鲢鱼肝脏中高分子CPIs提取方法的建立及其鉴定
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摘要
研究了不同浓度的丝氨酸蛋白酶抑制剂(PMSF)、热处理条件及pH值对鲢鱼肝脏中高分子半胱氨酸蛋白酶抑制剂(cysteine proteinase inhibitors,CPIs)提取过程中的作用,根据其对CPIs在TSK液相上高分子质量部分的蛋白峰值和比活力(荧光合成肽底物法)的影响,确定了最佳粗提条件为:以含5 mmol/L的苯甲基磺酰氟(phenylmethanesulfonyl fluoride,PMSF),100mmol/L的Tris,3 mmol/L的EDTA(pH 7.5)作为提取缓冲液,而后在pH 8.7碱处理条件下经90℃加热5 min后,再回调到pH 7.0。用该法制备的鲢鱼肝脏CPIs粗提物经制备型Sephacryl S-200分子筛层析,分离得到的高分子活性部分,进一步经过反相酶谱法鉴定得到一种高相对分子质量的活性CPI,推测其可能是与某些蛋白结合形成了复合物,或是高分子CPIs的二聚体形式。
In this study,different concentrations of the serine protease inhibitors phenylmethanesulfonyl fluoride(PMSF),different heat treatment and different pH were conducted to treat on the crude extraction of high-molecular-weight cysteine proteinase inhibitors(CPIs) from Silver carp liver,and we investigated their effect on the protein response values and specific activity of CPIs by HPLC with column of TSK-GEL G2000 SW.The optimum extraction conditions were as follows:the condition of extraction buffer is containing 5 mmol/L PMSF,100 mmol/L Tris,and 3 mmol/L EDTA at pH 7.5,followed by pH readjustment to 7.0 after alkali(pH 8.7) treatment at 90 ℃for 5 min.Then,the high-molecular-weight active CPI was obtained by chromatography on a Sephacryl S-200 column,and it was identified as a high-molecular-weight CPI by the reverse zymography method.Therefore,it may be conjugated with other protein or a dimeric protein.
In this study,different concentrations of the serine protease inhibitors phenylmethanesulfonyl fluoride(PMSF),different heat treatment and different pH were conducted to treat on the crude extraction of high-molecular-weight cysteine proteinase inhibitors(CPIs) from Silver carp liver,and we investigated their effect on the protein response values and specific activity of CPIs by HPLC with column of TSK-GEL G2000 SW.The optimum extraction conditions were as follows:the condition of extraction buffer is containing 5 mmol/L PMSF,100 mmol/L Tris,and 3 mmol/L EDTA at pH 7.5,followed by pH readjustment to 7.0 after alkali(pH 8.7) treatment at 90 ℃for 5 min.Then,the high-molecular-weight active CPI was obtained by chromatography on a Sephacryl S-200 column,and it was identified as a high-molecular-weight CPI by the reverse zymography method.Therefore,it may be conjugated with other protein or a dimeric protein.
引文
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[17]LI Shuhong,REN Yangyang,LI Yanfang,et al.Comparative study on three electrophoresis methods for identifying silver carp CPIs[J].Science and Technology of Food Industry,2013,34(21):56-64.(in Chinese)
[18]ZHANG Min,ZHANG Jun.A research review of low value freshwater fishes processingand their discards utilization[J].Journal of Food Science and Biotechnology,2006(5):115-120.(in Chinese)
[19]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1-2):248-254.
[20]ANASTASI A,BROWN M A,KEMBHAVI A A,et al.Cystatin,a protein inhibitor of cysteine proteinases.Improved purification from egg white,characterization,and detection in chicken serum[J].Biochemical Jourmal,1983,211(1):129-138.
[21]BARRETT A J,KIRSCHKE H.Cathepsin B,Cathepsin H and Cathepsin L[J].Methods in Enzymology,1981,80(41):535-561.
[22]LI D K,LIN H,KIM S M.Purification and characterization of a cysteine protease inhibitor from chum salmon(Oncorhynchus keta)plasma[J].Journal of Agricultural and Food Chemistry,2008,56(1):106-111.
[23]CAO Minjie,LI Yan,WENG Ling,et al.Study on myofibril-bound serine proteinase in silver carp[J].Food Science,2005(1):91-94.(in Chinese)
[24]UAN Shan,TANG Chengzheng,et al.Advances in researches of main endogenous proteases in surimi[J].Academic Periodical of Farm Products Processing,2014(3):52-61.(in Chinese)
[25]刘玲.鲢鱼卵中高分子CPIs的纯化鉴定及其对Ishikawa细胞影响的初探研究[D].雅安:四川农业大学,2014.
[2]DUBIN G.Proteinaceous cysteine protease inhibitors[J].CMLS Cellular and Molecular Life Sciences,2005,62:653-669.
[3]OCHIENG J,CHAUDHURI G.Cystatin superfamily[J].J Health Care Poor Underserved,2010,21:51-70.
[4]LALMANACH G,NNUDIN C,LECAILLE F,et al.Kininogens:More than cysteine protease inhibitors and kinin precursors[J].Biochimie,2010,92(11):1568-1579.
[5]ZHOU Liwei,MA Fei,LI Qingwei.Progress in the study on alternative splicing and functionsof kininogen genes[J].Hereditas,2006,28(12):1649-1655.(in Chinese)
[6]BAGLIA F A,BADELINO K O,LI C Q,et al.Factor XI binding to the platelet glycoprotein Ib-IX-V complex promotes factor XI activation by thrombin[J].The Journal of Biological Chemistry,2007,282(39):29067.
[7]LIU Y,PIXLEY R,FUSARO M,et al.Cleaved high-molecular-weight kininogen and its domain 5 inhibit migration and invasion of human prostate cancer cells through the epidermal growth factor receptor pathway[J].Oncogene,2009,28(30):2756-2765.
[8]SCHARFSTEIN J,SCHMITZ V,SVENSJO E,et al.Kininogens coordinate adaptive immunity through the proteolytic release of bradykinin,an endogenous danger signal driving dendritic cell maturation[J].Sandinavian Journal of Immunology,2007,66(2-3):128-136.
[9]SUN D,MCCRAE K.Endothelial-cell apoptosis induced by cleaved high-molecular-weight kininogen(HKa)is matrix dependent and requires the generation of reactive oxygen species[J].Blood,2006,107:4714-4720.
[10]XU Xiuyue,YANG Wei,WU Bin,et al.Effect of synthetic peptides derived from HKa D5 on proliferation and apoptosis of MDA-MB-231 cells[J].Journal of China Medical University,2008,37(4):494-499.(in Chinese)
[11]NORDAHL E A,RYDENGARD V,MORGELIN M,et al.Domain 5 of high molecular weight kininogen is antibacterial[J].The Journal of Biological Chemistry,2005,280(10):34832-34839.
[12]SONESSON A,NORDAHL E A,MALMSTEN M,et al.Antifungal activities of peptides derived from domain 5 of highmolecular-weight kininogen[J].International Journal of Peptides,2011.
[13]YLONEN A,RINNE A,HERTTUAINEN J,et al.Atlantic salmon(Salmo salar L.)skin contains a novel kininogen and another cysteine proteinase inhibitor[J].European Journal of Biochemistry/FEBS,1999,266(3):1066-1072.
[14]YLONEN A,HELIN J,BOQWALD J,et al.Purification and characterization of novel kininogens from spotted wolffish and Atlantic cod[J].European Journal of Biochemistry/FEBS,2002,269(11):2639-2646.
[15]ZHOU L,LIU X,JIN P,et al.Cloning of the kininogen gene from Lampetra japonica provides insights into its phylogeny in vertebrates[J].Journal of Genetics and Genomics,2009,36(2):109-115.
[16]MICHIAKI Y,SHIRO K.A comparison of cystatin activity in the various tissues of chum salmon Oncorhynchus keta between feeding and spawning migrations[J].Comparative Biochemistry and Physiology Part A:Physiology,1991,100(3):749-751.
[17]LI Shuhong,REN Yangyang,LI Yanfang,et al.Comparative study on three electrophoresis methods for identifying silver carp CPIs[J].Science and Technology of Food Industry,2013,34(21):56-64.(in Chinese)
[18]ZHANG Min,ZHANG Jun.A research review of low value freshwater fishes processingand their discards utilization[J].Journal of Food Science and Biotechnology,2006(5):115-120.(in Chinese)
[19]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1-2):248-254.
[20]ANASTASI A,BROWN M A,KEMBHAVI A A,et al.Cystatin,a protein inhibitor of cysteine proteinases.Improved purification from egg white,characterization,and detection in chicken serum[J].Biochemical Jourmal,1983,211(1):129-138.
[21]BARRETT A J,KIRSCHKE H.Cathepsin B,Cathepsin H and Cathepsin L[J].Methods in Enzymology,1981,80(41):535-561.
[22]LI D K,LIN H,KIM S M.Purification and characterization of a cysteine protease inhibitor from chum salmon(Oncorhynchus keta)plasma[J].Journal of Agricultural and Food Chemistry,2008,56(1):106-111.
[23]CAO Minjie,LI Yan,WENG Ling,et al.Study on myofibril-bound serine proteinase in silver carp[J].Food Science,2005(1):91-94.(in Chinese)
[24]UAN Shan,TANG Chengzheng,et al.Advances in researches of main endogenous proteases in surimi[J].Academic Periodical of Farm Products Processing,2014(3):52-61.(in Chinese)
[25]刘玲.鲢鱼卵中高分子CPIs的纯化鉴定及其对Ishikawa细胞影响的初探研究[D].雅安:四川农业大学,2014.