| Centipede comes from the arthropoda chilopoda plastic mesh centipede Branch, with the effects of xifengzhenjing, tongluozhitong, gongdusanjie and so on. There are many species of centipede, such as Scolopendra subspinipes mutilans L. Koch(mainly located in Jiangsu, Hunan, Hubei, Zhejiang and Sichuan), Scolopendra subspinipes mutilans L. Koch(mainly located in Hubei, Guangxi, Guangdong), Scolopendra mojiangica Zhang et Chi (mainly located in Yunnan) and Scolopendra negrocapitis Zhang et Wang(mainly located in Yunnan).Centipede mainly contains proteins, amino acids, fatty acids, trace elements and so on. Wherein proteins are the main ingredient in centipede, and pharmacological study has also found that the proteins in centipede have the activities of anticoagulant and anti-tumor. But up to now determination method of the proteins in centipede is almost based on kjeldahl, and there has no research on the identification method of proteins in centipede. Therefore, in order to provide a methodological basis for the study of active proteins in centipede and proteins in other animal drugs, quantitative and qualitative methods for proteins in centipede were established.The research work includes the following aspects:1 Study on quantitative methods for proteins in centipedeTo compare Bradford method, Lowry method and BCA method for the determination of protein content in centipede, SDS-PAGE method was used. Some false positives were found during the determination of proteins in centipede by Lowry and BCA methods. Bradford method was suitable for the quantitative analysis of proteins in centipede.Bradford method was chosen for the quantitative analysis of proteins in centipede and the methodology were investigated. It is found that the linear range was within 0.1671-1.0024 mg/mL, r= 0.9999 and the recovery was within 94.15%-99.41%. Protein content of different varieties and different areas centipedes were determined. The results showed that protein content of different varieties and different areas centipedes were different. Among them protein content of Scolopendra negrocapitis Zhang et Wang was higher then other varieties of centipedes, protein content of Scolopendra mojiangica Zhang et Chi was lower oppositely.2 Study on the qualitative methods for proteins in centipede by SDS-PAGESDS-PAGE method was used to analysis the proteins in centipede. The amount of proteins and qualitative analysis of proteins from different varieties and different origins of centipedes were studied. The results showed that protein bands with the amount from 7 to 14 μg can basically be showed on SDS-PAGE. According to SDS-PAGE, it was found that depth of protein bands basically had a positive correlation with the protein content; molecular weight distribution of proteins in centipedes from different varieties and origins were consistent, and mainly distributed around 70 kD,130 kD,120 kD,100 kD and 70 kD.3 To establish nanoLC-MS/MS method for the qualitative analysis of proteins in centipedeNanoLC-MS/MS method was established for qualitative identification analysis of proteins in centipede. NanoLC-MS/MS method coupled with SDS-PAGE method were developed for analyzing gel-based and gel-free digestion products of proteins in centipede and proteins database was used to identify proteins. NanoLC separation was performed on a Chrom XP CL enrichment and Chrom XP-C18 capillary column using a gradient elution program of 2% acetonitrile solution(containing 0.1% formic acid) and 98% acetonitrile solution (containing 0.1% formic acid) as the mobile phase with a sample flow rate of 5 μL/min and flow rate of 300 nL/min and the injection volume of 8μg. The analysis time was 90 min. Q-TOF-MS and ESI were applied for qualitative analysis of proteins in centipede under the IDA mode. Experimental data obtained from MS analysis were retrieved by ProteinPilot protein analysis software in arthropoda protein database (Arthropoda.fasta) download from uniprot.73 kinds of proteins from gel-based method and 96 kinds of proteins from gel-free method were identified, both containing 26 kinds of common proteins. Based on the gene ontological analysis, the proteins identified in centipede were differentially displayed according to cellular component, biological process and molecular functions. The function of proteins included binding activity, enzyme catalytic activity, motor activity and antioxidant activity, in which binding activity was the major molecular functions involved in the energy metabolism.Proteins in centipedes form different varieties and origins were identified by nanoLC-MS/MS method established previously. The results showed that there had no differences among proteins in different varieties of centipedes, but proteins in centipedes from different origins were different. Then different proteins from different varieties of centipedes were expressed through a list analysis.4 To explore separation and purification methods of anticoagulation proteins in centipedeTT, APTT, PT methods were used to detect the anticoagulant activity of proteins in centipede. The results showed that low protein concentration of centipede could extend the TT time, high proteins concentrations could extend APTT time and had no effects on extending PT time. TT method was chosen to screen anticoagulant activity of proteins in centipede.Ammonium sulfate precipitation method, ultrafiltration method and strong anion exchange chromatography methods were used to separate proteins in centipede respectively, and anticoagulant activity of the various parts of proteins in centipede were detected. (1)The results showed that proteins in centipede precipitated by different concentrations of ammonium sulfate had the same anticoagulant activity. (2) Anticoagulant activity of proteins with the molecular weight of less than 10 kD was higher then other segment proteins in centipede separated by ultrafiltration method. Compared to control group, anticoagulant activity of proteins in centipede with the molecular weight of 10～30 kD,30～100 kD and more than 100 kD were different, which suggested that the anticoagulant activity proteins in centipede were distributed widely in the light of molecular weight. (3)Strong anion exchange chromatography method was used to investigate anticoagulant activity proteins in centipede and 6 eluting peaks were obtained. Wherein the peak 2, peak 3 can significantly prolonged TT time, but need further study. |
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