超声和高压处理对牛血清白蛋白结构的影响

目的:研究牛血清白蛋白经过超声和高压处理前后的结构变化,以及自由基清除剂对其结构变化的影响.为进一步研究细胞破碎和乳化蛋白时超声和高压处理对蛋白的影响提供参考.方法:将BSA溶液经过超声和高压处理,然后利用高效液相色谱(HPLC)、动态光散射(DLS)、非变性聚丙烯酰胺凝胶电泳(nondenaturing SDS-PAGE)以及园二色谱(CD)等手段检测处理前后的结构变化.结果:发现超声使BSA严重聚集,加入自由基清除剂可以减少聚集的产生.而高压处理后的BSA未出现聚集,但二级结构发生了变化.结论:超声和高压这两种处理方法对蛋白损伤机理不同,所以选择合适的破碎方法要根据蛋白自身的性质而定.     

超声对胃蛋白酶，胰蛋白酶，过氧化氢酶作用的研究

以胃蛋白酶,胰蛋白酶,过氧化氢酶溶液在超声处理下的酶活变化为指标研究超声对蛋白质作用的机理和影响因素。结果表明超声对蛋白质的破坏程度随着功率的升高或处理时间的延长而增加;三种酶在超声作用下酶活变化形式和程度各不同;浓度是影响超声对酶作用效果的一个重要因素,可通过调整酶溶液浓度来减少酶所受到的破坏程度。自由基清除剂甘露醇和非离子表面活性剂吐温-80可以对酶活在超声作用下起到一定的保护作用,说明自由基和超声空穴是超声破坏酶结构的重要机理,研究结果同时表明对于不同的酶,超声的破坏作用可能有不同的发挥主导作用机理。     

类芽孢杆菌(Paenibacillus sp.)蛋白质组学研究中三种蛋白提取方法的比较分析

【目的】革兰氏阳性类芽孢杆菌（Paenibacillus sp.）本身细胞壁的结构特点导致其菌体全蛋白不易获得。本研究选取了3种破碎方法——溶菌酶联合超声破碎法（方法一）、溶菌酶联合SDS热处理破碎法（方法二）、液氮联合超声破碎法（方法三）进行革兰氏阳性菌的细胞破碎,以期获得适于样品菌株基于质谱技术进行蛋白质组学研究的制备方法。【方法】在蛋白样品的制备过程中,对3种不同破碎方法的蛋白提取得率和SDS-PAGE检测分析结果进行比较;随后将3种蛋白样品制备方法的样品用质谱技术进行鉴定,分析不同蛋白样品基于质谱技术鉴定蛋白的差异。【结果】在蛋白样品的制备提取过程中,不同破碎方法的蛋白提取率大致相同。用单因素方差比较3种提取方法质谱鉴定蛋白数的差异性,方法三鉴定的蛋白数最多（2 638个）,其次是方法一（2 452个）,方法二鉴定的蛋白数最少（2 003个）。进一步用韦恩图分析比较不同提取方法的蛋白鉴定通量差异,综合考虑蛋白提取效率的结果以及液氮研磨法提取蛋白的缺点,最终选取溶菌酶联合超声破碎法（方法一）提取菌株全蛋白作为该菌基于质谱分析其蛋白质组学研究中最适合的方法。最后,对质谱鉴定菌株蛋白包括分子量、等电点、疏水性的基本性质进行分析,发现3种破碎方法质谱鉴定的蛋白与模式菌株多黏类芽孢杆菌（Paenibacillus polymyxa）基因组中预测蛋白的各个组分分布占比基本一致,都保证了菌株蛋白质组数据信息的完整性。【结论】基于质谱技术开展革兰氏阳性类芽孢杆菌（Paenibacillus sp.）的蛋白质组学研究,溶菌酶联合超声破碎法是提取该菌株全蛋白最适合的方法。     

不同细胞破碎方法对无细胞蛋白表达系统细胞抽提物活性的影响

无细胞蛋白表达系统由于能够有效表达膜蛋白等有毒性蛋白,因此近二十年受到了关注,其蛋白表达产率有了显著的提高。细胞抽提物活性的高低是无细胞蛋白表达系统高效运行的关键,若找到简单易行的活性评估方法,将大大降低成本及时间。葡萄糖-6-磷酸脱氢酶 (glucose-6-phosphate dehydrogenase, G-6-PDH)是糖代谢的戊糖磷酸途径中的关键调控酶,该酶可以被用来评估细胞抽提物的活性。以G-6-PDH的活性为指标对无细胞蛋白表达系统中的抽提物活性进行评价,并利用G-6-PDH活性评价体系对机械破碎、高压破碎以及超声破碎三种破碎方法进行了比较,得出了三种破碎方法的最佳破碎条件。机械破碎最佳破碎条件是5 000r/min, 用直径0.1 mm玻璃珠,破碎6次;高压破碎的最佳破碎压力为1 300bar;超声破碎最佳破碎条件是功率强度为总功率的60%,破碎30次。酶活性测定结果显示机械破碎和超声破碎得到的抽提物活性比高压破碎得到的抽提物活性略高。     

乳液法静电纺丝PLGA组织工程缓释纤维的研究

目的：研究Dextran对蛋白药物的释放影响。方法：将模型蛋白BSA溶解于多糖溶液中,通过W/O乳液法静电纺丝制备缓释纤维。采用MicroBCA法测定该纤维体外释放行为,采用SEC-HPLC检测制备前后蛋白的聚集程度,并与不含多糖的BSA纤维做对照。结果：添加Dextran以后蛋白的包封率由52.68%提高到63.92%,第一天突释不大于药物载量的15%,对蛋白单体的保持达到85%以上。结论：Dextran可以改善一般组织工程纤维中蛋白药物的释放,提高蛋白药物在制剂、贮存、释放过程中的稳定性,增加纤维的载药量。     

超高压处理对蓝莓酚类物质生物可获得率及抗氧化性的影响

目的：探讨不同条件超高压处理对蓝莓酚类物质的生物可获得率以及抗氧化性的影响。方法：在不同压力下对蓝莓果实进行不同时间的超高压处理。分析测定不同处理条件下蓝莓中酚类物质的含量。通过测定1,1 二苯基 2 三硝基苯肼（DPPH）和2,2 联氮 双 3 乙基苯并噻唑啉 6 磺酸（ABTS）自由基清除能力评价超高压处理后蓝莓的抗氧化能力变化。利用超高效液相色谱 电喷雾串联四级杆质谱分析鉴定蓝莓中主要酚类物质及其含量变化情况。结果：不同压力与处理时间下,超高压处理均能提高蓝莓总酚、总黄酮和总花色苷的生物可获得率,其中在500MPa下处理5min效果最佳。DPPH和ABTS自由基清除能力实验结果表明,超高压处理可以显著提高蓝莓抗氧化性,在500 MPa处理5 min的蓝莓抗氧化能力最强。超高压前后共鉴定出9种酚类物质,其中7种为花色苷及其衍生物、2种类黄酮物质,这些物质中除了矮牵牛素外,其余物质在超高压处理后生物可获得率均显著增加。结论：超高压处理可以显著提高蓝莓中总酚、总黄酮及花色苷等酚类物质的生物可获得率,增强其清除自由基的能力,提高抗氧化性。     

60Co-γ射线对固体透明质酸分子量与抗氧化性的影响

利用60Co-γ射线对崮体透明质酸进行辐射,探讨60Co-γ射线对固体透明质酸分子量和黏度特性及抗氧化性的影响。结果表明：透明质酸的分子量和黏度特性随辐照剂量的增加而降低;对羟自由基（OH·）及超氧阴离子自由基（O2·）清除作用随着剂量的增大逐渐减弱,对DPPH·自由基清除作用和还原能力随着剂量的增大逐渐增强;辐照前后透明质酸外观品质没有明显的变化,流动性增强;辐照对透明质酸的紫外光谱结构没有明显的改变。     

ＴＮＦ—α基因在鱼腥藻７１２０中的表达及其产物的亲和层纯化

用转入ＴＮＦ－α基因并稳定有达了４年多的丝状体蓝藻－鱼腥藻７１２０（Anabaena sp.PCC7120),研究比较了在培养液中氨源（NaNO3)和碳源（NaAc)对转基因灌生长和ＴＮＦ－α基因的不同影响,氨源能稳定外源蛋白的表达,而碳源则影响细胞壁的合成。通过比较不同的破碎细胞的方法,发现超声破碎得到的可溶性总蛋白含量和ＴＮＦ－α含量均要比冻融法高出１倍左右,经过细胸破碎、硫酸铵分级沉淀、常压离子交换液相色析后,除去杂蛋白及大量容易堵柱的糖类等物质,最后通过亲和柱层析,分离出了纯的目标蛋白－重组肿瘤坏死因子（ＴＮＦ－α）。经凝胶电泳鉴定为单一的谱带,分子量为１７kD。     

硫醇基团的氧化还原过程对人正常朊蛋白聚集和纤维化特征的影响

本研究旨在通过体外对纯化的原核重组人正常朊蛋白硫醇基团的氧化还原过程,探究二硫键的改变对其生化特性的影响。蛋白沉淀实验显示重组正常人朊病毒蛋白经过硫醇基团的氧化还原过程明显增加了其聚集性;硫磺素T(Thioflavin T ,ThT)实验测定发现,经过硫醇基团的氧化还原过程重组PrP蛋白的纤维形成增多;圆二色谱（Circular Dichroism, CD）测定显示,处理后的重组PrP蛋白二级结构发生改变,其β-折叠结构比例显著增多;蛋白酶K消化实验也进一步显示硫醇基团的氧化还原后PrP的蛋白酶K抵抗能力有所增加。这些结果提示二硫键的形成可明显地改变PrP的二级结构,促进朊蛋白聚集和成纤维过程。     

大鼠肝细胞核膜脂质过氧化对核DNA影响的研究

采用大鼠肝脏细胞核为材料,研究了核膜脂质过氧化对核ＤＮＡ的影响。证实核膜脂质过氧化可以引起ＤＮＡ损伤,表现为ＤＮＡ的增色效应、熔解温度、从ＤＮＡ向ＥＢ的能量转移效率降低,圆二色谱发生显著变化。另外受损ＤＮＡ对ＤＮａｓｅⅠ产生明显抗性和琼脂糖凝胶电泳结果提示核膜脂质过氧化引起的ＤＮＡ损伤可能以交联为主,同时利用原子力显微镜（ＡＦＭ）直接观察到了交联ＤＮＡ的生成。各种自由基清除剂对ＤＮＡ损伤保护的差异说明脂类自由基可能在活性氧自由基引起的ＤＮＡ损伤中具有重要作用。     

The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in vitro models.

The OH(*) free radical scavenging properties of ascorbyl palmitate (AP), water-solubilized in the presence of a surfactant (Brij 35), were tested in various systems: (1) The inhibition of polymerization of bovine serum albumin by OH(*) free radicals generated by the Fenton reaction indicated AP exerts a considerable protective effect against polymerization by scavenging the OH(*) free radicals. (2) ESR spin trapping comparisons of DMPO with AP were conducted. Using the Fenton reaction as a source of OH(*) free radicals, AP was 1 order of magnitude faster in scavenging these radicals than DMPO. (3) Oxidative modification of BSA by (60)Co-gamma irradiation of 80 krad, results in a strong increase in protein carbonyl content. AP inhibits carbonyl formation very efficiently, indicating that AP may be utilized as a biological OH(*) free radical scavenger in human therapy.     

Effect of shear stress and free radicals induced by ultrasound on erythrocytes

The present study was undertaken to elucidate the mechanism of hemolysis induced by ultrasound. Ar or N2O gas was used to distinguish between cavitation with or without free radical formation (hydroxyl radicals and hydrogen atoms). Free radical formation was examined by the method of spin trapping combined with ESR. After sonication of erythrocyte suspensions, several structural and functional parameters of the erythrocyte membrane--hemolysis, membrane fluidity, membrane permeability, and membrane deformability--were examined. Although free radical formation was observed in the erythrocyte suspensions sonicated in the presence of Ar, no free radical formation was observed in the presence of N2O. However, the hemolysis behavior induced by ultrasound was similar in the presence of Ar or N2O. The membrane fluidity, permeability, and deformability of the remaining unlysed erythrocytes after sonication in the presence of Ar or N2O were unchanged and identical to those of the control cells. On the other hand, after gamma irradiation (700 Gy), the hemolysis behavior was quite different from that after sonication, and the membrane properties were significantly changed. These results suggest that hemolysis induced by sonication was due to mechanical shearing stress arising from cavitation, and that the membrane integrity of the remaining erythrocytes after sonication was the same as that of control cells without sonication. The triatomic gas, N2O, may be useful for ultrasonically disrupting cells without accompanying free radical formation.     

低强度超声促进放疗后犬下颌种植体骨结合的实验研究

目的：探讨放射线损伤犬的下颌骨行种植术后,低强度超声是否对其周围骨组织结合具有促进作用。方法：建立下颌骨放射线损伤的动物模型,并运用低强度超声对其进行治疗,通过Micro—CT、拔出实验等观察低强度超声治疗组（实验II组）与未用低强度超声治疗组（实验I组）,并与对照组对比,所得数据进行统计学分析。结果：成功的建立了放射线损伤的动物模型;实验II组骨小梁的各项指标均好于实验I组;拔出实验结果显示,实验II组拔出力值明显大于实验I组。统计结果显示各组之间的差异具有显著性的意义（P〈0．05）。结论：低强度超声对放射线照射后种植体周围的骨组织具有较好的修复作用     

Protein damage and degradation by oxygen radicals. III. Modification of secondary and tertiary structure

Proteins which have been exposed to the hydroxyl radical (.OH) or to the combination of .OH plus the superoxide anion radical and oxygen (.OH + O2- + O2) exhibit altered primary structure and increased proteolytic susceptibility. The present work reveals that alterations to primary structure result in gross distortions of secondary and tertiary structure. Denaturation/increased hydrophobicity of bovine serum albumin (BSA) by .OH, or by .OH + O2- + O2 was maximal at a radical/BSA molar ratio of 24 (all .OH or 50% .OH + 50% O2-). BSA exposed to .OH also underwent progressive covalent cross-linking to form dimers, trimers, and tetramers, partially due to the formation of intermolecular bityrosine. In contrast, .OH + O2- + O2 caused spontaneous BSA fragmentation. Fragmentation of BSA produced new carbonyl groups with no apparent increase in free amino groups. Fragmentation may involve reaction of (.OH-induced) alpha-carbon radicals with O2 to form peroxyl radicals which decompose to fragment the polypeptide chain at the alpha-carbon, rather than at peptide bonds. BSA fragments induced by .OH + O2- + O2 exhibited molecular weights of 7,000-60,000 following electrophoresis under denaturing conditions, but could be visualized as hydrophobic aggregates in nondenaturing gels (confirmed with [3H]BSA following treatment with urea or acid). Combinations of various chemical radical scavengers (mannitol, urate, t-butyl alcohol, isopropyl alcohol) and gases (N2O, O2, N2) revealed that .OH is the primary species responsible for alteration of BSA secondary and tertiary structure. Oxygen, and O2- serve only to modify the outcome of .OH reaction. Furthermore, direct studies of O2- + O2 (in the absence of .OH) revealed no measurable changes in BSA structure. The process of denaturation/increased hydrophobicity was found to precede either covalent cross-linking (by .OH) or fragmentation (by .OH + O2- + O2). Denaturation was half-maximal at a radical/BSA molar ratio of 9.6, whereas half-maximal aggregation or fragmentation occurred at a ratio of 19.4. Denaturation/hydrophobicity may hold important clues for the mechanism(s) by which oxygen radicals can increase proteolytic susceptibility.     

Fourier transform IR attenuated total reflectance spectroscopy studies of cysteine-induced changes in secondary conformations of bovine serum albumin after UV-B irradiation

Fourier transform IR spectroscopy equipped with attenuated total reflection was used to investigate the cysteine-induced alteration of the protein secondary structure of bovine serum albumin (BSA) in aqueous solution before and after UV-B irradiation. Several amino acids were also studied. The results indicate the unchanged IR spectra of BSA coincubated with amino acids, except cysteine, did not change after 72-h UV-B irradiation. There was no difference in the IR spectrum of the unirradiated BSA coincubated with cysteine. A shoulder at 1620 cm(-1) attributed to the intermolecular beta-sheet structure was observed for the IR spectrum of BSA coincubated with cysteine after 72-h UV-B irradiation. Moreover, the peak intensity at 1303 cm(-1) that is due the alpha-helix structure was reduced, but the peak intensity at 1247 cm(-1) corresponding to beta-sheet structures was increased. Longer UV-B exposure for a BSA solution coincubated with cysteine changed the BSA solution from clear to viscous to gel form in which a transparent gel and another white gel were simultaneously observed. A gradual IR spectral alteration was found for BSA coincubated with cysteine and subjected to increased UV-B irradiation. The longer UV-B irradiation yielded increased intensity at 1620 cm(-1). The second-derivative IR peaks at 1655, 1631, and 1548 cm(-1) were shifted to 1650, 1620, and 1544 cm(-1), respectively, by the increase of UV-B irradiation, suggesting a progressive transformation from an alpha-helix to an intermolecular beta-sheet structure for BSA coincubated with cysteine. This strongly implies that longer UV-B exposure time for the BSA solution in the presence of cysteine did alter the protein secondary structures of BSA more, thus inducing gel formation by protein aggregation.     

Reaction between protein radicals and other biomolecules

The present study investigates the reactivity of bovine serum albumin (BSA) radicals towards different biomolecules (urate, linoleic acid, and a polypeptide, poly(Glu-Ala-Tyr)). The BSA radical was formed at room temperature through a direct protein-to-protein radical transfer from H(2)O(2)-activated immobilized horseradish peroxidase (im-HRP). Subsequently, each of the three different biomolecules was separately added to the BSA radicals, after removal of im-HRP by centrifugation. Electron spin resonance (ESR) spectroscopy showed that all three biomolecules quenched the BSA radicals. Subsequent analysis showed a decrease in the concentration of urate upon reaction with the BSA radical, while the BSA radical in the presence of poly(Glu-Ala-Tyr) resulted in increased formation of the characteristic protein oxidation product, dityrosine. Reaction between the BSA radical and a linoleic acid oil-in-water emulsion resulted in additional formation of lipid hydroperoxides and conjugated dienes. The results clearly show that protein radicals have to be considered as dynamic species during oxidative processes in biological systems and that protein radicals should not be considered as end-products, but rather as reactive intermediates during oxidative processes in biological systems hereby supporting recent data.     

Effect of ionic strength on the binding of ascorbate to albumin

A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate. In the presence of BSA protein, the observed rate of the overall process is the sum of that from at least two reactions: the reaction between free ascorbate and free probe, and the reaction between bound ascorbate and bound probe. Our findings show that the observed rate is strongly dependent on the ionic strength of the medium. A corollary of this observation is the indication of a purely electrostatic interaction between ascorbate and the BSA protein. This conclusion was further corroborated by 1H NMR measurement of the transverse relaxation time, T(2), of ascorbate protons in BSA solutions. Ascorbate ion was released from the ascorbate/BSA ensemble in the presence of increasing concentrations of NaCl. Binding constants of AH(-) to BSA were calculated at different ionic strengths at pH 7.4. Furthermore, an increase in ionic strength did not affect the ability of albumin to protect ascorbate against autoxidation. This suggests that the protein's protective antioxidant effect may be attributed to BSA binding of trace quantities of transition-metal cations (rather than ascorbate binding to BSA). This conclusion is supported by ascorbate UV-absorption measurements in the presence of albumin and Cu(2+) ions as a function of ionic strength.     

糖基化终产物对人肾小球系膜细胞氧化应激及MCP-1表达的影响及机制

目的：探讨体外培养条件下糖基化终产物（AGEs）对人肾小球系膜细胞（HRMCs）中糖基化终产物受体（RAGE）、氧化应激及单核细胞趋化因子-1（MCP-1）表达的影响。方法：将HRMCs与不同浓度的糖化牛血清白蛋白（AGE-BSA）和牛血清白蛋白（BSA）共同培养,或与同一质量浓度的AGE-BSA和BSA共同培养不同时间,以中和抗RAGE抗体封闭细胞膜上RAGE;采用细胞免疫化学法检测AGEs对HRMCs中RAGE表达的影响,流式细胞术检测细胞内活性氧（ROS）,半定量逆转录-聚合酶链反应（RT-PCR）法检测MCP-1 mRNA的表达。结果：在HRMCs中AGE-BSA能够促进RAGE的表达,并以时间和剂量依赖方式促进HRMCs中ROS及MCP-1的表达;ROS及MCP-1的表达水平在加入不同浓度（50、100、200、400 mg/L）的AGE-BSA作用48 h后以及加入质量浓度为200 mg/L的AGE-BSA作用不同时间（12、24、48、72 h）后,较相应质量浓度或时间的BSA组和对照组均明显升高（P〈0.05）;抗RAGE抗体干预后能够部分抑制AGE-BSA诱导ROS及MCP-1的表达,而人IgG没有这种作用。结论：AGEs通过RAGE激活氧化应激效应诱导MCP-1的表达上调,是糖尿病肾病发生发展的可能机制。     

Planar supported bilayer polymers formed from bis-diene lipids by Langmuir-Blodgett deposition and UV irradiation

Substrate-supported lipid bilayers have been prepared from bis-diene functionalized phosphorylcholine (PC) lipids and polymerized by UV irradiation. The overall bilayer structure is largely preserved upon removal from water, although significant loss of material occurs from the upper leaflet of the bilayer, likely due to desorption at the air/water interface. The morphology and surface structure of the bilayer, as observed by AFM, indicate a substantially different arrangement of the lipids in the hydrated and dehydrated states, presumably due to the loss of water from the near surface region. These changes have been correlated with infrared spectral shifts sensitive to the conformation of the hydrocarbon chains. Protein adsorption studies show that rehydrated, polymerized bilayers retain a degree of resistance to BSA adsorption intermediate between model hydrophobic and fluid PC lipid bilayer surfaces. The degree of protein adsorption is correlated with desorption of material from the upper leaflet of the bilayer upon drying, which produces voids at which hydrophobically driven protein adsorption occurs.     

硬枝树花粗提物的抗氧化作用

采用DPPH和ABTS法对硬枝树花石油醚(30~60)、二氯甲烷、乙酸乙酯、甲醇等4个溶剂依次提取得到的浸膏物,进行抗氧化活性测定试验。结果表明:甲醇提取物,乙酸乙酯和二氯甲烷提取物在0.4~1.0 mg/mL检测浓度范围内对.DPPH的清除效果和质量浓度呈现一定的量效关系。甲醇提取物在1 mg/mL时,清除率高达93.99%,二氯甲烷提取物在1 mg/mL时清除率为92.32%,均高于同质量浓度的BHT。乙酸乙酯和二氯甲烷提取物在0.4~1.0mg/mL检测浓度范围内对ABTS.+自由基清除率与质量浓度呈现一定的量效关系,在1 mg/mL时,二氯甲烷提取物对ABTS.+自由基清除率为65.6%,乙酸乙酯提取物对ABTS.+自由基清除率为48%。