A Novel Protein Assay With an Azo Dye Using Rayleigh Light Scattering Technioque

ABSTRACT

The interaction of SPADNS [3-(4-sulfophenylazo)-4, 5-dihydroxy-2, 7-naphthalene disulfonic acid] with proteins in acidic solution was studied by spectrophotometric and Rayleigh light scattering (RLS) methods. SPADNS reacts rapidly with albumin at room temperature, and the intensity of the weak RLS of SPADNS was enhanced remarkably and quantitatively by this reaction.

Based on this observation, a novel RLS method for serum albumin determination was developed. The linear range is 0.125-14.9 μg/ml for BSA. The relative standard deviation (n=10) for 5.0 μg/ml BSA was 3.94%. The method was applied to the determination of proteins in human plasma, and the results were compared with the traditional proteins assay (CBB method). There is almost no interference from amino acids, metal ions and other coexistent substances. The reaction mechanism was also discussed.     

甲基紫共振瑞利散射光谱法测定透明质酸钠

在pH5的乙酸盐缓冲介质中,甲基紫与透明质酸钠作用形成结合产物时将导致溶液共振瑞利散射(RRS)大大增强并产生新的RRS光谱,其最大散射峰位于341 nm处。透明质酸钠在0~2.5 mg.L-1范围内其浓度与RRS强度成正比。该法具有高灵敏度,对透明质酸钠的检出限(3σ)为20.1μg.L-1,选择性较好。此法用于测定从鸡冠组织中提取的透明质酸的粗品,测定结果的平均相对标准偏差为2.3%,加标平均回收率为98.9%。     

用偶氮胂M分光光度法测定蛋白质

研究了蛋白质与偶氮胂 M的显色反应。在含 0 .0 5 0 % OP的 p H2 .5的缓冲溶液中 ,偶氮胂 M与蛋白质形成兰色复合物 ,λmax为 60 5 nm,ε为 4.5× 1 0 5L· mol- 1· cm- 1,蛋白质含量在 3.3～ 2 5 4 mg/L范围内 ,服从比耳定律。所提出的方法可直接用于血清、花生等生物样品中蛋白质含量测定     

高氯酸盐与十六烷基三甲基溴化铵作用的光散射

利用光散射光谱法研究了高氯酸根和阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)的作用。 在酸性条件下,高氯酸根和CTAB通过静电作用形成离子缔合物,导致体系光散射强度增强。 环境水样中的常见阴离子如Cl^-、Br^-、ClO₃^-、NO₃^-和PO₄^3-等与CTAB单独作用时其光散射强度很弱,而当它们与高氯酸根同时存在时,由于协同作用使体系散射强度发生改变。 以Cl^-为例,借助动态光散射测定,初步探讨了体系协同作用的机理。     

Clean analytical methodology for the determination of lead with Arsenazo III by cyclic flow-injection analysis

A clean analytical methodology, where the sample determined and the reagent used are both toxic, has been proposed. A cation exchange mini-column was incorporated after the flow-through cell for on-line regeneration of the main reagent and the accumulation of heavy metal ions. The method involves the spectrophotometric determination of lead with Arsenazo III, the accumulation of lead onto the cationic exchanger, and subsequent regeneration of the chromogenic reagent, which makes the system reversible and the reagents reusable. The developed method provides a satisfactory way to cut down on the toxic reagent consumption and the produced volume of waste, an important step towards the zero emissions research initiative concept. The excellent repeatability and reproducibility, and the simplicity of this method are well suited for continuous measurements. The method was successively applied to the assay of lead in high-octane gasoline.     

Determination of Trace Proteins by Rayleigh Light Scattering Technique with Indophenol Blue

The interaction of indophenol blue (IPB) with proteins in aqueous solution has been studied by optical absorption and Rayleigh light scattering (RLS) spectroscopy. At pH 3.8, the weak RLS of IPB is enhanced by proteins. Based on this phenomenon, a novel method for the determination of proteins at nanogram levels using the RLS technique is developed. The method is simple, practical and sensitive. The linear range is 0.25–20.9µgmL^–1 for BSA, and 0.25–17.6µgmL^–1 for HSA. The detection limits (S/N=3) are 23ngmL^–1 for BSA and 22ngmL^–1 for HAS. The results for the determination of proteins in human serum samples are very close to those obtained by an established clinical method. There is very little interference from amino acids, metal ions or other coexisting compounds.     

以偶氮胂K光度法测定蛋白质

研究了偶氮胂Ｋ（Ａｒｓｅｎａｚｏ Ｋ）与蛋白质的反应及利用此反应测定蛋白质的最佳条件。偶氮胂Ｋ与蛋白质生成的复合物的最大吸收波长为６１１ｎｍ,比偶氮胂Ｋ红移７１ｎｍ?对照性子。在室温下,于pＨ２．８～３．９的缓冲溶液中,反应迅速完成。表面活性剂ＴｒｉｔｏｎＸ－１００和溴化十六烷基吡啶（ＣＰＢ）均可提高反应的灵敏度复合物稳定,干扰甩。绘制了牛血清白蛋白（ＢＳＡ）、γ－球蛋白（γ－Ｇ）、人血清白蛋白（     

金纳米微粒与盐酸氯丙嗪相互作用的共振Rayleigh散射光谱研究及其分析应用

在pH 1.8~3.3的酸性介质中,金纳米微粒本身有一定的共振瑞利散射(RRS)强度,但盐酸氯丙嗪本身的RRS强度十分微弱,当二者共存时,溶液的RRS强度显著增强并出现新的RRS光谱,在280~368 nm之间产生强烈的散射带,其最大散射波长位于368 nm,并在284、440、498 nm处有明显的散射峰。在一定条件下,盐酸氯丙嗪在0~0.08 mg/L范围内与ΔIRRS强度成正比,方法具有较高的灵敏度,对盐酸氯丙嗪的检出限(3σ)达到1.75μg/L。本文考察了反应体系的RRS光谱特征,研究了适宜的反应条件、影响因素及分析化学性质,研究了共存物质的影响,表明方法具有较好的选择性,据此发展了一种用金纳米微粒作RRS探针测定盐酸氯丙嗪的新方法。     

三甲基品红与脱氧核糖核酸作用的共振光散射光谱的研究

在pH为0.85的三羟甲基氨基甲烷和盐酸 (Tris-HCl)缓冲溶液中 ,三甲基品红(NM)与脱氧核糖核酸(fsDNA,ctDNA)分子作用后共振光光散射增强 ,其强度增加值与DNA的浓度呈线性关系 ,据此 ,建立了一种测定DNA的共振光散射法。该方法线性范围分别为0.02～6.0mg·L -1,0.04～7.0mg·L -1,相关系数分别为0.9984、0.9987 ,检出限分别为18.3、21.8μg·L-1,用于DNA合成样品的测定获得了满意的结果。同时 ,对该反应机理进行了探讨 ,并用紫外 -可见光谱求出了其形成常数     

瑞利光散射技术快速测定萝卜红色素中痕量Pb(Ⅱ)

建立了快速测定Pb(Ⅱ)的瑞利散射(RLS)新方法。在Tris-HCl缓冲介质中,溴化十六烷基三甲基铵(CTMAB)增敏溴甲酚绿(BCG),使瑞利散射显著增强,最大瑞利散射峰位于345nm,CTMAB-BCG进一步与Pb(Ⅱ)结合后生成的三元复合物的瑞利散射增强程度(|ΔIRLS|)与0.006~0.41mg/L范围Pb(Ⅱ)的质量浓度呈线性关系,定量限为0.053mg/kg。方法用于萝卜红色素中Pb(Ⅱ)的测定,回收率为98.5%~102%,相对标准偏差为1.8%~2.5%。     

亮黄-曲通X-100体系共振瑞利散射法测定蛋白质

基于在TrtionX-100存在下，蛋白质对有机染料亮黄瑞利光散射的增强作用。拟定了测定蛋白质的瑞利光散射光，由于添加了TritonX-100，对不同的蛋白质，测定的灵敏度提高了5－11．8倍，线性范围在0－5．0mg/L之间，最低检测限为10．2μg/L。     

Evaluation of Calcium - Arsenazo III Stoichiometry in the Presence and Absence of Egta at Various pH

Abstract

The calcium titration experiments were performed with 9 uM and 100 uM concentrations of Arsenazo III (Az) at various pH°s in the presence as well as in the absence of ethylene glycol-bis-(2-amino ethylether)-N,N°-tetra-acetic acid (EGTA). Analysis of results indicated that in the presence of EGTA in 9 uM Az solutions the Ca-Az stoichiometry changes from 1:1 to two Ca binding with one Az (2:1) upon increase in pH from 6.55 to 7.26. In the same systems but without EGTA the binding stoichiometry remains 1:1 throughout the pH range 6.55 to 7.32. In case of 100 uM Az III, however, EGTA has no effect on Ca-Az stoichiometry and it is 1:1 in the above pH range. An estimate of dissociation constants (K_D) in EGTA containing systems showed a simultaneous decrease in K_D by three orders of magnitude with a change in stoichiometry from 1:1 to 2:1 in the case of 9 uM Az III. At 100 uM dye concentration, with no change in 1:1 binding stoichiometry, K_D also remained constant throughout the pH range studied. We suggest that at lower dye concentration a competition between Ca-Az and Ca-EGTA binding causes this change in stoichiometry. The discrepancy among the previous reports about Ca-Az stoichiometry appeared to be due to change in pH in the presence of EGTA.     

光散射技术在研究高分子溶液和凝胶方面的应用

从溶液中的高分子、凝胶粒子及微乳胶粒子形态结构的表征和凝胶化过程、微乳液聚合及大分子缔合等动态过程的跟踪等方面的研究综述了近十几年来光散射技术在高分子溶液和凝胶领域的应用,并简单介绍了光散射技术的基本原理、发展简史和仪器及使用方法。     

用共振光散射技术研究蛋白质与丽春红2R的相互作用

在pH3．50的Britton-Robinson缓冲溶液中，丽春红2R与蛋白质发生结合反应，使最大波长为352．5nm的共振散射光谱得到加强。据此建立了利用丽春红2R作探针，用共振散射光谱技术测定蛋白质的新方法。散射光强度与牛血清白蛋白(BSA)、人血清白蛋白(HAS)和免疫球蛋白(IgG)的浓度在0．25～17．5μg／mL范围内成正比。方法的稳定性好，快速、简便，绝大多数氨基酸、金属离子均不产生干扰，用于人血清样品中蛋白质的测定，获得了满意的结果，加标回收率为93．4％～100．1％。     

人血清白蛋白共振RayIeigh光散射检测氟离子

通过研究F^-离子对人血清白蛋白(HSA)溶液共振Rayleigh光散射(RLS)的增强效应,建立一种基于生物蛋白RLS增强测定环境中氟离子的新型检测方法。实验条件为λex=λem=368nm,pH=5．80,t=32℃。结果表明,应用含氟离子溶液与空白溶液在368nm处RLS差值(△／),标准曲线法测定样品中的氟离子,方法线性范围为4．78～16．2μg／mL,r=0．992,检出限为1．43μg／mL。方法精密度(RSD为1．31％～5．72％)和准确度(回收率R为91％～109％)较好。     

某些同多酸根与蛋白质相互作用的共振瑞利散射光谱研究

在酸性介质中,钼酸根、钨酸根和偏钒酸根等同多酸根与牛血清白蛋白（BSA）和人血清白蛋白（HSA）等蛋白质反应形成结合产物时,会导致溶液的共振瑞利散射（RRS）急剧增强,最大散射峰均位于470nm。反应的适宜酸度分别为pH0．9～1．3（钼酸根．BSA体系）,pH1．1—1．4（钨酸根-BSA体系）和pH0．6—0．9（偏钒酸根-BSA体系）。在一定的浓度范围内,不同的反应体系RRS强度与蛋白质浓度成正比,均可用于蛋白质的测定。反应具有很高的灵敏度,不同的同多酸对BSA的检出限（30σ／s）介于4．1—30．5μg/L之间,其中钼酸根体系的灵敏度最高。考察了共存物质的影响并研究了方法的分析应用。     

考马斯亮蓝-SLS体系共振瑞利散射法测定蛋白质

研究了考马斯亮蓝与蛋白质的结合反应。在十二烷基磺酸钠的存在下,于pH1.6的酸性介质中,蛋白质与考马斯亮蓝可形成复合物,使最大波长351 nm的共振光散射光谱得到加强,据此,用于蛋白质的定量测定。十二烷基磺酸钠的加入,可使灵敏度提高3.2倍。本法测定牛血清白蛋白、人血清白蛋白、卵蛋白、γ-球蛋白的线性范围分别为:0.04~0.8、0.03~0.5、0.04~1.00、.06~1.2 mg/L,检出限分别为:6.3、4.7、7.9、9.2μg/L。已用于人血清、牛奶、豆浆、尿液中蛋白质的测定,所得结果与经典的考马斯亮蓝法一致。     

Ru-PAA复合纳米粒子与蛋白质相互作用的光谱研究及其应用

以过硫酸钾作为引发剂,丙烯酸为单体,在超声作用下原位聚合制备聚丙烯酸(PAA)修饰Ru纳米复合颗粒(Ru-PAA).修饰后的Ru-PAA复合纳米颗粒,不仅水溶性和稳定性得到改善,同时具有良好的生物相容性.研究了Ru-PAA复合纳米颗粒与蛋白质的相互作用,并利用散射光谱法考察了光散射强度与蛋白质浓度之间的关系.结果发现: 在最佳的实验条件下,BSA浓度为0.05～1.2 mg/L、HSA浓度为0.05～3 mg/L时,其浓度与光散射强度均有良好的线性关系,检出限均为0.01 mg/L.该方法用于实际样品测定,得到了令人满意的结果.     

光散射技术在蛋白质晶体生长研究中的应用和进展

光散射技术广泛应用于生物大分子的晶体生长研究中,它包括静态光散射和动态光散射两种。利用静态光散射可以测定蛋白质溶液渗透的第二维里系数;利用动态光散射可以测定蛋白质溶液的平动扩散系数,获得溶液中蛋白质粒子的流体力学半径及分布情况,分离蛋白质结晶的成核与生长过程,研究大分子的聚集行为和晶体生长的动力学。借助光散射技术可以实现蛋白质晶体生长过程的动态控制。近些年光散射仪器向着小型化、轻便化的方向发展,光散射技术不断得到改进,日益完善,不仅用于地面实验,也应用于空间领域蛋白质晶体生长的研究中。     

Mutual Separation and Determination of Th(IV) and U(VI) Using Arsenazo III as a Dye Collector Reagent by Flotation‐Spectrophotometric Method

This paper reports a simple and highly selective method for the separation, preconcentration, and determination of trace amounts of thorium and uranium in some complex samples via staircase flotation. The method is based on the initial flotation of the Th(IV)‐arsenazo III complex in the presence of U(VI) from a solution of 5 mol dm^?3 HCl, then reduction of U(VI) to U(IV) and repetition of the flotation step. In both steps, the floated complex was dissolved in a 5‐mL portion of methanol and its absorbance was measured at 655 nm, spectrophotometrically. For a 30‐mL portion of the sample, Beer's law was obeyed over the concentration ranges of 3.40 × 10^?7to 3.06 × 10^?6 mol dm^?3 for Th(IV) and3.40 × 10^?7 to 3.40 × 10^?6 mol dm^?3 for U(IV) with the apparent molar absorptivity of 4.20 × 10⁵ dm³ mol^?1 cm^?1 and 3.59 × 10⁵ dm³ mol^?1 cm^?1, respectively. The RSDs (n = 7) corresponding to 1.7 × 10^?6 mol dm^?3 of Th(IV) and U(IV) were obtained as 1.7% and 1.87%. The detection limits (7 blanks) for both the metal ions were found to be 1.7 × 10^?7 mol dm^?3. The important benefit of the method is that the determinations are free from the interference of almost all cations and anions found in the complex matrixes, such as seawater samples. The proposed method was also applied to reference materials, and the determinations were shown to have good agreement with the certified values.