毛细管等电聚焦和电渗泵驱动聚焦区带分离蛋白质

建立了一种利用电渗泵驱动毛细管内的聚焦区带,实现毛细管电泳等电聚焦分离蛋白质的方法。通过控制电压来调节泵的输出流量,从而调节聚焦区带的迁移速度。适用于毛细管电泳等电聚焦两步法分离蛋白质等两性物质。考察了对牛血清白蛋白和溶菌酶两种粗提蛋白质混合物的分离,迁移时间的RSD分别为1.6%和1.3%,峰面积的RSD均为1.6%,证明方法可行。     

基于电迁移的蛋白质制备技术和方法新进展

基于电迁移的蛋白质制备技术是对一类分离和制备技术的统称,其特征是在电场的作用下对目标物质进行分离和纯化制备,这种技术在生物大分子和蛋白质组的研究中应用广泛。基于电迁移的制备技术主要包括制备型电泳、制备型电色谱、制备型等电聚焦和自由流电泳等。本文对每种制备型电迁移装置的设计、特点和基于该种装置的各种应用方法的优缺点进行了详细阐述,并列举了一些实例。另外,微量级制备型电泳因分离度高、回收率高以及高效快速的优点,在微量级生物样本分析中发挥着日益重要的作用,近年来备受关注,本文也着重关注了这方面的进展,并对基于电迁移的制备技术做了展望。     

毛细管电泳检测肺癌及癌旁正常组织蛋白质混合物差异

以建立的毛细管电泳(CE)-激光诱导荧光(LIF)检测蛋白质的方法对提取肺癌及癌旁正常组织蛋白质混合物(变 性/活性)差异进行检测. 采用异硫氰酸荧光素(FITC)为衍生剂, 电泳缓冲液为1×TBE (TBE为Tris-硼酸-EDTA, 变性电泳pH 10.0, 活性电泳为pH 8.3且含有2 mg/L考马斯亮蓝), 分离电压15 kV, 柱温15 ℃, 电动进样(10 kV×10 s), 激发波长/发射波长＝488/520 nm检测时, 肺癌及癌旁正常组织蛋白质混合物样品得到较好分离且有明显差异. 与目前常用蛋白分析方法: 变性SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)以及活性蓝绿温和胶电泳(BN-PAGE)进行比较. BN-PAGE结果显示肺癌组织相比正常组织有较明显蛋白种类差异|SDS-PAGE结果表明一些蛋白质表达量差异也是肺癌及癌旁正常组织的显著差别, 且主要集中在20～116 kDa. CE-LIF检测结果与PAGE结果大致相同, 且CE-LIF检测蛋白质的灵敏度高于PAGE, 能更准确反映肺癌及癌旁正常组织的蛋白质差异. 结论是CE-LIF可用于蛋白质差异检测, 时间短, 效果较好, 对活性蛋白质进行分析体现了其优点: 可提供较强的动力——电压, 及强动力下良好的温度稳定性.     

芯片毛细管电泳及其在生命科学中的应用

芯片毛细管电泳 (Chip CE)技术在近几年已取得了很大的进展。本文着重介绍芯片毛细管区带电泳技术 ,对等电聚焦、等速电泳、自由溶液电泳及胶束电动色谱等其它芯片电泳模式也有所提及。讨论了芯片材料和制作技术、芯片的几何形状、样品的操作和衍生、检测及芯片毛细管电泳技术的应用 ,特别是在核酸和蛋白质的分离分析中的进展     

新型固定化pH梯度毛细管等电聚焦方法用于蛋白分离

通过化学键合建立一种固定化pH梯度的方法,用于毛细管等电聚焦分离蛋白质.采用微流控泵驱动毛细管内的聚焦区带,通过调节泵的流量,从而调节聚焦区带的迁移速度.该方法避免了自由溶液聚焦时两性电解质所带来的影响,实现了高灵敏度及检测波长自由选择等优点,适用于两步法毛细管电泳等电聚焦分离蛋白质等两性电解质.本文考察了对牛血清白蛋白和血红蛋白两种蛋白质混合物的分离,证明了该方法可行.     

毛细管电泳-电喷雾质谱联用技术及其在蛋白质分析领域中的应用

综述了毛细管电泳与电喷雾质谱联用的接口技术、分离模式及其在蛋白质分析领域中的应用，特别是毛细管等电聚焦与电喷雾质谱联用在蛋白质组学中研究进展。     

Computer simulation on kinetics of primary process in photosynthesis of algae (V) --Excitation energy transfer in phycoerythrocyanins

Excitation energy transfer in phycoerythrocyanins (PEC) was studied by use of computer simulation. The results observed from the simulation are as follows: (i) The α84 is a more efficient sensitizing chromophore than β155 and donates the excitation energy into β84 and β155 while it scarcely emits fluorescence itself, (ii) Only the 1α84 →2β84 is the sub-picosecond process in a PEC trimer, therefore it is readily to obtain the time constant from fs-level time-resolved spectral measurement. (iii) The β84 and β155 chromophores in PEC behave quite differently from those in C-PC because of the changes in α84. It is observed that 1β156→6β155 is the dominant pathway linking two trimers and both of the chromophores possess much higher fluorescence fractions, and about 80% of the total fluorescence is emitted from the β84 chromophore. (iv) A far less mean number of transfer times is observed through the fast-transfer pairs in PEC compared with that in C-PC because of slow transfer rate for the path     

芯片国管电泳及其在生命科学中的应用

芯片毛细管电泳（Chip-CE)技术在近几年已取得了很大的进展。本文着重介绍芯片毛细管区带电泳技术，对等电聚焦、等速电泳、自由溶液电泳及胶束电动色谱等其它芯片电泳模式也有所提及。讨论了芯片材料和制作技术、芯片的几何形状、样品的操作和衍生、检测及芯片毛细管电泳技术的应用，特别是在核酸和蛋白质的分离分析中的进展。     

α-干扰素的液相色谱/毛细管电泳两维分离分析

用亲和色谱/反相色谱、亲和色谱/毛细管电泳和凝胶色谱/毛细管电泳等两维系统, 对白细胞提取液中的α-干扰素进行分离分析, 并对结果进行比较,充分肯定了亲和色谱/毛细管电泳联用在蛋白质类药物制备纯化中的显著作用,以及毛细管电泳作为此类药物分析工具的可能性。     

丁二酸二异辛酯磺酸钠对微乳液毛细管电泳分离蛋白质的影响

本研究探讨了在生理条件下,同时分离酸性和碱性蛋白质的毛细管电泳方法。在120mmol/L十二烷基硫酸钠(SDS) 5mmol/L丁二酸二异辛酯磺酸钠(AOT) 110mmol/L正辛烷 500mmol/L正丁醇 3mmol/LNa2B4O7 3mmol/LNaH2PO4(pH6.7)的微乳体系中,分离电压20kV,25min内完成核糖核酸A、细胞色素C、牛血清蛋白、α-淀粉酶和β-乳球蛋白5种不同酸、碱性蛋白质基线分离;分离效率为7.8×105～4.4×105/m;迁移时间的RSD为1.5%(n=11)。并讨论了蛋白质在AOT组成的复配微乳体系中的分离机制。     

Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.)

Glutelin, the major storage protein of rice seed, consists of microheterogenous subunits and partially exists in a macromolecular form that is polymerized by intersubunit disulfide bonds. In order to analyze the glutelin subunits using high-throughput CE, we first identified a sample preparation procedure suitable for CE. The polymerized glutelin treated with a reductant could not dissociate into its constituent monomer subunits when it was dissolved in an acidic solution. However, the glutelin dissociated into its subunits and component α and β polypeptides when it was denatured and reduced by an appropriate amount of urea and 2-mercaptoethanol at a specific incubation time and temperature. The molecular species of the completely dissociated α and β polypeptides were identified and quantitatively analyzed by CE using glutelin mutants. The CE analysis also demonstrated that the actual subunit variation in terms of the charge and/or size of the β polypeptides is much smaller than predicted when compared with that of α polypeptides, even under denaturing and reducing condition. Thus, the combined analytical system described here will be useful for basic and applied research, such as the kinetic characterization of higher-order structure and the quantitative evaluation of glutelin in a large number of diverse rice varieties.     

Melting behavior in blends of isotactic polypropylene and a liquid crystalline polymer

The influence of low contents of a liquid crystalline polymer on the crystallization and melting behavior of isotactic polypropylene (iPP) was investigated using electron and optical microscopy, differential scanning calorimetry, and X-ray diffraction. In pure iPP, the α modification was found, whereas for iPP/Vectra blends at Vectra concentration <5%, both α and β forms were observed. The amount of β phase varied from 0.23 to 0.16. Optical microscopy showed that Vectra was able to nucleate both α and β forms. Non-isothermal crystallization produces a material with a strong tendency for recrystallization of the α and β forms (αα′ and ββ′ recrystallization) leading to double endotherms for both crystalline forms in DSC thermograms. Melting thermograms after isothermal crystallization at low temperatures showed a similar behavior. At values of T_c > 119 °C for the α form and T_c > 125 °C for the β form, only one melting endotherm was observed because enough perfect crystals, not susceptible to recrystallization, were obtained. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 1949–1959, 2004     

A new way to understand quaternary structure changes of hemoglobin upon ligand binding on the basis of UV-resonance Raman evaluation of intersubunit interactions

The single residue vibrational spectra of tryptophan (Trp) and tyrosine (Tyr) residues in human adult hemoglobin (HbA), which play important roles in cooperative oxygen binding, were determined for the deoxy and CO-bound forms by applying UV resonance Raman spectroscopy to various variant Hbs. It was found that Trpβ37, Tyrα42, Tyrα140, and Tyrβ145 at the α(1)-β(2) subunit interface underwent transitions between two contact states (named as T and R) upon ligand binding, while Trpα14, Trpβ15, and Tyrβ35 displayed little changes. The corresponding spectral changes were identified only for the α(2)β(2) tetramer, but not the isolated α and β chains in the oligomeric forms, and therefore were exclusively attributed to a quaternary structure change. Ligand binding as well as allosteric effectors and pH altered only the number of the T-contacted Tyr and Trp residues without varying the two contact states themselves. A new method to semiquantitatively evaluate the amount of T-contacted Tyr and Trp residues in a given liganded form is here proposed, and with it a quaternary structure was determined for various symmetrically half-liganded forms obtained with ligand-hybrid, metal-hybrid, and valency-hybrid Hbs. It was found that ligand binding to the α or β subunits yielded different subunit contacts and that the contact changes of the Trp and Tyr residues were not always concerted. The contact changes at the α(1)-β(2) (α(2)-β(1)) interface are correlated with the proximal strain exerted on the Fe-His(F8) bond, which is noted to be much larger in the α than β subunits in the α(2)β(2) tetramer.     

Orthogonal test design for optimization of suitable conditions to separate C-phycocyanin from Spirulina platensis by high-speed counter-current chromatography using reverse micelle solvent system

High-speed counter-current chromatography (HSCCC) was applied to separate C-phycocyanin (C-PC) from Spirulina platensis in the article. The suitable conditions were optimized by an orthogonal test design (L(9)(3)(3)), including the stationary phase of reverse micelle solvent system (0.10 g/mL cetyltrimethylammonium bromide [CTAB]/isooctane-hexylalcohol), mobile phase A (0.05 mol/L sodium phosphate buffer, pH 4.0, containing 0.2 mol/L KCl) and mobile phase B (0.05 mol/L sodium phosphate buffer, pH 8.0, containing 0.4 mol/L KCl). Under the selected conditions, 78.7 mg protein was purified from 200 mg crude extract of S. platensis, and the purity of the product was 4.25 based on the absorbance ratio of A(620)/A(280) , which was increased 6.85 times compared with the crude extract. Then, the protein was identified to be C-PC by MALDI-TOF/TOF-MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis compared with the standard. The application of HSCCC used in the separation of C-PC from S. platensis was first reported in the article. Furthermore, three kinds of tumor cell lines including human hepatoma cell line SMMC-7721, human ovarian carcinoma cell line ES-2, and human lung adenocarcinoma cell line SPCA-1 were used to evaluate the anticancer activities of the separated product, and the results showed that the separated C-PC had excellent anti-tumor actions with the IC(50) values at 2.998, 4.854, and 8.423 μg/mL, respectively, for 48 h treatment. The outcome indicates that an effective method for C-PC purification by HSCCC has been established.     

Tetrafluoroaurate(III) der Lanthaniden M2F[AuF4]5 (M = Tb,Dy, Ho,Er)

Tetrafluoroaurates(III) of Lanthanoides M₂F[AuF₄]₅ (M = Tb, Dy, Ho, Er) Tetrafluoroaurates(III) M₂F[AuF₄]₅ with M = Tb, Dy, Ho, Er, all yellow, have been obtained. From single crystal data they crystallize triclinic, space group P1 -C¹_i (No. 2) with Tb: a = 1 194,34(7) pm, b = 798,46(6) pm, c = 902,02(7) pm, α = 89,033(7)°, β = 88,990(6)°, γ = 89,006(7)°; Dy: a = 1 191,66(9) pm, b = 796,33(8) pm, c = 899,65(9) pm, α = 88,956(8)°, β = 89,056(8)°, γ = 88,972(8)°; Ho: a = 1 189,06(10) pm, b = 795,46(6) pm, c = 896,81(7) pm, α = 88,912(8)°, β = 89,101(7)°, γ = 88,873(8)°; Er: a = 1 185,20(40), b = 793,98(14), c = 893,83(20), α = 88,751(23)°, β = 89,187(26)°, γ = 88,884(9)°     

丹酚酸B与牛血清白蛋白相互作用的电化学研究

在0.01 mol.L-1磷酸盐缓冲溶液(pH 7.4)中,用循环伏安法和方波伏安法研究了丹酚酸B与牛血清白蛋白相互作用前后在DNA修饰玻碳电极上的电化学行为。实验表明,丹酚酸B在此修饰电极上于0.100 V处产生良好的氧化峰,加入牛血清白蛋白后,丹酚酸B的电子转移系数α和表观电子传递速率常数ks均发生了变化,氧化峰电位正移,峰电流减小。根据氧化电流的变化求得丹酚酸B和牛血清白蛋白相互作用的结合常数β=1.00×108L.mol-1,结合数m=1.71,表明丹酚酸B与牛血清白蛋白生成了结合比约为2∶1的非电活性复合物。该结果与荧光光谱法的研究结果一致。     

离子交换色谱法分离纯化鸡卵黄免疫球蛋白

建立了高效、经济、大规模获得鸡卵黄免疫球蛋白(IgY)的生产方法。在对传统的水稀释法改良的基础上,结合聚乙二醇沉淀与离子交换色谱进行IgY的分离纯化。结果显示,用8倍无菌水稀释蛋黄液,用0.1 mol/L HCl调节pH为5.2,在4 ℃下静置8 h,于5000×g力离心可得上清粗IgY液,经测定回收率可达93.47%。然后用6%聚乙二醇沉淀后经DEAE-Toyopearl 650 M离子交换纯化,最佳的纯化条件: 0.05 mol/L磷酸盐缓冲液(PBS, pH 7)平衡上样,0.075 mol/L PBS(pH 7)洗脱。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果显示所得的IgY的纯度为95.02%,活性保持率高达73.77%。本研究弥补了传统分离方法不能同时达到高纯度和高回收率的缺点,且可用于大规模生产。     

Mechanism for the phase transition of poly(butylene terephthalate)

The α and β forms of poly(butylene terephthalate) transform reversibly by elongation and relaxation. The conformation change occurs in the tetramethylene glycol part, from GGTGG conformation to TSTS?T conformation. In this study, by using a doubly oriented sample, we measured the positions, intensities, and half‐widths of the (100) and (010) reflections of the α and β forms of poly(butylene terephthalate) with a position‐sensitive proportional counter system. During the transformation, the molecules translate only slightly. These slight molecular translations, or distortions, accumulate, and the crystallite of the α form breaks into the small crystallites of the β form as the α–β transformation proceeds, and the crystallite of the α form grows with the relaxation of the distortion accumulated in the crystal and amorphous regions and on the crystallite surface as the β–α transformation proceeds. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 765–771, 2002     

Subunit separation (alpha,alpha',beta) of cryptomonad biliproteins

Abstract— Phycocyanin 645, phycocyanin 612 and phycoerythrin 545 are biliproteins isolated from the cryptomonad algae, Chroornonas species, Hemiselmis virescens and Rhodomonas lens, respectively. The protein has α and β subunits. which are separated on an ion exchange column by using a urea gradient and omitting 2-mercaptoethanol from the solvent. This separation establishes that the α and β subunits are not joined by disulfide bonds. In addition it has recently been shown that mercaptoethanol can produce spurious results in the calculation of the chromophore contents of these biliproteins (Guard-Friar and MacColl, 1984). The mercaptoethanol-free experiments allow analysis of the chromophore content in a rapid and artifact-free manner. When the ion exchange chromatography of phycocyanin 645 is manipulated by changing the type of urea gradient, two distinct a subunit fractions are obtained. These two fractions have identical visible absorption spectra but different amino acid compositions. At least two different gene products are, thus. responsible for the a subunits. The sole chromophore on the two a subunits of phycocyanin 645 is the unique 697-nm bilin as seen in acidic urea. Its reactivity with mercaptoethanol is determined. The α subunits of phycoerythrin 545 have two different bilins: cryptoviolin and phycoerythrobilin. Phycocyanobilin is the chromophore on the α subunit of phycocyanin 612.     

Crystal Structures and Characterizations of Bis(pyrrolidinedithiocarbamato)Cu（Ⅱ） and Zn(Ⅱ） Complexes

The structures of [Cu (S₂CN (CH₂)₄)₂] (1) and [Zn₂(S₂CN‐(CH₂)₄)₄] (2) have been determined by X‐ray crystallography analysis. They are all isomorphous and triclinic, space group of P1^?, with Z = 1. The lattice parameters of compound 1 is: a = 0.63483(2) nm, b = 0.74972(3) nm, c=0.78390(1) mn, α = 75.912(2)°, β = 78.634(2)° and γ = 86.845(2)°; compound 2: a = 0.78707(6) nm, b=0.79823(6) nm, c = 1.23246(9) nm, α = 74.813(2)°, β = 73.048(2)° and γ = 88.036(2)°. The copper atom is located on a crystallographic inversion center and zinc atom lies across centers of symmetry. The Cu(II) ion has a square‐planar geometry while Zn(II) has a distorted tetrahedral geometry. The thermal gravity (TG) data indicate that no structural transitions in the two compounds were abserved and the decomposition products can adsorb gas. Also they all have a high thermal stability.