| The basal source of the Bungarus multicinctus is the dried body of the juvenile of the silver ringed snake Bungarus multicinctus Blyth,a member of the cobra family,it has the effect of relieving wind and spasm,and is effective in the treatment of rheumatism,numbness and constriction,stroke,staggering eyes and mouth,paralysis,convulsion and spasm,tetanus,leprosy,scabies,etc[1-4].At present,there is a lot of attention to the chemical components such as amino acids,nucleotides,trace elements,and lipids in Bungarus multicinctus,but there is still a lack of systematic research on the most abundant protein[5-7].In this study,we aimed to establish and characterize the optimized extraction process of the proteins of the Bungarus multicinctus,optimize the fingerprinting conditions of its proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）,identify the proteins by LC-MS/MS,and perform bioinformatics analysis;investigate the feasibility of using SDS-PAGE fingerprinting for the identification of the Bungarus multicinctus and its pseudo-herbs,and conducted a methodological study in order to provide new ideas and experimental bases for the discovery of the pharmacological basis of the herbs,the development and utilization of protein resources and molecular identification of the pseudo-herbs.The main results of the study are as follows:1.Firstly,the optimized extraction processes of acid-soluble,alkali-soluble and alcohol-soluble proteins of Bungarus multicinctus were established and characterized on the basis of single-factor tests using response surface methodology.The results showed that the optimal extraction conditions for the acid-soluble proteins of Bungarus multicinctus were 0.18mol/L sodium acetate,1:40（g/m L）material-to-liquid ratio,5.66 h extraction time and 40.01℃extraction temperature,and the best extraction rate was 3.76%at this time;SDS-PAGE showed that the molecular weight range was 18.4-80 k Da,containing 18.99 k Da,26.47 k Da,30.60 k Da,and the high abundance of protein,26.47 k Da,30.60 k Da of high protein abundance;HPLC showed that the retention times were 2.99,3.53,4.57 and 4.73 min,and FTIR revealed seven characteristic absorption peaks.Meanwhile,the optimal extraction conditions for the alkaline-soluble protein of Bungarus multicinctus were 0.20 mol/L Tris-HCl extraction solution（p H 7.88）,material-to-liquid ratio 1:41.67（g/m L）,extraction time 4.89 h,and extraction temperature 44.99℃,at which the optimal extraction rate was 5.95%;SDS-PAGE showed that the molecular weight range was 18.4-116 k Da,containing high abundance proteins of 18.4-19.5k Da,23.38-25.11 k Da,32.53-38.64 k Da and 58.62-65.96 k Da;HPLC showed that the retention times were 3.00,4.75,4.98 and 5.67 min;FTIR revealed seven characteristic absorption peaks.In addition,the optimal extraction conditions for the alcohol-soluble protein of Bungarus multicinctus were 40.11%ethanol concentration,1:16.94（g/m L）,3.87 h extraction time and41.61℃extraction temperature,at which the optimal extraction rate was 0.68%,SDS-PAGE showed that the molecular weight range was 26.69-88.02 k Da,containing 26.69-SDS-PAGE showed that the molecular weight range was 26.14-85.02 k Da,containing 26.69-28.34 k Da and70.84-88.02 k Da of high protein abundance;HPLC showed that the retention time was 2.928 and3.45 min;FTIR revealed eight characteristic absorption peaks.2.Secondly,the effects of the Separation gel concentration,electrophoresis system,dosing amount and electrophoresis time on the resolution of SDS-PAGE fingerprint were investigated to establish the optimized fingerprint of the proteins of Bungarus multicinctus and to identify them by LC-MS/MS.The results showed that,based on the previous chapter,alcohol-soluble,acid-soluble and alkali-soluble proteins were extracted using 40%ethanol solution,0.20 mol/L sodium acetate solution（p H 4.20）and 0.20 mol/L Tris-HCl extract（p H 8）in turn to obtain the whole proteins of Bungarus multicinctus.When the Glycine-SDS-PAGE system was used,the SDS-PAGE fingerprints of the acid-soluble,alkaline-soluble and alcohol-soluble proteins of Bungarus multicinctus were best clarified when the protein sample volume was 15μg,the concentration of the separation gel was 14%and the electrophoresis was performed at 87 V for4.50 h.The fingerprints obtained by the Tricine-SDS-PAGE system were clear and had better protein information.The fingerprints obtained by Tricine-SDS-PAGE system were clearer,richer in protein information,with better resolution,and more suitable for the analysis of small molecular weight proteins.A total of 16,328 spectra and 5610 peptides were obtained by LC-MS/MS for the identification of the whole proteins of the Bungarus multicinctus,and a total of 618 protein molecules were obtained by Protein Pilot database search and matching analysis.Bioinformatics analysis showed that these protein molecules were widely distributed in cellular organisms,intracellular cytoplasm,organelles,cell membranes and other sites,with various molecular functions such as catalytic activity,molecular regulation and binding function,while playing important roles in biological processes such as bioregulation,metabolic activities and multicellular biology.Most proteins are involved in signal transduction with 17.71%,11.18%in transformation,post-metabolic modifications and molecular chaperones,7.69%in carbohydrate transport,and a few proteins are involved in processes such as biological origin,cell growth and metabolism,translation processes,ribosome structure,energy production and conversion.These proteins act mainly through 15 signaling pathways including metabolic pathways,biosynthetic pathways of secondary metabolites,biosynthetic pathways of antibiotics,microbial metabolic pathways in multiple environments,metabolic pathways of carbon,ribosomal pathways,and cancer pathways.3.In addition,the proteins of Bungarus multicinctus and the common pseudo-snakes were extracted and analyzed by SDS-PAGE,and a method based on protein fingerprinting was established and investigated.The results showed that there were significant differences in the alcohol-soluble protein fingerprints,some differences in the acid-soluble protein fingerprints,and small differences in the alkali-soluble protein fingerprints of Bungarus multicinctus,Enhydris chinensis,Lycodon rufozonatus,indicating that the alcohol-soluble proteins could be identified based on the characteristic bands.The molecular identification of Bungarus multicinctus with two common pseudo-snakes was carried out.Compared with Enhydris chinensis,and Lycodon rufozonatus,the Bungarus multicinctus has a total of 12.03,18.08,26.25,40.33,51.49,61.61,85.28 KDa A total of 10 characteristic protein bands.Further analysis results show that the Bungarus multicinctus with 27.73,18.54 and 11.97 KDa bands,the Enhydris chinensis with 28.21,21.39 and 12.34 KDa bands,and the Lycodon rufozonatus with 29.03,18.08 KDa and two secondary bands were further analyzed with the mass spectrometry identification results,which may be related to the pharmacological activity of the Bungarus multicinctus.The results of the methodological investigation showed that the alcohol-soluble protein fingerprints had good stability and reproducibility.The above results provide an idea and a reference for the establishment of a molecular identification method based on the characteristic protein molecules. |
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