Highly sensitive spectrophotometric determination of human serum albumin with 3',4',5',6'-tetrachlorogallein-molybdenum(VI) complex

A highly sensitive spectrophotometric determination of human serum albumin (HSA) with 3',4',5',6'-tetrachlorogallein (T.Cl.Gall)-Mo(VI) complex in a Triton X-100 + polyvinyl alcohol micellar medium is proposed. This method can be used to determine up to ca. 150 micrograms/10 ml of HSA from the optical absorbance at 640 nm, and is superior in sensitivity to the other extremely sensitive spectrophotometric methods. The great sensitivity of this method results from the use of third-derivative spectrophotometry. The binding parameters of T.Cl.Gall-Mo(VI) complex to HSA are n = 77.3 and K = 1.05 x 10(4) M-1 as determined from dual double-reciprocal plots. It is suggested that the colored complex in this system may be the association complex between [HSA]m+ and [MoVI(T.Cl.Gall)2]n- involving hydrophobic interaction between HSA and T.Cl.Gall. The proposed method should also be useful for the detection and determination of some peptides (e.g. low molecular weight peptides containing basic amino acids), as well as proteins.     

亲和毛细管电泳法研究锌离子与人血清白蛋白的结合反应机制

建立了研究金属离子与人血清白蛋白(Human serum albumin,HSA)相互作用的亲和毛细管电泳(Affinity capillary electrophoresis,ACE)方法。生理条件下,构建配体(Zn2+)-受体(HSA)相互作用模型,以N,N-二甲基甲酰胺(N,N-Dimethylformamide,DMF)为内标物,基于Scatchard方程,依据有效淌度的变化,通过非线性模拟方程计算Zn2+-HSA结合反应的表观结合常数KB,定量表征了Zn2+-HSA相互作用的强度,并解析电泳谱图获得了Zn2+-HSA结合反应为一快平衡体系的结论。结果表明,建立的ACE方法简捷、有效,Zn2+-HSA相互作用的强度与Zn2+浓度之间存在明显的量效关系。     

Fluorimetric study of the interaction between human serum albumin and quinolones-terbium complex and its application

A highly sensitive spectrofluorimetric method is proposed for determination of human serum albumin (HSA) and some quinolone drugs. Using quinolones-terbium (Tb3+) complex as a fluorescent probe, in the buffer solution of pH 7.8, HSA can remarkably enhance the fluorescence intensity of the quinolones-Tb3+ complex at 545 nm and the enhanced fluorescence intensity of Tb3+ ion is in proportion to the concentration of HSA and quinolone drugs. Optimum conditions for the determination of HSA were also investigated. The linear ranges and limits of detection are 8.0 x 10(-9) to 8.0 x 10(-8) mol L(-1), 4.20 x 10(-9) mol L(-1) (for HSA); 1.0 x 10(-6) to 4.0 x 10(-6) mol L(-1), 1.87 x 10(-8) mol L(-1) (for norfloxacin) and 1.0 x 10(-7) to 1.0 x 10(-6) mol L(-1), 4.82 x 10(-8) mol L(-1) (for enoxacine), respectively. This method is simple, practical and relatively free interference from coexisting substances, as well as much more sensitive than most of the existing assays.     

Application of arsenazo III for the polarographic detection of proteins

In this paper, a diazo dye of arsenazo III (AAIII) was selected as a new electrochemical probe for the determination of proteins. In Britton-Robinson (B-R) buffer solution of pH 2.4, AAIII had a sensitive second order derivative linear sweep voltammetric reductive peak at ?0.39 V (vs. SCE). After the addition of human serum albumin (HSA) into AAIII solution, an interaction was taken place in the mixed solution and a biosupramolecular complex was formed, which resulted in the decreased reductive peak currents of AAIII. Based on the observed decrease in peak current, a sensitive electrochemical method was proposed for the determination of different proteins such as HSA, bovine serum albumin (BSA) and bovine hemoglobin (BHb). The optimal conditions for the interaction and the interfering effects of coexisting substances on the detection were investigated. The proposed method was successfully applied to the determination of HSA in synthetic samples with the recoveries in the range of 99.13–100.50%. The stoichiometry of HSA-AAIII biocomplex was calculated by voltammetric data with a binding number of 2 and a binding constant of 7.53 × 10⁹.     

A sensitive and rapid method for the determination of protein by the resonance Rayleigh light-scattering technique with Pyrogallol Red

The resonance Rayleigh light-scattering (RRLS) technique was used to develop a simple, sensitive and selective method for the determination of proteins. The method is based on the interaction between proteins and Pyrogallol Red (PR) in the pH range 3.6-4.2, which causes a substantial enhancement of the resonance scattering signal of PR in the wavelength range 300-450 nm with the maximum scattering peak located at 347 nm. With this method, 0.25-13 microg ml(-1) of bovine serum albumin (BSA), 0.25-10 microg ml(-1) of human serum albumin (HSA) and 0.25-13 microg ml(-1) of human immunoglobulin G (IgG) can be determined, and the detection limits, calculated as three times the standard deviation of nine blank measurements, for BSA, HAS and IgG were 51, 48 and 57 microg l(-1), respectively. Moreover, the method shows almost no protein-to-protein variability and is free from interference from many amino acids and metal ions. The method, with high sensitivity, selectivity and reproducibility, was satisfactorily applied to the determination of the total protein in human serum and saliva samples. Mechanism studies indicated that PR can bind to BSA depending mainly on electrostatic forces, and this interaction can encourage the J-aggregation of PR, which results in enhanced Rayleigh light-scattering in the PR-protein system.     

Spectrofluorimetric determination of human serum albumin using a doxycycline-europium probe

A new spectrofluorimetric method is proposed for determination of human serum albumin (HSA) with the limit of detection at ng levels. Using doxycycline (DC)-europium (Eu³⁺) as a fluorescent probe, in a buffer solution of pH 10.2, HSA can remarkably enhance the fluorescence intensity of the DC-Eu³⁺ complex at 612 nm and the enhanced fluorescence intensity of Eu³⁺ is proportional to the concentration of HSA. Optimum conditions for the determination of HSA are also investigated. The linear ranges for HSA are 0-9.2 and 9.2-34.5 μg ml⁻¹ with limits of detection of 64 and 115 ng ml⁻¹, respectively. This method is simple, practical and relatively free of interference from coexisting substances, as well as much more sensitive than most of the existing assays. The determination results for human serum and urine samples are identical to those by the AOAO method, with relative standard deviations of five determinations of 1.1-3.6%. By the Rosenthal graphic method, the binding number and association constant of human serum albumin with the probe are 1.8 and 3.71×10⁵ l mol⁻¹, respectively.     

Study of the interaction between daunorubicin and human serum albumin,and the determination of daunorubicin in blood serum samples

The interactions between daunorubicin and human serum albumin (HSA) were studied using the spectrofluorimetric method. The binding constants of daunorubicin with HSA were obtained at different temperatures. The effects of various metal ions on the binding constants of daunorubicin with HSA were also studied. The optimum conditions of fluorometric determination of daunorubicin were studied and the developed method was successfully applied to the determination of daunorubicin in serum samples.     

Electrochemical Study on the Interaction of Protein with Bromothymol Blue and Its Analytical Application

The interaction of bromothymol blue（BB） with human serum albumin（HSA） was studied by electrochemical techniques and a sensitive method for proteins assay was developed. When BB interacted with HSA, the voltammetric peak current value of BB decreased linearly with the concentration of HSA in a range of 1.0--40.0 mg/L, and the peak potential shifted negatively. Based on the results, a sensitive assay method for proteins, such as HSA, bovine serum albumin（BSA）, and egg albumin etc. was established. This method was further applied to determining the HSA in healthy human blood samples, and the results are not significantly different from those obtained by the classic Coomassie Brilliant Blue G-250 spectrophotometic method. The detecting conditions of this method were optimized and the interaction mechanism was discussed. The results show that the electrochemical parameters（formal potential E^0, standard rate constant of the electrode reaction ks, parameter of kinetic nα） of BB have no obvious changes before and after the interaction, which indicate that BB can interact with HSA, forming an electrochemical non-active complex. The equilibrium constant（βs） and the binding ratio（m） for this complex were calculated. The m is 4 and βs is 1.41 × 10^19. This method is fast, simple, highly sensitive, and has good selectivity, which can be used in clinical measurements.     

A rapid and sensitive method for the determination of trace proteins based on the interaction between proteins and Ponceau 4R

The interactions between proteins and Ponceau 4R (PR) in aqueous solution have been studied by the techniques of resonance light scattering (RLS) spectroscopy, the absorption spectroscopy, zeta potential assay and circular dichroism (CD) spectrum. The dry PR can assemble on the surface of protein via electrostatic and hydrophobic forces to produce an associated compound of protein-PR, this compound can enhance the RLS of protein. Based on this fact, a simple, rapid, and sensitive method has been developed for the determination of proteins at nanogram level by RLS technique with a common spectrofluorimeter. Under optimum conditions, the linear range is 0.10-39.2 μg mL⁻¹ for the determination of both bovine serum albumin (BSA) and human serum albumin (HSA). The detection limits (S/N = 3) are 6.96 ng mL⁻¹ for BSA and 5.71 ng mL⁻¹ for HSA, respectively. There is almost no interference from amino acids, most of the metal ions, and other coexistent substances. The method has been satisfactorily applied to the direct determination of the total protein in human serum.     

人血清白蛋白与氨基酸对映异构体的差异性识别研究

以人血清白蛋白为识别主体,利用表面等离子体共振(SPR)技术实时研究了其与5种氨基酸对映异构体相互作用过程中的动态行为。实验结果显示,每种氨基酸分子的L-和D-型异构体在与人血清白蛋白作用过程中均存在明显的动力学差异。动力学数据分析表明,虽然各种氨基酸的L-和D-型异构体与人血清白蛋白作用的结合速率常数、解离速率常数无统一规律,但在解离平衡常数或结合平衡常数上有一致性。每种氨基酸的L-型异构体与人血清白蛋白的亲和力均大于D-型,这表明人血清白蛋白在与氨基酸对映异构体作用的过程中基于手性识别作用而产生了明显的差异性结合。     

Protein binding of quinolonecarboxylic acids. II. Spectral changes on the interaction of cinoxacin, nalidixic acid and pipemidic acid with human and rat albumins

The interaction of cinoxacin (CINX), nalidixic acid (NA), and pipemidic acid (PPA) with human and rat serum albumins (HSA and RSA) was studied by UV difference absorption and circular dichroism (CD) spectroscopy. CINX and NA bound to the albumins and generated difference absorption and induced CD (ICD) spectra. The difference absorption spectral data explained reasonably our previous observations that CINX bound to HSA more weakly than NA, but to RSA as strongly as NA. We used a quantity delta epsilon/epsilon, designated as relative molar difference absorbance, at positions corresponding to the longest wavelength peaks in the difference spectra. The quantity was found to correlate linearly with percent bound to both HSA and RSA, but with different slopes, from which the binding site for CINX and NA in RSA was supposed to provide a much more nonpolar environment than that in HSA. The magnitude of ICD bands observed at 371 nm for CINX and at 342-348 nm for NA corresponded to the binding degrees of these drugs to both albumins. Anisotropy factors for the ICD bands at 350-271 nm for CINX and 320-348 nm for NA were approximately similar between HSA and RSA, suggesting a similar ability to generate the ICD spectra in these wavelength regions upon binding to the albumins. Spectral results for PPA in albumin solutions showed little or no binding of this drug to HSA and RSA. PPA existed as a betaine form in neutral solution and its positively charged group acted as an unfavorable factor for binding to both albumins.     

Rapid and sensitive determination of protein by light-scattering technique with Eriochrome Blue Black R

Based on the interaction between Eriochrome Blue Black R (EBBR) and proteins, which causes a strong light-scattering signal with the maximum scattering peak located at 398 nm, a simple, rapid, sensitive and selective method is developed for the determination of proteins by the light-scattering technique using a common spectrofluoremeter. Under proper experimental conditions, the protein determination can be performed in the range of 0.1-25, 0.1-20 and 0.25-25 μg ml⁻¹ for bovine serum albumin (BSA), human serum albumin (HSA) and human immunoglobulin G (IgG), respectively. The detection limit, calculated as 3 times the S.D. of nine blank measurements, are 33 μg l⁻¹ for BSA, 25 μg l⁻¹ for HSA and 38 μg l⁻¹ for IgG. Moreover, there is no significant difference among the scattering signals yielded by HSA, IgG and BSA, and almost no interference of many amino acids and metal ions. The method has been satisfactorily applied to the direct determination of the total protein in human serum, saliva and urine samples. The results obtained from the studies on the binding characteristics of EBBR to BSA indicated that an electrostatic force existed in the binding system, and the binding constant (K) and the number of the binding sites (n) at 25 °C are 1.69×10⁵ l mol⁻¹ and 0.946, respectively.     

高效亲和色谱法测定2种中药成分与人血清白蛋白的结合率

采用高效亲和色谱技术(HPAC)对中药成分与人血清白蛋白(HSA)的相互作用进行了研究。首先采用点击化学的方法制备了表面键合有HSA蛋白的硅胶固定相并装填成亲和色谱柱,根据药物在该色谱柱上与空白硅胶柱上的保留时间差计算得到药物与蛋白的结合率。利用该方法测得模型化合物华法令与HSA的结合率与文献中采用超滤法测得的结果基本一致,表明该方法可用于测定药物与HSA的结合率。在此基础上用该方法测定了葛根素和告依春两种中药成分与HSA的相对结合率分别为10.26%和10.20%。同时用超滤的方法测定了葛根素与HSA的结合率为14.25%。结果表明,HPAC可以作为研究药物与蛋白相互作用的一种简便可行的方法,其测定结果与超滤方法一致。     

A high performance method for thermodynamic study on the binding of human serum albumin with erbium chloride

Thermodynamics of the interaction between erbium(III) chloride, Er³⁺, with human serum albumin (HSA), was investigated at pH 7.0 and in phosphate buffer by isothermal titration calorimetry. Our recently, solvation model was used to reproduce the enthalpies of HSA interaction by Er³⁺ over a broad range of metal ion concentration. The solvation parameters recovered from our new model, attributed to the structural change of HSA and its biological activity. The binding parameters for the interaction of Er³⁺ and HSA indicate that the concentrations of Er³⁺ have no significant effects on the structure of HSA.     

A Thermodynamic Study on the Binding of Human Serum Albumin with Lanthanum Ion

Thermodynamics of the interaction between lanthanum(III) ion, La³⁺, and human serum albumin (HSA), was investigated at pH 7.0 and 300 K in Tris‐HCl buffer by isothermal titration calorimetry. A solvation model was used to reproduce the enthalpies of HSA interaction with La³⁺. The solvation parameters recovered from our new model were attributed to the structural change of HSA and its biological activity. The interaction of HSA with La³⁺ showed a set of two binding sites with negative cooperativity.     

Microdetermination of proteins with the arsenazo-DBN-Al(III) complex by Rayleigh light-scattering technique and application of the method

The determination of proteins with arsenazo-DBN and Al3+ by Rayleigh light-scattering (RLS) is described. The weak RLS of arsenazo-DBN and BSA can be enhanced greatly by addition of Al3+ in the pH range 5.3-7.0; this resulted in two enhanced RLS signals at 420-440 nm and 460-480 nm. The reaction between arsenazo-DBN, Al3+, and proteins was studied and a new method was developed for quantitative determination of proteins. This method is very sensitive (0.34-41.71 microg mL(-1) for bovine serum albumin, BSA, and 0.29-53.41 microg mL(-1) for human serum albumin, HSA), rapid (< 2 min), simple (one step), and tolerant of most interfering substances. The effects of different surfactants were also examined. When these proteins were determined in four human serum samples the maximum relative error was not more than 2% and the recovery was between 97 and 103%.     

A New Electrochemical Method for the Determination of Proteins with Cupferron‐Cadmium(II) Complex

A new electrochemical method was proposed for the determination of trace amounts of proteins based on the cupferron (Cup) and cadmium(II) complex [Cup‐Cd(II)] as the voltammetric probe. In the selected pH 6.5 Britton–Robinson (B–R) buffer solution, Cup can interact with Cd(II) to form a stable complex of [Cup‐Cd(II)], which had a sensitive linear sweep voltammetric reductive peak at ?0.654 V (vs. SCE). The addition of human serum albumin (HSA) into [Cup‐Cd(II)] complex solution could greatly decrease the reductive peak current without the change of the reductive peak potential, which indicated that HSA could interact with [Cup‐Cd(II)] complex to form a supramolecular biocomplex. The interaction mechanism was discussed and the decrease of reductive peak current was proportional to the concentration of HSA, which could be further used for the proteins determination. The optimal conditions of the binding reaction and the electrochemical detection were carefully investigated. Under the optimal conditions a new quantitative determination method for different kinds of proteins such as HSA, bovine serum albumin (BSA) and bovine hemoglobin (BHb) etc. was developed. The proposed method was simple, practical and relatively free from the interferences of coexisting substances, and it was further applied to the samples determination with satisfactory results. The binding constant (β_s) and the binding number (m) of HSA with [Cup‐Cd(II)] complex were calculated by the voltammetric data with the results as β_s=1.12×10⁶ and m=1.     

毛细管电泳法考察全氟辛酸与人血清白蛋白的相互作用

利用毛细管电泳（CE）技术建立了全氟辛酸（PFOA,C₈HF₁₅O₂）与人血清白蛋白（HSA）相互作用的分析方法。在生理条件下构建配体（PFOA）-受体（HSA）相互作用模型,通过淌度移动法、区段-区段动力学（plug-plug kinetic,PPK）法、简化的Hummel-Dreyer（HD）法研究其与HSA的相互作用。简化的HD法运用非线性方程、Scatchard方程和Klotz方程获得PFOA-HSA体系的相互作用参数,进而分析了模型适用度。结果表明,淌度移动法、PPK法、简化的HD法均适用于PFOA-HSA体系相互作用的分析,其中简化的HD法最优。模型适用度分析得出非线性回归方程为最适理论模型。相互作用参数测试表明PFOA-HSA相互作用体系之间发生的结合反应只有单一类型的结合位点且结合稳定。相关工作阐明了人血清白蛋白与PFOA的相互作用机制,可为PFOA毒理机制的深入研究提供有益参考。     

Determination of proteins by flow injection analysis coupled with the Rayleigh light scattering technique

A novel flow injection analysis (FIA) method with Rayleigh light scattering (RLS) detection was developed for the determination of protein concentrations. This method is based on the weak intensity of RLS of p-nitrohenzene-azo-3,6 disulfo-1-amino-8-naphthol-7-azo-benzene disodium salt (Amide Black-10B) which can be enhanced by addition of protein in weakly acidic solution. It has proved that the application of this method to quantify the proteins by using human serum albumin was available in real samples. In addition, this method is very sensitive (the determination limits are 0.11 μg/mL for human serum albumen (HSA) and 0.85 μg/mL for bovine serum albumen, BSA), simple, rapid and tolerance of most interfering substances. The FIA-RLS method was more stabile than the general RLS method and the average R.S.D. value of FIA-RLS less than general RLS. The effects of different interfering substances will be also examined. The amount of proteins in human serum sample was determined and the maximum relative error was no more than 3.00% as well as the recovery was between 94.9 and 105.9%.     

利福布汀与人血清白蛋白相互作用的光谱研究

用荧光光谱法和圆二色谱法研究了利福布汀(RB)与人血清白蛋白(HSA)的相互作用. 结果表明, RB与HSA之间的相互作用主要是疏水作用, 作用机制是静态猝灭与动态猝灭的结合. 其结合常数(Ka)在106数量级, 说明RB和HSA有很强的结合. 此外, 探讨了金属离子(Cu2+, Zn2+, Mg2+ 和Ca2+)对RB与HSA结合常数的影响. 同步荧光光谱和圆二色谱数据表明, RB可导致HSA的构象改变.