竹叶表(Trimeresurus stejnegeri)蛇毒凝血酶样酶的分离纯化及其理化性质的研究

用CM-Sephadex C-25分离竹叶青精毒,再经DEAE-Sephadex A-50和CM-Sephadex C-25纯化得到凝血酶样酶组分I。用DEAE-Sephadex A-50分离竹叶青粗毒,再经DEAE-Sepharose CL-63纯化得到凝血酶样酶组分Ⅱ。经鉴定,组分I在两种泳中均为一条带;组分Ⅱ在两种电泳中为二条带,经切割后鉴定证明组分Ⅱ的二条带都是凝血酶样酶。组分I和组分Ⅱ都是糖蛋白。图7表1参10

CHARACTERIZATION OF OHS1, AN ARGININE/LYSINE AMIDASE FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

In this paper, we present the results of purification and characterization of an arginine/lysine amidase from the venom of Ophiophagus hannah (OhS1). It was purified by Sephadex G-75 gel filtration and ion-exchange chromatography on DEAE-Sepharose CL-6B. It is a protein of about 43,000, consisting of a single polypeptide chain. It is a minor component in the venom. The purified enzyme was capable of hydrolysing several tripeptidyl-p-nitroanilide substrates having either arginine or lysine as the C-terminal residue. We studied the kinetic parameters of OhS1 on six these chromogenic substrates. OhS1 did not clot fibrinogen. Electrophoresis of fibrinogen degraded with OhS1 revealed the disappearance of the alpha- and beta-chains and the appearance of lower mel. wt fragments. OhS1 had no hemorrhagic activity. It did not hydrolyse casein, nor did it act on blood coagulation factor X, prothrombin and plasminogen. The activity of OhS1 was completely inhibited by NPGB, PMSF, DFP, benzamidine and soybean trypsin inhibitor, suggesting it is a serine protease. Metal chelator (EDTA) had no effect on it.

豌豆核酮糖1,5-二磷酸羧化酶/加氧酶亚基结合蛋白的纯化及特性

用~(35)S-Met在照光下与豌豆完整叶绿体保温,显示新合成的标记的RubisCO大亚基与结合蛋白形成一复合物,经ATP处理后解离为结合蛋白亚基,同时释放出的标记的RubisCO大亚基参与了RubisCO的装配。豌豆叶片提取液经热处理,硫酸铵分部,DEAE-Sepharose fast flow和Sephacryl S-300柱层析在ND-PAGE,SDS-PAGE上显示为一条带,估计纯度达90％以上,得率比以前报道的高12倍。纯化的结合蛋白表面巯基数经测定为12±1个,总巯基数为36±1个。远紫外CD

烙铁头(TRI MERESURUS MUCROSQUAMATUS)蛇毒中精氨酸酯酶的研究

采用DEAE－sephadex A-50、Sephadex G-100、CM-sepharose cl-6B三步柱层析，从烙铁头(T. mucrosquamatus)蛇毒中纯化得到的精氨酸酯酶在聚丙烯酰胺凝胶电泳(pH 8.3)和SDS－聚丙烯酰胺凝胼电泳中均呈现单一的蛋白带。其分子量为29000，等电点pI为5.2；由225个氨基酸残基组成，其中Gly,Asp和Glu的含量较高。它是一个糖蛋白，含有0.5%的中性已糖和0.75%的唾液酸，对热及酸碱变化较稳定。在280nm波长处有典型的蛋白质吸收峰，此时的消光数E0.1%/1cm为1.332。该酶具有较强的精氨酸酯酶活性和纤维蛋白原溶解活性，无出血活性、酪蛋白水解活性以及粗毒中含有的其它酶活性。从烙铁头(T. mucrosquamatus)蛇毒中纯化的具纤维蛋白原溶解活性的精氨酸酯酶（MFAE）是一丝氨酸蛋白酶，其活性可被PMSF抑制而不受EDTA的影响。MFAE酶促反应的最适pH为8.4，最适温度55 ℃; pH7.6、37 ℃时水解BAEER的米氏常数K_m为20 * 10~(-3)M。该酶能降解纯化人纤维蛋白原的Bβ链以及纯化牛纤维蛋白原的Aα和Bβ链，并有一定的纤维蛋白溶解活性。它能明显延长兔血浆的凝血酶时间和复钙时间。纯化的MFAE无出血活性、凝血酶样活性及血小板聚集活性，对ADP、AA、TMVA、Melittin等诱导的血小板聚集也无抑制或解聚作用。本文还测定了它对凝血酶及胞浆毒特异性合成三肽底物的水解活性。

竹叶青（Trimeresurus steinegeri）蛇毒凝血酶样酶研究

用MS-Sephadex C-25分离竹叶青粗毒，再经DEAE - Sephadex A-50和CM-Sephadex C-25纯化得到凝血酶样酶组分I。用DEAE-Sephadex A-50分离竹叶青粗毒，再经DEAE-Sepharose CL-6B纯化得到凝血酶样酶组分II。用聚丙烯酰胺凝胶电泳和SDS-聚丙烯酰胺凝胶电泳检查，组分I在两种电泳中均为一条带；组分II在两种电泳中均为二条带（靠得很近）。用聚丙烯酰胺凝胶电泳切割法证明了组分II的二条带都是凝血酶样酶。理化性质测定证明：组分I分子量为54，500，大约由261个氨基酸残基组成，其中Asp和Glu含量较高，含有14%的中性已糖，13.1%的已糖胺和14.7%的唾液酸，等电点为3.5，在230nm波长处有典型的蛋白质吸峰，在280nm处的消光系数为OD_(1cm)~(0.1%) = 0.855，组分I对温度对酸碱都有较好的稳定性。组分II用SDS-聚丙烯酰胺凝胶电泳测定，其分子量分别为54，000和47，000两种。凝胶电泳后用过碘酸-Schiff's试剂染色，证明组分II与组分I都是糖蛋白。

免疫亲和质谱法研究β_2-微球蛋白抗原表位

采用免疫亲和分离与质谱分析相结合的方法,对β2-微球蛋白抗原表位进行了系统研究.完整的抗原分子和已固定在载体(CNBr-activated Sepharose beads)上的单克隆抗体发生免疫亲和反应后,用Endoproteinase Glu-C,Trypsin,Aminopeptidase M和carboxypeptidase Y四种不同的蛋白酶依次酶解抗原分子,并采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对与抗体连接受保护而未发生水解的肽段进行了研究.结果表明:β2-微球蛋白抗原表位位于整个蛋白分子氨基酸序列的61～67位,即为SFYLLYY.通过合成肽段的分析,证明了SFYLLYY即为抗原表位,与亲和质谱方法分析结果一致.

Purification of R-phycoerythrin from Porphyra haitanensis (Bangiales, Rhodophyta) using expanded-bed absorption

R-phycoerythrin (R-PE) was purified from leafy gametophyte of Porphyra haitanensis T. J. Chang et B. F. Zheng (Bangiales, Rhodophyta) by a simple, scaleable procedure. Initially, phycobiliproteins were extracted by repeated freeze-thaw cycles, resulting in release from the algal cells by osmotic shock. Next, R-PE was recovered by applying the crude extract with a high concentration of (NH4)(2)SO4 salt directly to the expanded-bed columns loaded with phenyl-sepharose. An expanded-bed volume twice the settled-bed volume was maintained; then low (NH4)(2)SO4 concentration was used to develop the column. After two rounds of hydrophobic interaction chromatography (HIC), R-PE was purified by anion-exchange column. The method was also successful with free-living conchocelis of P. haitanensis. The purified R-PE was identified with electrophoresis, and absorption and fluorescence emission spectroscopy. The results were in agreement with those previously reported. The yield with a spectroscopic purity (OD565/OD280) higher than 3.2 (the ratio of A(565)/A(620) <= 0.02) was 1.4 mg . g(-1) of leafy gametophyte of P. haitanensis. For the free-living conchocelis of P. haitanensis extract, R-PE could be purified successfully with only one round of HIC. The yield with a spectroscopic purity (OD565/OD280) higher than 3.2 (the ratio of A(565)/A(620) <= 0.02) was 5.0 mg . g(-1) of free-living conchocelis of P. haitanensis. The method described here is a scaleable technology that allows a large quantity of R-PE to be recovered from the unclarified P. haitanensis crude extract. It is also a high protein recovery technology, reducing both processing costs and times, which enhances the value of this endemic Porphyra of China.

Arginine kinase from Litopenaeus vannamei: Cloning, expression and catalytic properties

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. in this paper, the full-length cDNA of AI( was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LIPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Eschetichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps. (C) 2009 Elsevier Ltd. All rights reserved.

One-step chromatography method for efficient separation and purification of R-phycoerythrin from Polysiphonia urceolata

Phycoerythrins have been widely used in food, cosmetics., immunodiagnostics and analytical reagents. An efficient one-step chromatography method for purification of R-phycoerythrins from Polysiphonia urceolata was described in this paper. Pure R-phycoerythrin was obtained with an absorbance ratio A(565)/A(280) of 5.6 and a high recovery yield of 67-33%, using a DEAE-Sepharose Fast Flow chromatography with a gradient elution of pH, alternative to common gradient elution of ionic strength. The absorption spectrum of R-phycoerythrin was characterized with three absorbance maxima at 565, 539 and 498 mum, respectively and the fluorescence emission spectrum at room temperature was measured to be 580nm. The results of native-PAGE. and SDS-PAGE showed no contamination by other proteins in the phycoerythrin solution. which suggests an efficient method for the separation and purification of R-phycoerythrins from Polysiphonia urceolata. (C) 2004 Elsevier B.V. All rights reserved.

Isolation of the main light-harvesting chlorophyll a/b-protein complex from thylakoid membranes of marine alga, Bryopsis corticulans by a direct method

The main chlorophyll a/b light-harvesting complex (LHC 11) has been isolated directly from thylakoid membranes of marine green alga (Bryopsis corticulans Setch.) by two consecutive runs of anion exchange and gel-filtration chromatography. LHC 11 proteins in the membrane extracts treated with 3% n-Octyl-b-D-glucopyranoside (OG) obtained specific binding ability on Q Sepharose column, and thus were isolated from the thylakoid membranes in a highly selective fraction. The monomeric, trimeric and oligomeric subcomplexes of LHC 11 have been obtained by fractionation of the LHC 11 mixes with sucrose density gradient ultracentrifugation. The SDS-PAGE analysis of peptide composition and absorption spectrum showed that LHC 11 monomers, trimers and oligomers prepared through this work were intact and in high purity. Our report is the first to show that it is possible to purify LHC If directly from thylakoid membranes without extensively biochemical purification.

Large-scale isolation and purification of R-phycoerythrin from red alga Palmaria palmata using the expanded bed adsorption method

R-phycoerythrin, a light-harvesting protein in some marine algae, and can be widely used in medicine, was isolated and purified from a red alga, Palmaria palmata (Lannaeus) Kuntze, using the streamline column (expanded bed adsorption) combined with ion-exchange chromatography. Because the crude extract was applied to the column upwardly, the column would not be blocked by polysaccharides usually very abundant in the extract of marine alga, this kind of blockage could hardly lie overcome in ordinary chromatographic column. After applying the crude extract containing 0.5 mol/L (NH4)(2)SO4, (NH4)(2)SO4 solution of different concentrations (0.2 mol/L, 0.1 mol/L and 0.05 mol/L) was used to elute the column downwardly and the eluates were collected and desalted. The desalted eluates were then applied onto all ion-exchange chromatographic column loaded with Q-sepharose for further purification of the R-phycoerythrin. Through these two steps, the purity (OD565/OD280) of the R-phycoerythrin from P. palmata was up to 3.5, more than 3.2, the commonly accepted criterion for purity, and the yield of the purified R-phycoerythrin could reach 0.122 mg/g of frozen P. palmata, much higher than that of phycobiliproteins purified with the previous methods. The result indicated that the cost of R-phycoerythrin will drop down with the method reported in this article.

Purification and characterization of agarases from a marine bacterium Vibrio sp. F-6

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn2+, Mg2+ or Ca2+ increased AG-a activity, while Mn2+, Cu2+ or Ca2+ increased AG-b activity. However, Ag+, Hg2+, Fe3+, EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.

Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography

C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A(620)/A(280) ratio) of C-phycocyanin isolated with STREAMLINE (TM) Column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A(620)/A(650) index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin. (c) 2006 Elsevier B.V. All rights reserved.

Method for large-scale isolation and purification of R-phycoerythrin from red alga Polysiphonia urceolata Grev

R-phycoerythrin was isolated and purified from a red alga, Polysiphonia urceolata Grev, using Streamline column combined with ion-exchange chromatography or hydroxyapatite chromatography. The purity of R-phycoerythrin isolated by Streamline column was up to 1.66 and the yield of R-phycoerythrin could be as high as 0.68 mg/g frozen P. urceolata. All the eluates from Streamline column were divided into two equivalent parts, respectively. One part was pumped into the ion-exchange column loaded with Q-Sepharose and the other was applied to the adsorption column loaded with hydroxyapatite. The purities of R-phycoerythrin purified using these two methods were both up to 3.26, more than 3.2 the commonly accepted criterion. The yield of purified R-phycoerythrin from the ion-exchange chromatography was 0.40 mg/g frozen P. urceolata and that from the hydroxyapatite chromatography could reach 0.34 mg/g frozen P. urceolata. The purified protein had three absorption peaks at 498, 535, and 565 nm and displayed a fluorescence maximum at 580 nm, which was consistent with the typical spectrum of R-phycoerythrin. The purified R-PE was also identified with electrophoresis. Only one single protein band appeared on native-PAGE with silver staining. SDS-PAGE demonstrated the presence of one 20 kDa major subunit, and one low intensity band corresponding to 33 kDa subunit. The results indicate that using the expanded bed adsorption combined with ion-exchange chromatography or hydroxyapatite chromatography, R-phycoerythrin can be purified from frozen P. urceolata on large scale. (c) 2006 Elsevier Inc. All rights reserved.

两种大型水母毒素的分离纯化及其蛋白酶活性和抗氧化活性的研究

水母在我国分布广，种类多，数量大，是一种丰富的海洋生物资源。已研究的水母毒素主要分布于触角和刺丝囊中，是一类结构新颖独特的肽类毒素。水母种类的不同，其毒素的结构、生物活性往往不同。本文首次报道了海蜇（Rhopilema esculentum Kishinouye）毒素 和白色霞水母（Cyanea nozakii Kishinouye）毒素的提取、纯化、理化性质及蛋白酶和抗氧化方面的生物学活性，主要结果如下： 1.用DEAE Sepharose Fast Flow分离海蜇毒素，用含0.4 mol/L NaCl 的PBS (0.02 M，pH 8.0) 进行阶段洗脱时，得到的组分是组蛋白H4。 2.水母毒素的蛋白酶活性实验结果为：海蜇毒素和白色霞水母毒素都有蛋白酶活性。该两种毒素的蛋白酶活性都受理化因素的影响，其中0.5%甘油和1%邻菲罗啉可以有效的抑制海蜇毒素的蛋白酶活性。 3.对海蜇毒素蛋白的体外抗氧化活性研究表明：蛋白样品均具有抗氧化性，它们对超氧阴离子和羟自由基具有显著的清除作用。对白色霞水母毒素蛋白的体外抗氧化活性研究表明：白色霞水母蛋白具有较高的清除羟自由基的能力，白色霞水母蛋白对超氧阴离子的清除作用较弱。白色霞水母蛋白有较强的还原能力，但没有螯合能力。 通过本文的研究表明水母毒素具有蛋白酶活性和抗氧化活性，能够清除氧自由基。这些研究结果为开发利用水母资源和毒素的合理、综合利用打下了坚实的基础。