Time-resolved fluorescence analysis of the mobile flavin cofactor in p -hydroxybenzoate hydroxylase

Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the ‘in’ conformation with the isoalloxazine ring located in the active site, and the ‘out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the ‘in’ and ‘out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes.     

Element-selective detection using capillary gas chromatography with atomic emission detection

Capillary GC coupled to an atomic emission detector (AED) provides a powerful new hyphenated technique for the separation and characterization of complex mixtures and compounds. The AED provides simultaneous and truly specific multi-element detection. The specificity of detection reduces the need for the complex sample pretreatment procedures which are necessary to reduce the interference from co-eluted substances which is experienced with detectors such as the FID and the ECD. A range of environmentally significant problems has been studied, including PCB analysis, the characterization of the reaction products of a novel waste treatment process, and the profiling of sulfur-containing species formed by the pyrolysis of various types of coal.     

On the surface modification of microchannels for microcapillary electrophoresis chips

This paper presents systematic investigation of the microchannel surface properties in microCE chips. Three popular materials for microCE chips, polydimethylsiloxane (PDMS), quartz, and glass, are used. The zeta potentials of these microchannels are calculated by measuring the EOF velocity to evaluate the surface properties after surface modification. The hydrophobic PDMS is usually plasma-treated for microCE applications. In this study, a new method using a high-throughput atmospheric plasma generator is adopted to treat the PDMS surface under atmospheric conditions. In this approach, the cost and time for surface treatment can be significantly reduced compared with the conventional vacuum plasma generator method. Experimental results indicate that new functional groups could be formed on the PDMS surface after treatment, resulting in a change in the surface property. The time-dependent surface property of the plasma-treated PDMS is then measured in terms of the zeta potential. Results show that the surface property will reach a stable condition after 1 h of plasma treatment. For glass CE chips, two new methods for changing the microchannel surface properties are developed. Instead of using complicated and time-consuming chemical silanization procedures for CE channel surface modification, two simple and reliable methods utilizing organic-based spin-on-glass and water-soluble acrylic resin are reported. The proposed method provides a fast batch process for controlling the surface properties of glass-based CE channels. The proposed methods are evaluated using PhiX-174 DNA maker separation. The experimental data show that the surface property is modified and separation efficiency greatly improved. In addition, the long-term stability of both coatings is verified in this study. The methods proposed in this study show potential as an excellent solution for glass-based microCE chip surface modification.     

Use of a Dual-Marker Form of Analysis to Estimate Binding Constants Between Receptors and Ligands by Affinity Capillary Electrophoresis

This work describes the use of a dual-standard analysis approach termed the time-average ratio (TAR) in affinity capillary electrophoresis (ACE) to estimate binding constants of receptors to ligands. In this form of analysis the TAR is the migration time of the receptor divided by the average of the sum of the migration times of two non-interacting standards. This change in TAR as a function of the concentration of ligand yields a value for the binding constant. This concept is demonstrated using three model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, vancomycin (Van) and ristocetin (Rist) from Streptomyces orientalis and Nocardia lurida, respectively, and d-Ala- d-Ala terminus peptides. Three ACE techniques are used to examine the three systems: standard ACE, flow-through partial-filling ACE (FTPFACE), and on-column derivatization coupled to ACE. The findings described here demonstrate that ACE data analyzed using the TAR form of analysis yield binding constants between receptors and ligands comparable to those estimated using other ACE forms of analysis. A comparison to three other forms of analysis is described.     

Conformational studies of soluble and immobilized frog epidermis tyrosinase by fluorescence

Fluorescence spectra and soluble quenching of intrinsic protein fluorescence were used as indexes of conformational changes suffered by frog epidermis tyrosinase. The activation process and the immobilization of the enzyme involving either free amino groups or its carbohydrate moiety were studied. The conformational changes resulting from denaturation of each one of the protein derivatives, as well as the effect of active center copper extraction, were followed by fluorescence studies. The results showed that: (a) both activation and immobilization were accompanied by conformational changes of the protein leading to more unfolded states; (b) neither enzyme nor immobilized enzyme were fully unfolded upon denaturation although enzymic activity was lost; (c) the enzyme immobilized through its carbohydrate moiety was more unfolded upon denaturation than the enzyme immobilized through amino groups, thus pointing to a higher conformational stabilization in the last situation; and (d), that tryptophyl residues moved to a localization near the active site upon activation.     

Capillary Zone Electrophoresis of some organic acids in milk whey

This paper describes a method for analysing some acids of milk whey by Capillary Zone Electrophoresis. After eliminating the whey proteins by ultrafiltration, the whey underwent electrophoretic separation in the presence of anodic electroosmotic flow. The following analytes were detected: citric, orotic, uric, and hippuric acids. A procedure is described for sample preparation and the operating conditions for electrophoretic capillary separation established. Finally, orotic acid is quantitatively determined.     

原子荧光光谱法测定砷过程中还原反应条件的优化

自然界中砷和砷的化合物一般可通过水、大气和食物等途径进入人体,危害人体健康,原子荧光光谱法测定砷含量被广泛应用。为准确测定样品中砷的含量,以原子荧光光谱法(AFS)测定土壤中总砷含量为例,重点研究高价态砷(Ⅴ)还原为低价态砷(Ⅲ)的实验条件,采用正交实验进行条件优化,建立适合的实验方法。通过设计L9(34)正交实验,高价态砷的还原反应与四个因素有关,即与反应温度、反应时间、盐酸加入量和硫脲的用量呈一定的正相关,结果表明,四个因素中硫脲用量的影响最为显著,其次是盐酸加入量、反应时间和反应温度。通过对四个因素的多水平检验,优化后的实验条件为：硫脲用量2.0 mL、盐酸加入量10%、反应时间20 min、温度20～35 ℃(即普通室温)。采用土壤标准物质验证优化后条件的可行性,得出砷的测定值在标准值范围内,RSD在2.2%～4.2%,精密度和准确性均满足质控要求。优化后的实验条件使得操作简便易行,提高了工作效率。     

X射线荧光光谱(XRF)法测定绿泥石中镁、铝、硅、磷、钾、钙、钛、铁元素含量

建立了X射线荧光光谱法测定绿泥石中镁、铝、硅、磷、钾、钙、钛、铁元素含量的快速分析方法。以标准物质及标准物质与基准试剂氧化镁、氧化钙人工混合配制标样的方法建立标准工作曲线,重点讨论了熔剂和脱模剂的选取。最佳熔样条件：采用8.0g四硼酸锂和偏硼酸锂混合熔剂(质量比67: 33)+0.8g样品并添加溴化锂作为脱模剂,熔样温度1100℃,熔样时间10min。该方法相对标准偏差(n=12)均小于5.57%,绿泥石样品测定结果与化学法一致,硅酸盐标准物质测定结果均满足不确定度要求。     

粉末压片-X荧光光谱法测定过磷酸钙中硫含量

硫是过磷酸钙中重要营养指标之一,为准确快速测定过磷酸钙中硫的含量,试验采用粉末压片-X荧光光谱法,将过磷酸钙试样充分干燥后研磨至粒度小于74 μm,采用硼酸镶边,在压力18 Mpa条件下保压30 s,制成样片。通过在过磷酸钙样品中添加不同质量的纯物质硫酸钙（质量分数范围1.52 %～17.21 %）,经过专用混匀设备混合均匀后,与试样压片相同条件下压制标准样片,作为过磷酸钙中硫的标准样品,建立硫标准曲线,曲线线性相关系数R2为0.9995,采用经验系数法校正干扰,建立了粉末压片-波长色散X射线荧光光谱法测定过磷酸钙中硫含量的方法, 方法检出限为0.002 %。对3个不同硫含量的过磷酸钙样品采用本实验方法重复测量7次,RSD在1.4 %~3.1 %,方法精密度性好,同时用高温燃烧红外光谱法和电感耦合等离子发射光谱法对比,三者测量结果相对极差小于2.0 %,测量结果无显著性差异。此方法不需要对样品进行熔融或溶解,样品制备简单,数据准确度和稳定性好,分析效率高,适合大批量样品中硫的测定。     

锗元素测定方法综述

锗在国防工业、航空航天和通信等领域中的战略性,锗含量的测定对于保证材料质量和满足国际标准至关重要。本文综述了锗含量测定方法的多种技术,包括分光光度法、原子荧光光谱法、原子吸收光谱法、电感耦合等离子体原子发射光谱法、电感耦合等离子体质谱法以及滴定法。在每个检测方法的介绍中,详细探讨了方法的原理、前处理步骤以及应用范围,并分别总结了各个方法的优势和不足。最后,强调了锗含量测定方法的意义,特别是在满足出口监管和促进科学研究方面的作用。同时对锗元素的测定方法进行了展望,为未来的发展提供了参考方向。