高速逆流色谱仪分离纯化芦荟多糖的研究

采用紫外-可见分光光度计法进行了高速逆流色谱技术分离芦荟多糖的溶剂系统研究,得出了高速逆流色谱分离芦荟多糖的溶剂系统为w(PEG600)∶w(KH2PO4)∶w(K2HPO4)∶w(H2O)=5∶15∶15∶65,加入NaCl的质量分数为2%。在水浴温度30℃,转速600 r/min,下相流速为2 mL/min的条件下,采用高速逆流色谱技术成功分离出芦荟多糖粗品,得到APS-1和APS-2两个组分,经Sephadex G-100凝胶柱层析技术和高效凝胶渗透色谱技术初步分析:APS-1和APS-2均为单一组分。     

大豆磷脂组成的研究——Ⅱ.总脂肪酸及各种磷脂组分疏水侧链脂肪酸组成的气相色谱分析

在前文用单向薄层色谱解决大豆磷脂各种磷脂组分分析问题的基础上,本文又分析了大豆磷脂的总脂肪酸组成。并通过薄层色谱和气相色谱分析了大豆磷脂各种磷脂组分(包括溶血磷脂酰胆碱、磷脂酰胆碱、磷脂酰肌醇、磷脂酰乙醇胺、磷脂酸、磷脂酰甘油和双磷脂酰甘油等)疏水侧链的脂肪酸组成。     

大豆磷脂组成的研究——Ⅱ.总脂肪酸及各种磷脂组分疏水侧链脂肪酸组成的气相色谱分析

在前文用单向薄层色谱解决大豆磷脂各种磷脂组分分析问题的基础上,本文又分析了大豆磷脂的总脂肪酸组成。并通过薄层色谱和气相色谱分析了大豆磷脂各种磷脂组分(包括溶血磷脂酰胆碱、磷脂酰胆碱、磷脂酰肌醇、磷脂酰乙醇胺、磷脂酸、磷脂酰甘油和双磷脂酰甘油等)疏水侧链的脂肪酸组成。     

入侵植物牛膝菊种子萌发对PEG模拟干旱胁迫的响应

采用浓度分别为5%、10%、15%、20%和25%的PEG-6000溶液模拟干旱条件,测定不同干旱胁迫条件下牛膝菊的种子萌发时间、萌发率、日相对萌发率、发芽势、发芽指数、活力指数及幼苗的初生根与胚芽的长度等指标,研究了干旱胁迫对牛膝菊种子萌发及幼苗生长的影响,探讨了经PEG浸种预处理后种子的萌发恢复能力。结果表明:随着水分胁迫的加重,牛膝菊种子萌发和幼苗生长受到抑制程度增加,牛膝菊种子的日相对萌发率、发芽率、发芽势和发芽指数、活力指数及幼苗的初生根长和胚芽长均呈下降趋势;与对照组相比,牛膝菊种子首次萌发时间延迟越长,当PEG浓度为20%、25%时牛膝菊种子未见萌发;本研究建立了水分胁迫PEG浓度与种子萌发率的线性回归方程,得出牛膝菊种子萌发的PEG浓度临界值为12.9%;经PEG浓度为20%、25%处理的种子在适宜条件下可以恢复萌发,浸种时间相同条件下,20%PEG浓度对牛膝菊种子伤害较小,其恢复性较强,25%PEG浓度对牛膝菊种子伤害较大,其恢复性较弱。     

干旱胁迫对远志种子萌发及幼苗生长和生理特性的影响

以远志(Polygala tenuifolia Willd.)为研究对象,采用不同浓度(2.5%~25%)聚乙二醇(PEG-6000)模拟不同程度的干旱胁迫,探讨干旱胁迫对远志种子萌发及幼苗生理生化特性的影响。结果表明:(1)随着干旱胁迫强度的增加,远志种子的发芽启动时间推迟,发芽率、发芽势、发芽指数和活力指数降低,但种子发芽率在2.5%~15%PEG胁迫下与对照无显著性差异,而在20%PEG胁迫下均显著低于对照,在25%PEG胁迫下种子不能萌发;在干旱胁迫条件下,远志幼苗生物量降低,胚芽生长受到显著抑制,胚根长度则先伸长后缩短。(2)远志幼苗叶绿素含量在2.5%~10%PEG范围内随胁迫强度的增加和时间的延长而持续上升,在15%和20%PEG胁迫下则表现为先上升后下降,在10%PEG胁迫处理第15天时含量最高,为对照的1.34倍。(3)幼苗叶片的游离脯氨酸、可溶性糖和可溶性蛋白含量随PEG胁迫强度的增加和时间的延长而增加,各指标均在20%PEG胁迫处理第15天时含量最高,分别为对照的1.99倍、1.53倍和1.50倍。(4)随着PEG胁迫时间的延长,远志幼苗叶片超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性先上升后下降,并在10%PEG胁迫处理第10天时活性最强;过氧化物酶(POD)活性随着胁迫时间的延长表现出先上升后下降又上升的特性,并在20%PEG胁迫处理第5天时活性最强;叶片丙二醛(MDA)含量在15%和20%PEG胁迫处理下持续上升,在2.5%~10%PEG胁迫范围内先上升后又有所下降。研究发现,远志种子在轻、中度干旱胁迫下仍可正常萌发,而且幼苗能通过调节自身生长、渗透调节物质含量和抗氧化酶活性主动适应干旱环境,对干旱环境表现出较好的适应能力。     

干旱胁迫对鹿角杜鹃种子萌发和幼苗生理特性的影响

为探明鹿角杜鹃种子萌发和幼苗生长期的耐旱性,以鹿角杜鹃干种子和90d苗龄幼苗为材料,采用聚乙二醇(PEG-6000)模拟不同程度的干旱胁迫,研究干旱胁迫对其种子萌发、早期幼苗生长及幼苗的细胞膜透性、MDA含量、有机渗透调节物质和抗氧化酶活性的影响,并对种子萌发率、早期幼苗生长量与PEG胁迫浓度间进行了回归分析。结果表明:(1)5%~25%PEG胁迫范围内,随着干旱胁迫程度的增加,鹿角杜鹃种子的发芽启动时间推迟,发芽持续时间延长,发芽率、发芽势、发芽指数、活力指数和幼苗生长量显著降低;重度干旱胁迫(25%PEG)下,鹿角杜鹃种子完全未萌发。(2)发芽率、发芽势、发芽指数、活力指数以及幼苗生长量的变化均与干旱胁迫程度呈极显著负相关关系,回归分析求得鹿角杜鹃种子萌发的半致死PEG干旱胁迫浓度为15.68%、半致矮PEG干旱胁迫浓度为15.37%。(3)随着PEG胁迫浓度的增加,鹿角杜鹃幼苗叶片SOD活性呈先升后降的趋势,但各胁迫处理仍显著高于CK(0%PEG);细胞膜透性、MDA、脯氨酸、可溶性糖含量、POD和CAT活性则在中度(15%~20%PEG)和重度胁迫下显著升高,与干旱胁迫程度呈极显著正相关关系。研究表明,干旱胁迫显著抑制了鹿角杜鹃种子萌发和早期幼苗生长,使其细胞膜受到损伤,同时鹿角杜鹃可通过体内渗透调节物质和抗氧化酶活性的增加来适应干旱环境,使得自身受抑制、损伤程度降到最低。     

草鱼细胞融合及早熟凝集染色体的诱导

用聚乙二醇（PEG）诱导草鱼ZC-7901细胞株融合,测定了不同浓度的PEG诱导草鱼组胞的融合率,从而得出了PEG诱导草鱼细胞融合的适宜浓度为45%（W/W）。用45%PEG诱导间期细胞（I期细胞）与分裂期细胞（M期细胞）融合,早熟凝集染色体（PCC）的诱导率是18.85%。观察PCC形态,G1-PCC呈单股染色体纤维形;G2-PCC呈双股染色纤维形,较分裂期细胞染色体细长;S-PCC呈“粉末状”染色体片段。     

用聚乙二醇(PEG)／无机盐双水相体系从枯草杆菌发酵液中提取a-淀粉酶的研究

本文报道分别用聚乙二醇(PEG)／磷酸盐和PEG／(NH·)2SO4双水相体系从枯草轩菌发酵液中提取a-淀粉酶。研究了PEG的平均分子量、PEG浓度,成相的盐的浓度和Nacl的浓度对a-淀粉酶和蛋白质的分配系数以及相比的影响,确定了最佳的操作条件。实验表明在180%PEG 1500／10％磷酸盐／0．05M NaCl的体系中,a-淀粉酶的分配系数为6.62,蛋白质分配系数为1．14,相比为2．5,a-淀粉酶总收率为94．3％,在16％PEG 1500／12％(NH₄)₂SO₄／O．05M NaCl体系中,a-淀粉酶分配系数为82,蛋白质分配系数为5．2,相比为O．92,酶收率为99％,结果表明用PEG／无机盐双水相体系直接从含有菌体的发酵液中提取a-淀粉酶是可行的。     

朱唇种子吸水特性及其在干旱胁迫下萌发特性

种子粘液质是植物在长期适应环境过程中形成的,该物质对于种子的扩散、定居、生存力的改善、萌发、幼苗生存乃至抵御有毒化学物质毒害等都具有重要的生态学意义。朱唇为唇形科鼠尾草属多年生草本植物,原产美洲热带地区,现已广泛栽植于世界各地。为了理解朱唇种子表面的粘液物质吸水特性和种子在干旱胁迫下的萌发特性,该研究以朱唇种子为材料,运用光学显微镜和扫描电子显微镜观察以及种子萌发试验的方法,对种子和粘液层的形态结构、粘液质对种子萌发的影响进行了研究。结果表明:朱唇种子为卵形,表面为负网状结构,千粒重为(1.611±0.0084)g,无粘液种子吸水倍数为3,粘液种子吸水倍数为25,粘液层吸水倍数为122。粘液和无粘液种子及粘液层的重量都随吸水时间的延长而增长,但脱水过程要远长于吸水过程。朱唇种子吸水2 h达到饱和,经过36 h可干燥失水恢复原重。不同浓度PEG对朱唇种子的萌发均有影响,发芽势随PEG浓度升高而显著降低。朱唇种子在5%PEG胁迫下发芽率最高达(90.00±8.66)%,20%PEG胁迫下发芽率最低为(76.67±10.41)%,低浓度PEG对朱唇种子萌发有一定促进作用。这说明朱唇种子为速萌型种子,其粘液质在种子吸水过程中起到举足轻重的作用,能保证短时间内有充足的水分供其萌发。     

PEG预处理对青稞种子萌发和幼苗生理特性的影响

选择国内外28份青稞品种材料幼苗第一片展开叶,分别测定其相对含水量和失水率,并选择其中对水分胁迫敏感与不敏感的材料各1份,研究不同浓度(5%～30%)聚乙二醇(PEG)预处理对青稞种子萌发、幼苗生长和生理特性的影响,探讨短期水分胁迫对青稞生长发育的调节作用。结果显示:(1)28个青稞材料中‘旱地紫青稞’幼苗叶片的相对含水量最高(60.16%)、‘大麻青稞’最低(38.98%),而离体失水率‘旱地紫青稞’最低(8.80%)、‘大麻青稞’最高(20.20%)。说明‘大麻青稞’对水分胁迫最敏感,而‘旱地紫青稞’最不敏感。(2)随着胁迫程度的增加,青稞种子的萌发率和生根率,幼苗的根长、苗高和鲜重均呈先增加后降低的趋势,‘旱地紫青稞’和‘大麻青稞’种子分别在15%和10%PEG处理下发芽和生根最佳且均与对照差异显著(P<0.05),两品种的幼苗根长、苗高和鲜重均在10%PEG处理下表现最佳,但‘大麻青稞’与对照差异不显著。(3)‘旱地紫青稞’幼苗叶片可溶性蛋白和叶绿素含量随PEG处理浓度增加而逐渐增加,而丙二醛含量和相对电导率则逐渐降低,并在30%PEG处理下效果差异极显著(P<0.01);‘大麻青稞’叶片可溶性蛋白和叶绿素含量随PEG处理浓度增加呈先增加后降低的趋势,丙二醛含量和相对电导率呈先降低后增加的趋势,并在20%PEG处理下最佳。研究表明,短时间低浓度PEG处理对青稞种子萌发、幼苗生长及可溶性蛋白、叶绿素、丙二醛含量和相对电导率等生理指标的改善均有一定的促进作用;高浓度PEG处理却具有抑制作用,且高浓度PEG胁迫条件下,耐旱性强比耐旱性弱品种的自我调控能力更强。     

A continuous-flow high-yield process for preparation of lipid-free hemoglobin

Hypotonic hollow-fiber dialysis of bovine red blood cells followed by ultrafiltration through 0.1-micron pore hollow fibers provides a simple method for isolation of lipid-free hemoglobin. Hemoglobin (Hb) isolated by comparative techniques were all contaminated with membrane stroma. HPLC analysis of Hb revealed a protein peak of 99.6% purity and sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis revealed a single band. The process requires hypoosmotic dialysis of bovine RBC to a final 160-180 mosmol/kg osmotic pressure. Additional reduction in osmotic pressure causes irreversible cell lysis which leads to lipid contamination of the Hb. Processing of 1/2 liter of packed red blood cells requires 4-5 h, resulting in an average of 90% hemoglobin recovery.     

Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography

The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded plasmid DNA. RNA, denatured genomic and plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled plasmid, were retained and separated from nonbinding plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded plasmid was recovered after HIC. RNA and denatured plasmid in the final plasmid preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.     

Development of an isotope dilution mass spectrometry assay for HbA1c based on enzyme-cleaved peptide analysis

The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R(2) = 0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed.     

Influence of tryptophan tags on the purification of cutinase, secreted by a recombinant Saccharomyces cerevisiae, using cationic expanded bed adsorption and hydrophobic interaction chromatography

During cationic bed adsorption (EBA), with cutinase with varying length tryptophan tags (WP)(2)and (WP)(4), 33% and 10% of adsorption capacity and 80% and 32% eluted specific activity were observed in relation to wild type (wt)-cutinase in the conventional process. Therefore, as the hydrophobicity of the protein increases, it is important to integrate the EBA step with a hydrophobic interaction chromatography (HIC) process. As the length of the hydrophobic tag-(WP) increases from n = 2 to n = 4, the purification factor obtained by HIC was 1.8 and 2.2-fold higher than wt-cutinase. However, the recovery yield obtained in HIC decreases substantially as the length of hydrophobic tag increases (97%, 84% and 70% for wt-cutinase, cutinase-(WP)(2) and cutinase-(WP)(4)). The integration of two purification steps, EBA followed by HIC, resulted in the highest overall purity level for cutinase-(WP)(2), and the highest overall recovery yield for wt-cutinase. When optimizing the design of a hydrophobic tag fused to a protein secreted by Saccharomyces cerevisiae it must be considered that the cultivation parameters could impair the downstream process, and consequently the optimum tag is not necessarily the one that presents the highest purification factor in HIC.     

Mechanism of haemolysis by Vibrio vulnificus haemolysin

The haemolytic action of Vibrio vulnificus haemolysin (VVH) was compared to that of streptolysin O (SLO). Both were cholesterol-binding haemolysins, but differed in the release of haemoglobin (Hb). In the first step of haemolysis, the haemolysins were temperature-independently bound to the cholesterol site on the target erythrocyte membrane. This was followed by the rapid release of K+, which is an intra-erythrocyte marker. Hb was then released, in different ways. In the case of VVH, Hb was released slowly after a relatively long lag, whereas with SLO, Hb was released as rapidly as K+. Haemolysis by VVH was inhibited by the addition of 30 mM-dextran 4 (mean Mr 4000), which is considered to be an effective colloid-osmotic protectant. The results therefore indicated that haemolysis by VVH (like that by Escherichia coli alpha-haemolysin and Staphylococcus aureus alpha-toxin) was caused by a colloid-osmotic mechanism. Both K+ and Hb release caused by VVH proceeded temperature-dependently, and the membrane fluidity of liposomes prepared with lipids extracted from sheep red blood cell membranes increased above 20 degrees C. These results suggest that the temperature-dependence of the haemolysis by VVH is due to the requirement for an increase in the membrane fluidity during the formation of a transmembrane pore.     

Interaction of hemoglobin with enterobacterial lipopolysaccharide and lipid A. Physicochemical characterization and biological activity.

The interaction of hemoglobin (Hb) with endotoxins [i.e. with enterobacterial deep rough mutant lipopolysaccharide (LPS) Re and the "endotoxic principle" of LPS, lipid A] was investigated using a variety of physical techniques and with two biological assays, tumor necrosis factor (TNF)-alpha induction in human mononuclear cells and the Limulus amebocyte lysate (LAL) assay. Fourier-transform IR-spectroscopic experiments indicate nonelectrostatic binding to the hydrophobic moiety with a slight rigidification of the lipid A acyl chains, and an increase in the inclination of the lipid A backbone with respect to the membrane surface from 35 degrees to more than 40 degrees due to Hb binding, but no change of the predominantly alpha-helical secondary structures of Hb due to LPS binding. From isothermal titration calorimetry, the molar [Hb] : [endotoxin] binding ratio lies between 1 : 3 and 1 : 5 molar. Synchrotron radiation X-ray diffraction measurements indicate a reorientation of the lipid A aggregates from one cubic structure to another, the final structure belonging to space group Q224. The LPS-induced TNF-alpha production of mononuclear cells is enhanced by Hb, whereas in the LAL assay an LPS concentration-dependent increase or decrease was observed. Although a detailed mechanism of action cannot be given, the enhancement of LPS bioactivity can be understood in the light of the previously presented conformational concept; Hb induces an increase in the conical shape of the lipid A moiety of LPS, higher cross-section of the hydrophobic than the hydrophilic part, and of the inclination angle of the diglucosamine backbone with respect to the direction of the acyl chains.     

Microspectrophotometric and scanning microphotometric studies of carp (Cyprinus carpio L.) erythrocytes

Carp (Cyprinus carpio) hemoglobin readily autoxidizes in blood smears. Quantification of Soret-band absorbance in individual erythrocytes by means of scanning cytophotometry therefore requires more elaborate methods of preparation of blood samples. Of the fixatives that have been tested, suspension of whole blood in isotonic salt solutions containing glutaraldehyde was most suitable. Glutaraldehyde-fixed red blood cells are totally resistant to hemolysis. In the course of fixation, hemoglobin is transformed to methemoglobin. Spectrophotometry indicated extensive similarities between glutaraldehyde-fixed carp methemoglobin and human methemoglobin. In aqueous solutions, the intensity of the Soret-peak was pH-dependent. The allosteric modifier organic polyphosphate caused an R----T transition, resulting in increased molar extinctions. Dried preparations showed Soret-spectra that were not influenced from either pH or organic polyphosphate concentration of the aqueous suspensions in which the erythrocytes had been stored. The same was true for slide preparations of cyanomethemoglobin, easily derived from methemoglobin on addition of potassium cyanide. In the absence of oxygen fresh blood cells from carp slowly transform their hemoglobin into deoxyhemoglobin. Spectra of the intermediate stages of deoxygenation, Hb4(O2)3, Hb4(O2)2 and Hb4(O2), as well as mixtures of these intermediates, could be monitored.     

Purification of Concentrated Hemoglobin Using Organic Solvent and Heat Treatment

A simple method for obtaining a purified and concentrated hemoglobin (Hb) solution (25 g/100 ml) from human red blood cells has been established. To prevent MetHb formation during the purification procedure, Hb in red blood cells was carbonylated in advance, and then washed red blood cells were mixed with organic solvents such as diethyl ether or dichloromethane for hemolysis and removal of stroma. The Hb solution was isolated by centrifugation (1 900g) with the high removal efficiency of phospholipid (>99.8%). After the solution was heated (60°C, 1 h), the precipitates were removed by centrifugation. The purity of Hb was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing and oxygen-binding properties of the obtained Hb solution demonstrated its purity and showed no denaturation of globin. This purification procedure is applicable to large-scale production of the purified Hb.     

Depletion of highly abundant proteins in blood plasma by hydrophobic interaction chromatography for proteomic analysis

The proteomic analysis of plasma is extremely complex due to the presence of few highly abundant proteins. These proteins have to be depleted in order to detect low abundance proteins, which are likely to be of biomedical interest. In this work it was investigated the applicability of hydrophobic interaction chromatography (HIC) as a plasma fractionation method prior to two-dimensional gel electrophoresis (2DGE). The average hydrophobicity of the 56 main plasma proteins was calculated. Plasma proteins were classified as low, medium and highly hydrophobic through a cluster analysis. The highly abundant proteins showed a medium hydrophobicity, and therefore a HIC step was designed to deplete them from plasma. HIC performance was assessed by 2DGE, and it was compared to that obtained by a commercial immuno-affinity (IA) column for albumin depletion. Both methods showed similar reproducibility. HIC allowed partially depleting α-1-antitrypsin and albumin, and permitted to detect twice the number of spots than IA. Since albumin depletion by HIC was incomplete, it should be further optimized for its use as a complementary or alternative method to IA.     

Mechanism of oxidative damage to fish red blood cells by ozone

The present study was conducted to elucidate the adverse effects of ozone exposure on rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs). We evaluated whether hemoglobin (Hb) or Hb-derived free iron could participate in the RBC damage using an in vitro ozone exposure system. Ozone exposure induced hemolysis, formation of methemoglobin, and RBC membrane lipid peroxidation. This RBC damage was not suppressed by the addition of a specific iron chelator (deferoxamine mesilate) to the medium but was suppressed by carbon monoxide (CO) treatment before ozone exposure. Generation of hydrogen peroxide (H2O2) in RBC was observed upon ozone exposure but was significantly suppressed by CO treatment before ozone exposure. Thus the Hb status (i.e., Hb redox condition) and H2O2 generation in RBC should play important roles in mediating RBC damage by ozone exposure. In other words, neither ozone nor its derivative directly attacked from the outside of the cell, but ozone that penetrated through the membrane derived the reactive oxygen species from Hb inside of the cell.