Nereistoxin Detection In Purification Process Of N-V Proteinase

Summary:Cardial and cerebral vascular thrombosis disease with its high morbidity will lead to high mortality rates and disability. Clinically, the principal method of clinical treatment is to cut down fibrinogen in the blood in order to reduce deaths and disability and to improve the quality of life. It is an important topic to study and research this kind of drug in the medicine domain. Nereis virens (N-V) proteinase, a kind of proteolytic enzyme in Nereis succinea, had been extracted successfully in our laboratory. N-V proteinase is a kind of aminopeptidase with plasminogen and plasminogen activator activity, which can be developed for defibrase and thrombolytic drugs as a therapeutic approach for heart and brain infarction, for treating and preventing heart and brain thrombus diseases. This thesis reported the purification process of the N-V proteinase.N-V proteinase is a special kind of protein which uses for medical therapy. The key point of purification is the detection of purity of the protein and pollutants in it. There is a S-N compounds in nereis, known as nereis toxins(NTX), which is often used as a pesticide. NTX is a kind of poisonous impurities that may contain in N-V proteinase. We must detect NTX contained in raw materials, different samples from the course of purification and N-V proteinase.This paper summarizes the experiments, erected a series of methods that can detect NTX contained in N-V proteinase, and detected different samples from the course of purification. At present, the purification technology of N-V proteinase is more mature. So we use different methods to detect the quantity of NTX contained in proteinase, in order to monitor the effect of extraction process to remove impurities for further improve of the purification technology. And to prepare conditions for the declaration of the new drug. Experimental research:1. Summary of purification of N-V proteinase.Salting-out and chromatography were used to purify N-V proteinase. Dialysis was adopted to desalt. And PAGE proves the high purity of N-V proteinase in the course of time.2. Detection of nereistoxin in N-V proteinase agent(1) ultraviolet and visible spectrophotometryUV wavelength scan showed that the maximum absortion of NTX in methanol is 210nm. We detect the different concentrations of the standard solution in this wavelength, and calculated linear regression equation:y=0.0320x+0.0199,n=7,R2=0.9975. The detection limit was 0.125μg/mL.But during the further detecting of N-V proteinase samples, we find there are still some impurities that affect the results, which make them do not match with the facts. So this method can measure the content of NTX in standard substance exactly. But it is not suitable for NTX in N-V proteinase.(2) thin layer chromatographyWe use high-performance thin-layer silica gel G thin layer chromatography plate detection to detect NTX. The developing agent is methanol: acetoacetate: water (5:4:1), the chromogenic agent is calcein-palladium chloride. The detection limit is 0.5μg/mL.After extraction, a great deal of NTX can be detected in the raw materials. The content of NTX in the extract decreased with the continuous extraction process, especially after the second time of chromatography. No NTX is detected in N-V proteinase agent finally. This method is simple. Only UV lamps is needed, and just required few of samples. This method is sensitive and can rapid qualitative analysis of the existence of a small number of NTX, but not specific quantitative tests. It should be combined with other method to test the exact content NTX.(3)High-performance liquid chromatographyNTX is a kind of free amine with small molecule, which is difficult to retain in the C8 column. So we chose C18 reverse column for detection. According to the characteristics of C18 column, methanol and water were chosen as the mobile phase. And through pre-test, and ultimately determine the column temperature was 20℃, flow rate 1ml/min, detect wavelength 227nm. We detect the different concentrations of the standard solution in this wavelength, and calculated linear regression equation: y = 1.7707x + 22.182, n = 9, R2 = 0.9996. NTX minimum detection limit is 0.06125μg/mL, the accuracy rate was 95.6%—97.4%.High-performance liquid chromatography is sensitive, rapid, high resolution, reproducible and less demanding on the equipment. It is an effective way to detect NTX in N-V proteinase Experimental resultN-V proteinase has been purified and extracted after salting-out and chromatography. After extraction, a great deal of NTX can be detected in the raw materials. The content of NTX in the extract decreased with the continuous extraction process, especially after the second time of chromatography. No NTX is detected in N-V proteinase agent finally. The method, thin layer chromatography and high-performance liquid chromatography, can detect NTX in samples fast and sensitively. Particularly high-performance liquid chromatography can detect the quantity of NTX accurately.This paper summarizes the experiments, erected a series of methods that can detect NTX contained in N-V proteinase We use different methods to detect the quantity of NTX contained in proteinase, in order to monitor the effect of extraction process to remove impurities for further improvement of the purification technology. And to prepare conditions for the clinical application of the new drug.