尖吻蝮(Agkistrodon acufus)蛇毒凝血酶样成分的研究 Ⅱ: 酶学与血液学活性的研究

对尖吻蝮蛇毒四个凝血酶样成分(TLP)的酶学性质的比较研究表明,它们都具有凝结(血)活性和精氨酸酯酶活性。其凝结活力TLP_(4)>TLP_(3 )>TLP_(1)>TLP_(2),Ca~(++)有激活作用,但不被肝素、EDTA所抑制; 精氨酸酯酶活力以TLP_(1)、TLP_(2)较高, Ca~(++)、EDTA无明显的激活或抑制作用;TLP_(1)对热比较稳定,TLP_(4)对于酸碱变化比较稳定。经动物体内试验表明,尖吻蝮蛇毒与美洲矛头蝮蛇毒一样,同时含有去纤酶和蛇毒凝血酶。图1表3参9

湖南尖吻蝮蛇毒出血毒素的圆二色性和化学修饰

用远紫外CD谱研究了湖南产尖吻蝮蛇毒的两个出血毒素(DaHT-1、DaHT-2)的溶液构象, 计算得DaHT-1的#alpha#螺旋、#beta#折叠、无规卷曲的含量分别为36.9%, 35.5%, 27.6%; DaHT-2的#alpha#螺旋、#beta#折叠、无规卷曲分别为23.4%, 31.3%, 45.3%。随pH的增大或减小。温度和pH对CD谱的影响相似。0.02mol/L EDTA便导致两个出血毒素呈极度的无序状态。胰蛋白酶不影响它们的出血活性。图4表4参10

尖吻蝮蛇蛇毒出血毒素DaHT-3的纯化与性质

通过Sephadex G-75,DEAE-Sephadex A-50,Sephadex G-200和两次PBE聚焦层析,从尖吻蝮蛇(Dienagkistrodon acutus)蛇毒中纯化到一个分子量为56 000的出血毒素(DaHT-3),经氨基酸组成测定计算,由487个氨基酸残基组成。此成分在SDS-PAGE上显示出一条均一的蛋白染色带,用等电聚焦电泳测定,其pI为5.50。该出血成分的最小出血剂量是2.6#mu#g,具有蛋白水解酶活力,其活力为3.68,但没有精氨酸酯酶和磷脂酶A_(2)活力。用红外光谱仪研究DaHT-3在溶液中酰胺Ⅰ带的吸收谱,该毒素含有31.8%的#alpha#螺旋、56.1%的#beta#折叠和12.1%的转角;当加入EDTA螯合剂去除金属离子后,它们的#alpha#螺旋、#beta#折叠、转角和无规卷曲分别变为11%、26.4%、46.2%和16.5%,而出血活力和蛋白水解酶活力均丧失,表明该出血毒素是金属蛋白酶。

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.

环境因子对铜绿微囊藻7820胞外多糖的影响

研究了多种环境因子对铜绿微囊藻7820可溶性胞外多糖(extracellular polysaccharide,EPS)合成的影响.在18 d内,较高浓度的NO3-,较高的pH和光强,均显著提高了EPS的合成,其中,NO3-对EPS的合成影响最大,其最大产率为5.255μg.L-1.d-1.而KH2PO4,CaCl2,MgSO4等大量元素、以及微量元素FeCl3和EDTA对EPS的合成无明显影响;除在实验后期20℃时EPS有较大增加外,温度对其无明显影响.在各种环境因子影响下,EPS产量均随时间延长而增加

营养条件对水华蓝藻铜绿微囊藻的胞外多糖产生的影响

当自然条件下的群体微囊藻转入实验室培养后,它们的胶被多糖往往急剧下降,形态由群体解聚为单细胞。M icrocystis aeruginosaDCA(群体)和M.aeruginosaDCS(单细胞)是同源而形态不同的两株微囊藻,对它们的研究在一定程度上可展示微囊藻群体解聚机制。研究显示两株微囊藻在正常培养基中的生长和EPS产量差异很大,单细胞微囊藻生长快但EPS产量少,群体微囊藻正相反。以单细胞微囊藻为材料研究N,P,S,Fe和EDTA等营养元素对EPS产生的影响,实验浓度为正常培养基中营养元素的5倍和1/

鱼类饲养对地下水中溶解态磷酸酶的影响

比较了鱼类养殖前后 ,地下水中正磷酸盐 (o P)浓度、碱性磷酸酶活性 (APA)在不同大小颗粒之间的分布、溶解态APA对pH、温度、CuSO4、ZnSO4、EDTA 2Na与表面活性剂 (CTAB与TritonX 10 0 )的应答方式及其动力学特征。养鱼之后 ,玻璃缸水中碱性磷酸酶表现出明显较高的活性 ,且以溶解态为主要存在形式 ,这种效应与鱼类的品种有关 ,溶解态APA的最大反应速度 (Vmax)与米氏常数 (Km)均明显提高 ,最适温度与pH值以及对于Zn2 + 的应答方式亦发生明显改变 ,颗粒结

湖北钉螺细胞原代培养的初步研究

目的 研究湖北钉螺组织细胞的原代培养。方法 无菌处理并解剖钉螺成螺及螺胚,分别获得软体、肝脏、外套膜及胚胎组织。将软体、肝脏及胚胎组织剪碎,用0.25％胰蛋白酶和0.02％乙二胺四乙酸(EDTA)混合液4℃下消化数小时,所得细胞按三宅的湿润系统固定法接种于钉螺的外套膜组织。培养液为1/2浓度的RPMI1640含20％小牛血清附加常量抗生素(青霉素100IU/ml,链霉素100μg/ml),温度为27℃～28℃,pH7.2～7.4。结果 钉螺的胚胎组织冷消化后,获大量游离细胞。接种培养5d,见有贴壁细胞,扁

银鲫卵黄原的诱导、分离纯化及其生化鉴定

以雄性银鲫为实验材料 ,通过雌二醇 (Estradiol- 1 7β,E2 )的多次诱导 ,使得 E2 诱导产物成为血清中的主要蛋白。而后 ,在快速蛋白液相色谱 (FPL C)系统上 ,利用高交换量的阴离子交换层析 Q柱 ,成功的从血清中提纯了与雌性特异蛋白相一致的 E2 诱导产物。糖、磷、脂蛋白分析表明 ,它是一类糖磷脂蛋白大分子。同时 ,它能被 Mg2 + - Ethylenediamine tetraaceticacid(Mg2 + - EDTA)部分沉淀 ,这进一步证明 ,与雌性特异蛋白相一致的

对磷酸氢钙(HG2636-94)检测方法的改进建议

本文对饲料级磷酸氢钙标准(HG2636-94)检测方法中钙含量的测定、氟含量的测定存在的问题进行了探讨,并提出了解决方法.利用分散剂消除磷酸根对钙测定的干扰,EDTA直接络合滴定钙,方法快速准确;控制适当的试科量以保证氟检测结果的准确度.

蓝藻球形体的分离、培养及再生

在高渗溶液中,用0.05%溶菌酶和2—5mmol·1~(-1)EDTA 处理蓝藻柱孢鱼腥藻、多变鱼腥藻和组囊藻细胞。5—8h 后,70—90%的细胞转为对渗透压敏感的球形体(Spheroplast),又称原生质球。研究了藻的不同培养条件对球形体形成率的影响。测定了 EDTA 处理藻纽胞后外膜脂多糖的释放量。在高渗溶液中,藻细胞和经酶处理获得的球形体的光合放氧活性明显下降,固氮种类的固氮活性失去。饲养层法、固体混合法和含有0.5mg·1~(-1)BA 的液体悬滴培养的柱孢鱼腥藻的球形体,9天后出现再生藻落;

鱼腥藻细胞外膜完整性与防氧的关系

暴露鱼腥藻7120(Anabaena 7120)细胞于膜扰乱剂EDTA或Tris(pH 8.0,37℃,5—10分钟),导致细胞释放外膜脂多糖和蛋白成分。Tris处理后,细胞对结晶紫和洗涤剂(SDS,Triton X-100)的敏感性增加,整细胞碱性磷酸酶活性增强,暗示外膜结构被修饰,透过性增大;与此同时,被处理细胞固氮酶活性在空气氧中大大降低,而在厌氧条件下活性不受影响。将细胞释放物质与处理后的细胞重组时,可明显恢复固氮活性,表明外膜结构与固氮防氧保护有密切关系。EDTA处理的细胞,虽然释放的脂多糖和蛋

Extraction of genomic DNA using a new amino silica monolithic column

A new amino silica monolithic column was developed for DNA extraction in a miniaturized format. The monolithic column was prepared in situ by polymerization of tetraethoxysilane (TEOS) and N-(beta-aminoethyl)-gamma-aminopropylmethyldimethoxysilane (AEAPMDMS). DNA was loaded in 50 mM tris(hydroxylmethyl)aminomethane-EDTA buffer at pH 7.0 and eluted with 300 mM potassium phosphate solution at pH 10.0. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 56 ng DNA with an extraction efficiency of 71 +/- 5.2% (X +/- RSD). When the amino silica monolithic column was applied to extract genomic DNA from the whole blood of crucian carp, an extraction efficiency of 52 +/- 5.6% (X +/- SD) was obtained by three extractions. Since the chaotropic-based sample loading and organic solvent wash steps were avoided in this procedure, the purified DNA was suitable for downstream processes such as PCR. This amino silica monolithic column was demonstrated to allow rapid and efficient DNA purification in microscale.

Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01 M EDTA-Na-2-5% acetic acid, followed by a solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5 mu g g(-1) dry weight [DW]) and ranged from 91 to 103% for MC-LR, and from 65.0 to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0 mu g g(-1) DW, with a limit of quantification (LOQ) of 0.02 mu g g(-1) DW. The limit of detection (LOD) of the method was 0.007 mu g g(-1) DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR. (c) 2007 Elsevier B.V. All rights reserved.

Simultaneous determination of chromium(VI) and chromium(III) of bioavailable fraction in bottom mud by flow injection-atomic absorption spectroscopy

A rapid and sensitive method for separation and determination of Cr(VI) and Cr(III) in bottom mud of lake by flow injection on-line preconcentrtion system and GFAAS was developed. The available Cr(VI) and Cr(III) were extracted by HOAc or EDTA + NH4 NO3 and adsorbed simultaneously by an anion and a cation resin microclummn and then eluted simultaneously by 2 mol/L NH4 NO3 + 0.05 mol/L ascorbate and 2 mol/L H2SO4, respectively. The elution was performed for 50 s after adsorption for 2 min, and the efficiencies of elution were 85.4% - 94.8% and 96.7% - 106% for Cr(VI) and Cr(M) respectively. The detection limits of the method were 0.9 mu g/L and 2.7 mu g/L with relative standard deviations of 3.5% and 6.4% for the determination of Cr(VI) and Cr(III) in sample, respectively.