Voltammetric Investigation of Zinc Release from Metallothioneins Modulated by the Glutathione Redox Couple and Separated with a Porous Membrane

Glutathione (GSH), in addition to serving as a redox buffer in cellular environment, has been suggested as a modulator in metal regulation and homeostasis by metallothioneins (MTs). The interactions of MTs with both GSH and its oxidized form GSSG have been shown to govern the direction of metal transfer. Common methods for the determination of zinc release from MTs modulated by GSH/GSSG either involve radioactive species or enzymes or are labor‐intensive. In this study, upon separation of Zn²⁺ from the reaction mixture of MTs and GSH with a centrifugal filter membrane, differential pulse voltammetry (DPV) was used for the Zn²⁺ quantification. The same approach is extended to the studies of metal transfer between Zn₇MT with a GSH/GSSG mixture and that between Zn₇MT with GSSG. The concomitant conversion between the free thiol and disulfide bonds was confirmed with UV‐vis spectrophotometry. The results demonstrate that GSSG, GSH, and the GSH/GSSG mixture all modulate zinc release from Zn₇MT. The percentage of zinc release increases in the order of GSH, GSSG, and the GSH/GSSG mixture. The new approach is demonstrated to be well suited for investigation of redox regulation of MT and its reaction with zinc‐containing enzymes.     

Aggregation of the Adsorbed Proteins—in Solution or on the Surface？

The adsorption of bovine serum albumin(BSA) on porous polyethylene(PE) membrane was studied based on adsorption and desorption measurements as well as FTIR analysis. A different mechanism was roposed which showed that a critical concentration existed in the adsorptional process. Below this concentration, the adsorption seems to be conducted in a normal side-on way; above this concentration,the adsorption is in an aggregation way.     

Voltammetric Studies of Cadmium‐ and Zinc‐Containing Metallothioneins at Nafion‐Coated Mercury Thin Film Electrodes

Voltammetric studies of rabbit liver metallothioneins (MTs, containing both Zn and Cd ions) and Zn₇‐MT were carried out at Nafion‐coated mercury film electrodes (NCMFEs). The accumulation of MT molecules into the NCMFEs enhances the voltammetric signals and the electrostatic interaction between the Nafion membrane and MT facilitates facile electron transfer reactions. Two well‐defined redox waves, with reduction potential (E_pc) values at ?0.740 and ?1.173 V, respectively, were observed. The peak at E_pc =?0.740 V is attributable to the reduction of the Cd‐MT complex, whereas that at E_pc=?1.173 V was assigned to the reduction of the Zn‐MT complex. Zn₇‐MT exhibits only one redox wave with E_pc=?1.198 V. The NCMFE was found to be more advantageous than thin mercury film electrode (MFE), because the pristine metal ions in MTs (e.g., Cd²⁺ and/or Zn²⁺) are not significantly replaced by Hg²⁺. The NCMFE is also complementary to Nafion‐coated bismuth film electrode in that it has a greater hydrogen overpotential, which allows the reduction of the Zn‐MT complex to be clearly observed. Moreover, intermetallic compound formation between Cd and Zn appears to be less serious at NCMFEs. Consequently, the amounts of Cd and Zn deposited into the electrode upon the reduction reactions can be quantified more accurately.     

Studies on the Interaction of Beryllon Ш with Proteins by Voltammetric Technique and its Application

A new quantitative determination method of proteins using beryllon Ш by voltammetric technique was developed in this paper. In pH 3.5 Britton-Robinson (B-R) buffer solution, beryllon Ш can bind with human serum albumin (HSA) to form an electro-inactive supermolecular complex. Beryllon Ш has a well-defined voltammetric reduction peak at -0.38 V (vs. SCE) and the addition of protein in this solution can cause the decrease of the reductive peak current. Based on the decrease of the reduction peak current, a new electrochemical method for the determination of HSA was established with linear range of 1.0~40.0 mg/L and the detection limit of 1.0 mg/L. This method is further applied to the determination of real sample of healthy human serum.     

环丙沙星与牛血清白蛋白相互作用的研究

研究了不同酸度条件下,环丙沙星(CPFX)与牛血清白蛋白(BSA)间的相互作用,讨论了药物对BSA构象的影响,证实了二者间相互作用为单一的动态猝灭过程,求出了猝灭常数,并依据能量转移理论确定了药物与蛋白的最近距离.     

次甲基蓝与牛血清白蛋白作用的光谱特性研究

应用荧光及紫外光谱法研究了次甲基蓝(MB)与牛血清白蛋白(BSA)相互作用的光谱特性。测定了19℃、29℃、39℃三个温度下的结合常数KA和结合位点数n。结果表明:MB对BSA内源荧光的猝灭机理主要为静态猝灭;以疏水作用与BSA相互作用;同步荧光技术研究了MB对BSA构象的影响,表明BSA的荧光主要源于色氨酸残基,MB对BSA的构象有影响;利用Frster偶极-偶极非辐射能量转移理论,计算了MB与BSA的作用距离及19℃、29℃、39℃三个温度下的能量转移。     

Voltammetric Studies on Chromotrope 2B‐Protein Interaction and Its Analytical Application

In this paper the interaction of chromotrope 2B (Ch2B) with proteins was studied by the electrochemical method. Ch2B is an azo dye and shows irreversible electrochemical responses on the mercury electrode in a pH 3.0 Britton‐Robinson (B‐R) buffer solution. After the addition of human serum albumin (HSA) into the Ch2B solution, an interaction took place, and a supramolecular complex was formed in the mixed solution. The electrochemical parameters of the Ch2B‐HSA interaction system were calculated and compared. The results showed that in the absence and presence of HSA in Ch2B solution, the electrochemical parameters such as the formal potential E⁰, the electrode reaction standard rate constant k_s, etc. showed no significant changes, which indicated that an electro‐inactive supramolecular biocomplex was formed. The free concentration of Ch2B in reaction solution was decreased, and this resulted in the decrease of the peak current. The binding constant and the binding ratio were calculated as 7.85 × 10⁹ and 1:2, respectively, and the interaction mechanism was discussed. Based on the decrease of the peak current, this new electrochemical method was proposed for the determination of HSA in the concentration range of 2.0?25.0 mg/L with the linear regression equation as ΔIp′ (nA) = 50.56C (mg/L) — 6.72 (γ = 0.995). This method was further used to determine other different kinds of proteins, such as bovine serum albumin (BSA), oval albumin, etc‥ The new method was successfully applied to detect the content of albumin in healthy human serum samples with the results in good agreement with the traditional Coomassie Brilliant Blue G‐250 spectrophotometric method.     

锌试剂与牛血清白蛋白作用机理的研究

采用UV光谱法研究了锌试剂与牛血清白蛋白(BSA)在弱酸性溶液中的结合反应,研究了溶液吸光度与BSA浓度的关系。测得其表观摩尔吸光系数ε_P=1.3×10⁶L·mol^-1·cm^-1,最大结合数n=278,表观结合常数K_c=8×10⁷.研究了小分子探针与蛋白质的反应机理及在蛋白质上的结合部位及结合力类型。它们之间主要是以分子间的静电引力结合反应。离子强度对结合反应有显著的影响;不同类型的表面活性剂均以不同的程度和形式对反应有影响。讨论了此结合反应的模式,认为该反应基本符合Scatchard模型。     

Interaction Between <Emphasis Type="Italic">Trans</Emphasis>-resveratrol and Serum Albumin in Aqueous Solution

The interaction between trans-resveratrol (TR) with bovine serum albumin (BSA) in aqueous solution was investigated by means of fluorescence, synchronous fluorescence and infrared spectroscopy. The fluorescence of BSA can be quenched remarkably by TR in aqueous solution. A notable red-shift of the maximum emission of BSA from 340 to 353 nm together with appearance of an isoemissive point at 395 nm were observed. The results indicate that TR binds to BSA, forming a TR–BSA complex. The TR–BSA binding distance was determined to be less than 7 nm, suggesting that energy transfer from BSA to TR may occur. The interaction process is spontaneous. Based on the obtained thermodynamic parameters, electrostatic forces may play a major role in this process. Both synchronous fluorescence and FT-IR spectra confirmed the interaction, and indicate the conformational changes of BSA.     

pH对氟喹诺酮药物与BSA之间相互作用影响的研究

采用毛细管区带电泳法,通过测定在不同pH值、不同牛血清白蛋白(BSA)浓度缓冲溶液的条件下药物迁移时间的变化,并分别计算出了pH为6.8、7.4和8.0时培氟沙星、左氧氟沙星、氧氟沙星、环丙沙星等四种氟喹诺酮类药物与BSA相互作用的结合常数.结果表明:pH对结合常数有较大影响,四种药物分子结合常数的最大值均出现在pH=6.8时,并随着pH的增大,结合常数值明显下降.根据实验结果,还对四种氟喹诺酮类药物与BSA之间相互作用的类型、作用位置进行了分析探讨.研究结果对于进一步阐明药用机理并迅速开发出更高效的广谱抗菌药物具有较强的理论意义.     

Electrochemical Studies of Inclusion Complex Formed Between Glutathione and β‐cyclodextrin‐modified Carbon Electrodes and its Application for Determination of Glutathione

In this work, the electrochemical determination of glutathione (GSH) using β‐cyclodextrin (β‐CD) modified carbon electrodes was carried out. Different methodologies were used to modify the electrodes. In the first part of this paper, we analyze and compare the ability of the electrodes to determine GSH using the different β‐CD‐modified electrodes and cyclic voltammetry. We found that the carbon paste electrode modified by potential sweeping was the best electrode for GSH determination; in addition, we found that an inclusion complex formed between β‐CD deposited on the electrode surface and GSH. The formation constant for this complex was 2498.54 M^?1 at 25 °C. Furthermore, we have also calculated thermodynamic parameters for the formation of the inclusion complex. In the second part of this paper, we analyze the effect of sweep rate and pH on the determination of GSH. The best results were obtained at a rate of 50 mV s^?1 and a pH of 2.2. The β‐CD‐modified carbon paste electrode exhibits a linear response in a concentration range of 20 to 157 µM with a sensitivity of 1083.65 µA mM^?1cm^?2 and a detection limit of 3.92 µM. Finally, the electrode was used to determine the GSH concentration in Eichhornia crassipes root extract, and the concentration determination accuracy was validated by a well‐known spectroscopic method.     

Electrodeposition of Silver Amalgam on Thin Gold Film Electrodes for Voltammetric Detection of 4‐Nitrophenol and DNA Labeled with Osmium Tetroxide‐Bipyridine Complex

Alternative electrode materials suitable to prepare novel working electrode applicable in detecting biopolymers such as nucleic acids, proteins or glycoproteins, represent a significant contribution to bio‐electroanalysis. Herein, electrodes made of vapor‐deposited thin gold films (vAuE) were used as an alternative substrate for the electrodeposition of silver amalgam particles (AgAPs), next to indium tin oxide and pyrolytic graphite, which are already used. The conditions and parameters of double pulse chronoamperometry were optimized for the most‐sensitive voltammetric detection of 4‐nitrophenol (4‐NP). The resulting electrodes were characterized by scanning electron microscope with energy dispersive X‐ray spectroscopy. While 4‐NP could not be detected by bare nonactivated vAuEs at all, their electrochemical activation offered a limit of detection (LoD) of 25 and 5 μmol.l^?1 by means of CV and DPV, respectively. AgAP electrodeposited on vAuE, offered 2.5‐times lower LoDs 10 μmol.l^?1 by CV and comparable LoD 5 μmol.l^?1 by DPV. Advantageously, AgAPs could be repeatedly deposited on and anodically dissolved from the vAuE with a relative standard deviation 13 % of the ten‐times repeated DPV signal of 4‐NP (100 μmol.l^?1). In comparison to vAuE, the vAuE‐AgAP offered about 400 mV broader potential window, which allowed detection of single strand DNA fragment labeled by osmium tetroxide?bipyridine complex down to 2 ng.μl^?1 by means of DPV.     

Synthesis,Crystal Structure,Thermal Stability,Magnetic Property,and Biological Activity of a New Copper(II) Complex Based on 1,2,4‐Benzenetricarboxylic Acid and 2,2′‐Bipyridine

Based on an asymmetric 1,2,4‐benzenetricarboxylic acid (H₃btc) and 2,2′‐bipyridine (bpy), a new Cu^II complex, Cu₂(H₂btc)₄(bpy)₂ · 8H₂O ( 1 ), was synthesized and structurally characterized by single‐crystal X‐ray diffraction, hirshfeld surface (HS) analysis, IR spectroscopy, powder X‐ray analysis, thermal gravimetry analysis (TGA), magnetic susceptibility, EPR measurement, and UV/Vis spectrometry. Complex 1 shows a dinuclear copper structure. The Cu^II of each dinuclear moiety are in a slightly distorted square‐pyramidal environments. Magnetic susceptibility of 1 shows a ferromagnetic coupling between both metal atoms. The interaction of 1 with bovine serum albumin (BSA) is investigated using UV/Vis, fluorescence spectroscopic methods. The Cu^II complex shows strong binding propensity in albumin binding study.     

Polythymine Oligonucleotide‐Modified Gold Electrode for Voltammetric Determination of Mercury(II) in Aqueous Solution

Polythymine oligonucleotide (PTO)‐modified gold electrode (PTO/Au) was developed for selective and sensitive Hg²⁺ detection in aqueous solutions. This modified electrode was prepared by self‐assembly of thiolated polythymine oligonucleotide (5′‐SH‐T₁₅‐3′) on the gold electrode via Au? S bonds, and then the surface was passivated with 1‐mercaptohexanol solution. The proposed electrode utilizes the specific binding interactions between Hg²⁺ and thymine to selectively capture Hg²⁺, thereby reducing the interference from coexistent ions. After exchanging the medium, electrochemical reduction at ?0.2 V for 60 s, voltammetric determination was performed by differential pulse voltammetry using 10 mM HEPES; pH 7.2, 1 M NaClO₄ as supporting electrolyte. This electrode showed increasing voltammetric response in the range of 0.21 nM Hg²⁺, with a relative standard deviation of 5.32% and a practical detection limit of 60 pM. Compared with the conventional stripping approach, the modified electrode exhibits good sensitivity and selectivity, and is expected to be a new type of green electrode.     

Attachment of Amine‐ and Maleimide‐Containing Ferrocene Derivatives onto Self‐Assembled Alkanethiol and Alkanedithiol Monolayers: Voltammetric Evaluation of Cross‐Linking Efficiencies and Surface Coverage of Electroactive Groups

Ferrocene derivatives containing primary amines and maleimide groups were attached covalently onto N‐hydrosuccinimidyl (NHS)‐terminated alkanethiol self‐assembled monolayers (SAMs) and SAMs of alkanedithiol. The surface coverage and efficiencies of the two cross‐linking reactions were evaluated with cyclic voltammetry. All the ferrocene derivatives attached onto the alkanethiol or alkanedithiol SAMs exhibit reversible redox waves. The surface coverage of the aminated ferrocene groups was compared to that of N‐hydrosuccinimidyl (NHS)‐terminated alkanethiol SAM. The covalent attachment of β‐ferrocenylethylamine onto a 11,11′‐dithio‐bis(succinimidylundecanoate) SAM yielded an efficiency as high as 63.1%. The cross‐linking efficiency of this reaction was found to increase with the nucleophilicity of the amino groups. SAMs of longer alkyl chains favor the attachment of a greater number of ferrocene derivatives. As for the Michael‐type electrophilic addition between the sulfhydryl groups of the alkanedithiol SAMs and the ferrocenyl maleimide, the cross‐linking efficiencies were found to range from 6.5% to 25.7%, depending on the alkanedithiol chain length. The difference in the efficiencies between the two types of cross‐linking reactions might be partially attributable to the steric hindrance imposed by the SAMs and the relative sizes of the functional groups.     

卡马西平与牛血清白蛋白结合反应的热力学特性研究

用荧光光谱法研究了卡马西平与牛血清白蛋白的结合反应的热力学特性,并发现卡马西平对牛血清白蛋白有较强的荧光猝灭作用。从荧光猝灭光谱数据,由stern-volmer方程、double-reciprocal方程和热力学公式,得到了25℃时结合反应的结合常数为2.047×104mol.L-1,其△Hθ、△Gθ和△Sθ分别为-14.46 kJ.mol-1-、32.88kJ.mol-1和-24.03 J.K-1。     

A Homogenous Assay of FAD Using a Binding Between Apo‐Glucose Oxidase and FAD Labeled with an Electroactive Compound

A homogenous assay of FAD using a binding between glucose oxidase (apo‐GOD) and FAD labeled with an electroactive compound was developed. Because daunomycin is sensitively detected with voltammetry, daunomycin was connected to FAD with a cross‐linker. The peak current decreased due to the apo‐GOD‐labeled FAD binding. Competitive reaction to the apo‐GOD between FAD and the labeled FAD produces the increase of peak current. Accordingly, FAD is detected on the basis of the reaction. The merit of this method is that the influence from FMN and riboflavin in the measurement of FAD can be suppressed by the high selective binding.     

Interaction Between Levamisole Hydrochloride and Bovine Serum Albumin and the Influence of Alcohol: Spectra

The interaction between levamisole hydrochloride (LH) and bovine serum albumin, BSA, has been studied by a spectral method under physiological conditions. For 1:n complexes, the relationship between fluorescence quenching intensity and concentration of the quenchers can be deduced on the basis of the modified Stern–Volmer equation. The binding constants and corresponding thermodynamic parameters ΔH _m, ΔG _m and ΔS _m at different temperatures were calculated. The experimental results demonstrated that the combination reaction of LH and BSA was a static quenching process because a 1:1 complex was formed, and the main dominant binding forces were hydrogen bonding and van der Waals forces. Meanwhile, the polarity of the tyrosine residue (Tyr) or tryptophan residue (Trp) micro-region was not obviously affected by the interaction. Furthermore, the binding constant increase when alcohol was added.     

Electroanalytical Studies and Simultaneous Determination of Amlodipine Besylate and Atorvastatine Calcium in Binary Mixtures Using First Derivative of the Ratio‐Voltammetric Methods

The electrochemical behavior of atorvastatin and amlodipine at a glassy carbon electrode has been studied using different voltammetric techniques. First derivative of the ratio voltammetric methods for determination of amlodipine and atorvastatin in tablets in the presence of the other compound has been described. This technique depends on the measuring of first derivative of the ratio voltammograms of each concentration as a function of the increased concentrations. DP and SW voltammetric methods depend on first derivative of the ratio‐voltammetry by measurements of the selected potentials for amlodipine and atorvastatin. The linear response was within the range of 4×10^?6–1×10^?4 M for amlodipine and 2×10^?6–1×10^?4 M for atorvastatin. The proposed methods have been extensively validated.