气相色谱法测定人白蛋白制品中辛酸钠含量

本文报导用气相色谱法测定人白蛋白制品中辛酸钠含量。样品用正庚酸作内标,经酸化、氯仿抽提、浓缩后,通过ＨＰ－ＩＮＮＯＷａｘ柱,以氢火焰检测器测定。本法色谱峰形好,平均回收率及变异系数分别为９８．５４％、１．３９％。测定线性范围在１０７μｇ／０．２５ｍｌ～７２０μｇ／０．２５ｍｌ。最小检测限为７．４１０μｇ。实验操作简便,样品及溶剂用量少,可作为人白蛋白制品中辛酸钠含量的检测方法。     

人血清白蛋白基因的打靶研究

为获得整合有人血清白蛋白(HSA)基因的猪胎儿成纤维细胞克隆,从猪基因组文库中杂交筛选得到猪血清白蛋白(PSA)基因全序列35kb并克隆了人血清白蛋白cDNA序列,扩增猪血清白蛋白基因5′调控序列7.2kb片段及第一内含子至第四内含子2.8kb的片段;构建了含有neo及tk正负筛选标记基因的人血清白蛋白基因打靶载体,并验证了neo基因的有效性。将线性化的打靶载体通过电击转染的方法整合到猪胎儿成纤维细胞基因组中,利用G418及GANC进行细胞克隆的抗药性筛选,PCR及Southern blot鉴定抗药性细胞克隆,最终获得3个发生同源重组的细胞克隆。这为下一步进行体细胞核移植制备生产人血清白蛋白转基因猪奠定了基础。     

Ligand Binding to the FA3-FA4 Cleft Inhibits the Esterase-Like Activity of Human Serum Albumin

The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of K _s, k ₊₂, and k ₊₂/K _s obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k ₊₃ << k ₊₂). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pK _a-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>_a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.     

Stabilization of Colicin E2 by Bovine Serum Albumin

Colicin E2 was partially purified from Escherichia coli W3110. This preparation was remarkably stabilized by bovine serum albumin in a solution at neutral pH, as shown by dilution experiments and tests on heat stability of colicin. One killing unit of colicin E2 was estimated to correspond to one molecule of colicin E2, on the assumption of a molecular weight of 60,000.     

Characterization of Sulfhydryl Heterogeneity in Human Serum Albumin and Recombinant Human Serum Albumin for Clinical Use

Since human serum albumin has one sulfhydryl group and 17 disulfides, reactive sulfhydryl groups give rise to heterogeneity. The present paper presents a comparison of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use. Low molecular weight sulfhydryl compounds were identified from both sources. The recombinant albumin had a much higher sulfhydryl content than plasma serum albumin.     

人血清白蛋白与镍离子相互作用的热力学研究

1　Introduction　　Serumalbuminproteinsareamongthemosthighlystudiedandappliedinbiochemistry[1～ 4].Albuministhemostabundantproteininbloodplasmaandoneofitsmainfunctionsisbasedonauniqueabilitytobindnumerousendogenousandexogenouscompounds.Duetoitsligandbindingpropertiesalbuminservesasacirculatingdepotofsomemetabolites.Thisdepoteffectisoftenmadeuseofindrugtherapy.　　Humanserumalbumin(HSA)isasinglepeptidechainconsistingof 5 85aminoacids( 6 6 5ku)asdeterminedbyaminoacidsequencestudies[5] andasde…     

Resveratrol Binding to Human Serum Albumin

Abstract

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419–426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclo- oxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents μM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure.

Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K_Res = 2.56× 10⁵ M^?1. The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of α-helix from 57% (free HSA) to 62% and a decrease of β-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.     

重组人血清白蛋白生产工艺研究进展

人血清白蛋白是一种重要的临床药物 ,人源血浆白蛋白可能会传染病原体将逐步被重组白蛋白所代替。对用巴斯德毕赤酵母生产重组人血清白蛋白的表达系统、发酵方法、分离纯化技术以及重组白蛋白的理化及生理特性进行了综述。     

人血清白蛋白荧光的红际激发效应

研究了人血清白蛋白中色氨酸的荧光红际激发效应,阐明了人血清白蛋白的红际激发效应与ＰＨ,温度及尿素浓度存在一定的关系,外界条件的改变使得色氨酸残基所处的微环境发生了变化,使得色氨酸残基的基态和激发态的能级产生不同的分布,从而得到不同的红际激发效应。     

胡椒碱分子键合人血清白蛋白位点的表征

利用荧光光谱法、紫外光谱法并结合计算机模拟技术在分子水平上研究了胡椒碱与人血清白蛋白(human serum albumin HSA)的键合作用.同步荧光及紫外光谱图表明,胡椒碱对HSA微环境有影响.位点竞争试验证明,胡椒碱分子键合在HSA的位点Ⅱ区.通过荧光光谱滴定数据求得不同温度下(300K 310K和318 K)药物与蛋白相互作用的结合常数及结合位点数.分子模拟的结果显示了胡椒碱与HSA的键合区域和键合模式,表明药物与蛋白有较强的键合作用;维持药物与蛋白质的相互作用力主要是疏水用,兼有氢键(位于氨基酸残基Arg 257,Arg 222及Arg218位).通过实验数据计算得到的热力学参数(ΔH0与ΔS0的值分别为原33.11 kJ·mol-1和原18.90 J·mol原1·K-1)确定了胡椒碱与HSA分子的相互作用力类型主要为氢键兼范德华力.     

人血清白蛋白纯化技术研究进展

本文综述了国内外关于血浆人血清白蛋白（pHSA）和基因重组人血清白蛋白（rHSA）分离纯化的进展和发展趋势。以冷乙醇沉淀为主的pHSA分离方法仍是目前多数工业采用的工艺,但近年来发展的离子交换色谱、凝胶过滤色谱、亲和色谱等多种色谱分离技术具有自动化程度高、生产周期短、更符合GMP等特点,正在逐步取代传统的冷乙醇沉淀法。当前用基因工程技术表达的人血清白蛋白对分离纯化提出了新的挑战。为获得高纯度、安全、稳定的产品,各种色谱分离技术得到更多的应用,其中扩张床离子交换色谱可以省去离心、过滤等传统固液分离操作。尽管rHSA的纯化技术已有一定的发展,但纯化过程仍需进一步优化以提高产品纯度及收率。     

芦丁与人血清白蛋白相互作用的紫外可见光谱特性研究

本文通过测定芦丁与HSA相互作用前后的紫外可见吸收光谱、圆二色性及人血清白蛋白(HSA)的荧光特性,研究了芦丁与HSA结合作用。结果表明,芦丁在紫外区有三个特征的吸收峰(264.0、285.5及354.5nm)、在330～300 nm及300～230 nm处显示圆二色性,HSA引起芦丁紫外可见吸收光谱波峰红移;芦丁与HSA相互作用后,不引起HSA二级结构的改变,但对其三级结构有影响,同时对HSA荧光激发及发生光谱最大峰位及幅度有影响。     

人和牛血清白蛋白的极谱测定

人或牛血清白蛋白溶液调至ｐＨ１０并在４℃左右放置３０ｈ,诱导出重复性良好的极谱峰。然后把试样溶液调至适当ｐＨ并记录极谱电流。在浓度低于１．０×１０－５ｍｏｌ／Ｌ时,峰电流对浓度作图是直线,试样浓度可从图上直接读出。对影响白蛋白测定的诸因素作了详细研究和讨论。结果对富含硫的蛋白质的极谱研究有参考价值。     

The Investigation of the Binding of 6-Mercaptopurine to Site I on Human Serum Albumin

6-Mercaptopurine (6-MP) is one of a large series of purine analogues which has been found active against human leukemias. The equilibrium dialysis, circular dichroism (CD) and molecular docking were employed to study the binding of 6-MP to human serum albumin (HSA). The binding of 6-MP to HSA in the equilibrium dialysis experiment was detected by measuring the displacement of 6-MP by specific markers for site I on HSA, warfarin (RWF), phenylbutazone (PhB) and n-butyl p-aminobenzoate (ABE). It was shown, according to CD data, that binding of 6-MP to HSA leads to alteration of HSA secondary structure. Based on the findings from displacement experiment and molecular docking simulation it was found that 6-MP was located within binding cavity of subdomain IIA and the space occupied by site markers overlapped with that of 6-MP. Displacement of 6-MP by the RWF or PhB was not up the level expected for a competitive mechanism, therefore displacement of 6-MP was rather by non-cooperative than that the direct competition. Instead, in case of the interaction between ABE and 6-MP, when the little enhancement of the binding of ABE by 6-MP was found, the interaction could be via a positively cooperative mechanism.     

重组人血清白蛋白表达研究进展

本文综述了重组人血清白蛋白在细菌,酵母,植物和动物等表达系统中表达的研究进展。用酵母表达系统,尤其是毕赤酵母,表达的重组白蛋白产量高且提取工艺简单,是其产业化最具有前途的表达系统。同时在转基因动物、植物中培养表达也具有诱人的前景。     

The Use of Macro-Aggregates of Radioiodinated Human Serum Albumin in Brain Scanning

Intra-arterially injected macroaggregates of radioiodinated human serum albumin were used in the detection of brain tumours in mice and in human subjects. The mean size of particle used was 29 μ. Brain-to-tumour ratios in ependymoblastoma-bearing mice ranged from 43.5:1 to 2.8:1. Autoradiographs of brain and tumour from sacrificed mice indicated that most of the macroaggregates were trapped within cerebral and cerebellar grey matter. Tumour retained little activity. Particles within small vessels did not cause complete obstruction, although temporary vessel spasm, cessation and reversal of flow were observed. Twelve patients with clinically evident brain tumours received carotid or vertebral injections of macroaggregates. Ten had histologically proved tumours, and six of these were correctly localized by external scanning. The quality of the scans was not superior to that obtained with other tracers currently in use. Because four patients developed transient neurological complications that could be attributed to the procedure, the clinical pilot study was terminated.     

人血清白蛋白融合技术研究进展

结合笔者所在课题组的实验研究成果,综述人血清白蛋白融合技术研究进展。人血清白蛋白融合技术是近些年来应用较多的用 于改造小分子蛋白和多肽药物的手段。但是众多的研究结果发现将小分子蛋白/多肽和人血清白蛋白融合后会出现一些共性的问题,例 如目标蛋白产率低、易降解等。这些共性问题可以通过融合蛋白的优化设计、蛋白酶缺陷宿主的构建、融合基因多拷贝以及与分子伴侣 共表达得以解决     

重组人血白蛋白临床研究进展

人血白蛋白是人血浆中最丰富的蛋白质,具有许多重要的生理特性,用途广泛。目前主要以毕赤酵母作为宿主表达的重组人血白蛋白,开发了重组人血白蛋白的纯化技术,同时对重组人血白蛋白结构进行了分析,结果表明与人血浆白蛋白基本一致。临床研究结果表明重组人血白蛋白与人血浆白蛋白有着几乎相同的疗效和安全性。综述了重组人血白蛋白的性质结构分析及酵母表达系统;重点介绍了重组人血白蛋白在临床方面研究进展。     

Existence of Different Structural Intermediates on the Fibrillation Pathway of Human Serum Albumin

The fibrillation propensity of the multidomain protein human serum albumin (HSA) was analyzed under different solution conditions. The aggregation kinetics, protein conformational changes upon self-assembly, and structure of the different intermediates on the fibrillation pathway were determined by means of thioflavin T (ThT) fluorescence and Congo Red absorbance; far- and near-ultraviolet circular dichroism; tryptophan fluorescence; Fourier transform infrared spectroscopy; x-ray diffraction; and transmission electron, scanning electron, atomic force, and microscopies. HSA fibrillation extends over several days of incubation without the presence of a lag phase, except for HSA samples incubated at acidic pH and room temperature in the absence of electrolyte. The absence of a lag phase occurs if the initial aggregation is a downhill process that does not require a highly organized and unstable nucleus. The fibrillation process is accompanied by a progressive increase in the β-sheet (up to 26%) and unordered conformation at the expense of α-helical conformation, as revealed by ThT fluorescence and circular dichroism and Fourier transform infrared spectroscopies, but changes in the secondary structure contents depend on solution conditions. These changes also involve the presence of different structural intermediates in the aggregation pathway, such as oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates. We suggest that fibril formation may take place through the role of association-competent oligomeric intermediates, resulting in a kinetic pathway via clustering of these oligomeric species to yield protofibrils and then fibrils. The resultant fibrils are elongated but curly, and differ in length depending on solution conditions. Under acidic conditions, circular fibrils are commonly observed if the fibrils are sufficiently flexible and long enough for the ends to find themselves regularly in close proximity to each other. These fibrils can be formed by an antiparallel arrangement of β-strands forming the β-sheet structure of the HSA fibrils as the most probable configuration. Very long incubation times lead to a more complex morphological variability of amyloid mature fibrils (i.e., long straight fibrils, flat-ribbon structures, laterally connected fibers, etc.). We also observed that mature straight fibrils can also grow by protein oligomers tending to align within the immediate vicinity of the fibers. This filament + monomers/oligomers scenario is an alternative pathway to the otherwise dominant filament + filament manner of the protein fibril's lateral growth. Conformational preferences for a certain pathway to become active may exist, and the influence of environmental conditions such as pH, temperature, and salt must be considered.