DBC-偶氮羧共振光散射法测定蛋白质

基于蛋白质对DBC-偶氮羧光散射增强的效应,拟定了一种测定蛋白质的新方法.在pH 4.1的Britton -Robinson(B-R)缓冲溶液中,蛋白质与DBC-偶氮羧结合,产生强烈的共振光散射.在362 nm处,共振光散射强度较大,且光散射强度与加入的蛋白质浓度在一定范围内呈线性关系,其中牛血清白蛋白(BSA)在0.05～7 mg·L-1、人血清白蛋白(HSA)在0.05～8 mg·L-1、溶菌酶(Lyso) 在0.05～7 mg·L-1.该法用于人血清总蛋白含量的测定,结果令人满意.     

吡咯红Y-SDS-蛋白质体系的共振光散射光谱研究

在硫酸存在下,微量蛋白质的加入可使吡咯红Y-SDS体系弱的共振光散射信号得到极大的增强。在λ=364 nm处,光散射强度最大,并且光散射增强强度与蛋白质的质量浓度在一定范围内呈线性关系,由此建立了一种测定蛋白质的新的分析方法。对牛血清白蛋白(BSA)和人血清白蛋白(HSA)进行测定,其线性范围分别为0.15~3.6μg/mL和0.06~4.8μg/mL,检测限分别为21.0 ng/mL和12.0 ng/mL。该方法用于人血清样中蛋白总含量的测定,结果令人满意。     

邻苯二酚紫共振光散射光谱法测定蛋白质的研究

基于人血清白蛋白(HSA)对邻苯二酚紫的共振光散射的增强效应，拟定了一种测定HSA的新的共振光散射体系。在pH2．56的Brltton-Robinson缓冲溶液中，邻苯二酚紫与HSA结合导致共振光散射增强，在λ=515．0nm处共振光散射有最大的强度，并且共振光散射强度与HSA的浓度在0．0～20．0μg／mL范围内有良好的线性关系，检测限可达0．18μg／mL。该方法简便、快速，用于人体血清样品的测定并与经典的考马斯亮蓝法比较，结果满意。     

茜素红共振散射法测定硫酸庆大霉素

在pH5.5的Britton-Robinson介质中,茜素红和硫酸庆大霉素(GEN)相互作用形成缔合物,使体系的共振散射信号(RLS)增强,最大散射峰位于312 nm处,且增强的RLS强度与GEN浓度成正比,据此建立了一种测定硫酸庆大霉素含量的新方法。测定硫酸庆大霉素的线性范围是0.15~1.5 U/mL,检出限为0.029 U/mL。将方法用于硫酸庆大霉素注射液中硫酸庆大霉素含量的测定,结果满意。     

曙红Y共振光散射法测定水中十六烷基三甲基溴化铵

利用共振光散射(RLS)技术,通过曙红Y与阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)缔合产生强烈共振光散射增强效应,建立了一种测定阳离子表面活性剂CTAB的方法.实验结果表明,在pH=4.96的柠檬酸钠-盐酸缓冲介质中,采用吐温80作为曙红Y -CTAB体系的稳定剂,痕量CTAB的加入导致曙红Y在440～ 600 nm波长内的共振光强度增加,最大RLS峰位于538 nm处,其强度与CTAB的质量浓度在0～40 mg·L-1内成正比,检出限为0.012 mg·L-1.方法具有较高的灵敏度和良好的选择性,可用于水样中CTAB的测定.     

双波长瑞利光散射测定药物中的地奥司明

建立了以亮绿作探针测定药物中地奥司明的快速、准确、高灵敏瑞利光散射(RLS)新方法。在pH 9. 17的碱性溶液中,亮绿与地奥司明作用生成二元离子缔合物,使瑞利光散射信号明显增强并产生具有2个明显RLS峰的新瑞利散射光谱。在370 nm和469 nm波长处,地奥司明的质量浓度在一定范围内与体系的RLS增强强度的绝对值(|ΔIRLS|)呈线性关系,检出限分别为0. 003 8 mg/L(370 nm)和0. 004 3 mg/L(469 nm)。当用双波长RLS法测定地奥司明时,其检出限为0. 002 0 mg/L。地奥司明的质量浓度在0. 005～0. 85 mg/L范围内与单波长法或双波长法的|ΔIRLS|呈线性关系。该法用于市售地奥司明药物中地奥司明的测定,加标回收率为98. 4%～102%,相对标准偏差RSD(n=5)为1. 2%～1. 7%。     

吖啶红-碘化钾体系共振光散射法测定水中痕量铅

在20 mmol/L的硫酸溶液中,铅(Ⅱ)与过量的碘化钾形成[PbI4]2-配阴离子,再与碱性阳离子染料吖啶红形成离子缔合物,产生稳定增强的共振光散射,其最大RLS波长位于420 nm处,在5.16×10-8～8.0×10-7mol/L范围内,Pb(Ⅱ)浓度与RLS强度ΔI成正比,检出限为1.55×10-8mol/L。该方法灵敏、反应条件温和、易操作,适用于环境水样中铅的测定。     

钨酸钠共振光散射法测定维多利亚蓝B

在pH=5.33缓冲溶液中,维多利亚蓝B(Victoria blue)与钨酸钠作用并产生以281 nm和373 nm为特征峰的共振光散射(RLS)增强信号。在上述特征波长下测定的增强共振光散射强度(ΔIRLS)与维多利亚蓝B浓度在一定范围内呈线性关系。当钨酸钠浓度为4.0×10-5mol/L时,维多利亚蓝B的检出限可分别达8.74 nmol/L和10.77 nmol/L。据此建立了痕量维多利亚蓝B的共振光散射分析方法。     

铬天青B共振光散射技术测定药物中的维生素B12

探究了十六烷基溴化吡啶鎓(HPB)存在下铬天青B与维生素B₁₂的缔合反应,建立了测定药物中维生素B₁₂的高灵敏共振光散射(RLS)新方法。在pH 7.69的弱碱性溶液中,维生素B₁₂与铬天青B-HPB反应,以静电引力相互作用生成三元离子缔合物,使RLS信号明显增强在最大RLS峰371 nm波长处体系的RLS增强强度(△I(RLS)与0.003～2.71 mg/L范围内的维生素B₁₂的质量浓度呈很好的线性关系,检出限为0.002 5 mg/L。该方法简便、快速、准确、灵敏用于药物中维生素B₁₂的测定加标回收率为98.2%～102%相对标准偏差RSD(n=5)为1.5%～1.8%。     

聚甲基丙烯酰氧乙基苄基二甲基氯化铵与核酸相互作用的共振光散射光谱及分析应用

用核酸与聚阳离子聚甲基丙烯酰氧乙基苄基二甲基氯化铵(PMBDAC)的相互作用导致共振光散射(RLS)增强的现象来测定核酸。考察了pH值、PMBDAC浓度和离子强度对体系共振光散射强度的影响。在优化条件下,建立了用RLS光谱测定微量核酸的新方法。方法的抗干扰能力较强,可允许大部分的常见金属离子、核苷酸、氨基酸、糖、蛋白质等干扰物质的存在。     

A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G

The therapeutic application of small recombinant antibody molecules is often limited by a short serum half-life. In order to improve the pharmacokinetic properties, we have investigated a strategy utilizing fusion with an albumin-binding domain (ABD) from streptococcal protein G. This strategy was applied to a bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. This novel tri-functional fusion protein (scDb-ABD) was expressed in mammalian cells and recognized both antigens as well as human and mouse serum albumin. scDb-ABD was capable to retarget T cells to CEA-expressing target cells in vitro and to activate the effector cells as measured by stimulation of IL-2 release. Although activity was reduced 3-fold compared with scDb and further reduced 4-fold in the presences of human serum albumin, this assay demonstrated that scDb-ABD is active when exposed to all three antigens. Compared with scDb, the circulation time of scDb-ABD in mice was prolonged 5- to 6-fold similar to a previously described scDb-HSA fusion protein. This strategy, which adds only a small protein domain (46 amino acids) and which utilizes high-affinity, non-covalent albumin interaction, should be broadly applicable to improve serum half-lives of small recombinant antibody molecules.     

阳离子型Gemini表面活性剂共振光散射法测定DNA及相互作用机理的研究

在酸性介质(pH=3.51)中,脱氧核糖核酸(DNA)能显著地增强阳离子型Gemini表面活性剂(G14-4-14)的共振光散射信号,据此提出了G14-4-14共振光散射法测定DNA的新方法,并对介质酸度、离子强度、G14-4-14浓度及共存物质干扰等实验条件进行了优化。在最佳条件下绘制了工作曲线,该方法线性范围为0～2760μg.L-1,检出限为7.79μg.L-1,用于合成样品的测定,结果令人满意。以芘(pyrene)为荧光探针,应用稳态荧光法探讨了G14-4-14与DNA的相互作用,并运用红外光谱及共振光散射光谱初步探讨了G14-4-14与DNA相互作用的机理。     

荧光法研究Gemini表面活性剂与BSA的相互作用

应用荧光法研究了Gemini表面活性剂(G14-3-14)与牛血清白蛋(BSA)在不同温度条件下的荧光猝灭现象,利用荧光猝灭双倒数图计算了G14-3-14与BSA之间的结合常数,并根据热力学参数确定了二者之间的作用力类型.用内源荧光法求得它们在30℃和35℃温度下的结合常数分别为K1=1.01×105 L·mol-1和...     

淀粉、蛋白质及聚丙烯酸之间相互作用的共振光散射光谱研究

基于共振光散射光谱(RLS)探索了牛血清白蛋白(BSA)与淀粉及聚丙烯酸(PAA)三者之间的相互作用机理,并分析了BSA与淀粉之间质量比率(MR=MBSA/MStarch)对三者之间相互作用的影响。结果表明,淀粉和BSA通过各自与PAA之间氢键作用而形成一种交联三元复合物,且结合过程优先发生在PAA与BSA之间。淀粉、BSA和PAA是构成此三元复合物的初级元素,淀粉/PAA和BSA/PAA是此三元复合物的二级结构单元。此外,它们之间的相互作用还与BSA溶液浓度的高低有着密切关系。     

金橙G共振光散射法测定脱氧核糖核酸

研究了生物染色荆金橙G（OG）与脱氧棱糖核酸（DNA）相互作用的共振光散射光谱。结果发现,DNA对OG的共振光散射有增强效应,且在特定波长（358nm）下,其增强值（△I）与加入DNA的浓度呈良好的线性关系,据此建立了一种定量测定DNA的共振光散射法。该方法线性范围为0．053-1．20μ·mL^-1,检出限为0．0159μ·mL^-1,回收率为98．6％-102．3％。该方法简便、快速,用于合成样品中DNA的测定,结果令人满意。     

Albumin Suppresses Human Hepatocellular Carcinoma Proliferation and the Cell Cycle

Many investigations have revealed that a low recurrence rate of hepatocellular carcinoma (HCC) is associated with high serum albumin levels in patients; therefore, high levels of serum albumin are a major indicator of a favorable prognosis. However, the mechanism inhibiting the proliferation of HCC has not yet been elucidated, so we investigated the effect of serum albumin on HCC cell proliferation. Hep3B was cultured in MEM with no serum or containing 5 g/dL human albumin. As control samples, Prionex was added to generate the same osmotic pressure as albumin. After 24-h incubation, the expressions of α-fetoprotein (AFP), p53, p21, and p57 were evaluated with real-time PCR using total RNA extracted from the liver. Protein expressions and the phosphorylation of Rb (retinoblastoma) were determined by Western blot analysis using total protein extracted from the liver. For flow cytometric analysis of the cell cycle, FACS analysis was performed. The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACScalibur (BD) with appropriate software (ModFit LT; BD). The cell proliferation assay was performed by counting cells with using a Scepter handy automated cell counter (Millipore). The mRNA levels of AFP relative to Alb(−): Alb(−), Alb(+), and Prionex, were 1, 0.7 ± 0.2 (p < 0.001 for Alb(−)), and 1 ± 0.3, respectively. The mRNA levels of p21 were 1, 1.58 ± 0.4 (p = 0.007 for Alb(−) and p = 0.004 for Prionex), and 0.8 ± 0.2, respectively. The mRNA levels of p57 were 1, 4.4 ± 1.4 (p = 0.002 for Alb(−) and Prionex), and 1.0 ± 0.1, respectively. The protein expression levels of Rb were similar in all culture media. The phosphorylation of P807/811 and P780 of Rb protein was reduced in Alb(+). More cells in the G0/G1 phase and fewer cells in S and G2/M phases were obtained in Alb(+) than in Alb(−) (G0/G1: 60.9%, 67.7%, 61.5%; G2/M: 16.5%, 13.1%, 15.6%; S: 22.6%, 19.2%, 23.0%, Alb(−), Alb(+), Prionex, respectively). The same results were obtained in HepG2. Cell proliferation was inhibited in 5 g/dL albumin medium in both HepG2 cells and Hep3B cells in 24 h culture by counting cell numbers. The presence of albumin in serum reduces the phosphorylation of Rb proteins and enhances the expression of p21 and p57, following an increase in the G0/G1 cell population, and suppresses cell proliferation. These results suggest that albumin itself suppresses the proliferation of hepatocellular carcinoma.     

Effect of adsorbed metal ions and buffer nature on IgG separation from human plasma by column chromatography using an ion exchange resin,Amberlite IRC‐718

Fractionation of human plasma on ion exchanger resin was performed on Amberlite IRC‐718 saturated with metal ions. Depletion of human immunoglobulin G was carried out by column chromatography using Tris‐HCl, pH 7 at different concentrations. Results showed that, when Cu⁺² and Ni⁺² were adsorbed on the resin, one or two fractions of purified IgG were obtained, respectively. Whereas Fe⁺² and Zn⁺², both retain IgG and serum albumin or serum albumin alone. Furthermore, the Ni⁺²‐resin retention of serum proteins is too strong that the use of 700 mMTris‐HCl cannot liberate any other proteins than nonadsorbed serum albumin. In conclusion, this investigation demonstrates that immobilized metal ion affinity chromatography with Cu²⁺, Ni²⁺, and Fe²⁺ immobilized on Amberlite IRC‐718 has the potential to be developed as part of a process to purify IgG out of untreated human plasma as acceptable adsorption and elution levels of IgG could be achieved. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

苯甲酸与牛血清白蛋白相互作用的荧光光谱研究

采用荧光光谱、三维荧光光谱、同步荧光光谱研究了苯甲酸与牛血清白蛋白(BSA)分子之间的相互作用机制、特征及苯甲酸对牛血清白蛋白构象的影响。根据不同温度下苯甲酸对牛血清白蛋白的荧光猝灭作用,利用Stem-Volmer和Lineweaver-Burk方程和热力学方程分别处理实验数据,求得它们之间的结合常数K为1.096×103L/mol、结合位点数n为1.12(20℃),ΔH=-31.8 kJ/mol,ΔG=-17.1 kJ/mol,ΔS=-50.2 J.mol-1.K-1。研究发现苯甲酸对BSA内源性荧光的猝灭作用属于静态猝灭,苯甲酸与BSA分子之间的相互作用是一个吉布斯自由能降低的自发过程,两者之间的主要作用力类型为氢键力或范德华力。     

Simultaneous depletion of albumin and immunoglobulin G by using twin affinity magnetic nanotraps

For biomarker discovery, simultaneous removal of albumin and immunoglobulin G (IgG) become more important for both concentrating other species including potential biomarkers and getting rid of their masking effect. In this study, we have proposed a covalent and photosensitive cross-linking conjugation of the biomolecules on nanostructures for the depletion of target proteins from aqueous solution and serum. The effect of concentration, pH, temperature and ionic strength on the adsorption of twin affinity nanotraps based on Cibacron Blue F3GA and Protein A were investigated. The efficiency of albumin and IgG depletion from human serum was performed using sodium dodecylsulfate–polyacrylamide gel electrophoresis.     

Moderator Effect of Hypoalbuminemia in Volume Resuscitation and Plasma Expansion with Intravenous Albumin Solution

Intravenous administration of crystalloid or colloid solutions is the most common intervention for correcting hypovolemia in intensive care unit patients. In critical illness, especially sepsis and severe trauma, vascular wall permeability increases, and trans-endothelial escape of serum albumin, the major oncotic plasma constituent, contributes to the development of hypoalbuminemia and edema formation. The volume effects of intravenous human albumin solution exceed those of crystalloid solutions. If hypoalbuminemia is an effect moderator, the crystalloid-to-albumin ratio of fluid resuscitation volumes is not well characterized. Randomized controlled trials have confirmed that intravenous administration of human albumin solutions for volume resuscitation results in a lower net fluid balance compared with crystalloids, and smaller infusion volumes may be sufficient for hemodynamic stabilization when human albumin solutions are used. This narrative review summarizes the current evidence and conclusions drawn regarding the role of hypoalbuminemia in volume resuscitation. In the ‘Saline versus Albumin Fluid Evaluation’ study using 4% human albumin solution or saline, the saline-to-albumin ratio of study fluids was significantly higher in patients with baseline serum albumin concentrations of 25 g/L or less as compared to patients with baseline serum albumin concentrations of more than 25 g/L. In patients receiving renal replacement therapy, intravenous administration of 20–25% human albumin solution reduces intradialytic hypotension and improves fluid removal better than saline if serum albumin levels are similarly reduced. These data suggest that hypoalbuminemia acts as an effect moderator in volume resuscitation and plasma expansion with albumin solution. The volume effectiveness of intravenous human albumin solution in resuscitation appears to be greater when the serum albumin levels are low. In clinical situations, serum albumin concentrations per se may inform when and how to include intravenous albumin in fluid resuscitation if large amounts of crystalloids are needed, which requires further studies.