅡ,Ⅲhave identical immunogenicity, the other is different from them. But three compositions have identical antigen determinant group which was proved by antigen-antibody reactions. Results of the analysis of the amino acids sequences for the ten peptide sections are as follows: NVVAVK, INL, QAPNYSTASY, FLSTNNK, LYIHDTGVR, AVYLAGMK, NFPNYYINLY, VYLAANPTASS, QTFNSDTL, VYILDTGI, it is suggested that the N-V protease is new protease by alignment of amino acid sequence, this conclusion is uniform with PMF. The N-V protease is a kind of serine proteinase according to the analysis of the amino acid sequence and enzyme inhibitor experiments. It possess both fibrinoclase and kinase activity, but the main function is direct fibrinolysis. The optimal temperature of the protease is 50℃, the optimal pH is 8-9.The enzymology properties of three components of N-V protease are similar. ComponentⅠshares same function, similar enzymology properties, homology and variability in structure, and has different antigenicity with componentsⅡ,Ⅲ, which indicates they are isoenzymes.3. Pharmacodynamic avtivity of the N-V protease N-V protease possessed the fibrinolytic activity in vitro and vivo proved by its function of decreasing the content of Fg, D-Dimer and shortening ELT without influence on the APTT, PT, and the effects on blood coagulation function are not observed. It was found that N-V protease inhibited platelet aggregation with different revulsant in rats and reduced the maximum aggregation rate; N-V protease reduced whole blood viscosity, and had no effects on plasma viscosity, blood sedimentation rate, and volume of packed red cells in rats; N-V protease could effectually inhibit the thrombogensis of rats in body and in vitro, the thrombogensis of rabbits in vitro. N-V protease possesses strong effect against platelet aggregation, improves hemorheological characteristics, and exerts remarkable effect against thrombosis.