腺苷与人血清白蛋白间相互作用的荧光光谱及分子模型法研究

在模拟人体生理条件下,结合紫外光谱和分子对接模型运用荧光光谱研究了腺苷与人血清白蛋白(HSA)间的键合作用。腺苷有较强的能力猝灭人血清白蛋白的内源荧光,且根据Stern-Volmer方程判断出猝灭机制为静态猝灭。本文运用相应的荧光值和Vant’Hoff热力学方程求得了不同温度下的结合常数(K)以及一些热力学参数,如焓变(ΔH)和熵变(ΔS)。结果表明：键合过程中疏水作用力对新化合物的稳定性起主要作用,这与分子对接模型方法研究的结果基本一致。另外还研究了常见离子对结合常数的影响。     

基质辅助激光解吸电离飞行时间质谱技术研究人血清转铁蛋白稳定性及裂解产物

制备质谱纯人血清转铁蛋白(HTF),供分子结构分析。选用SDS-PAGE、胶外酶解、基质辅助激光解吸/电离质谱技术(MALDI-TOF)、数据库检索和比对技术鉴定铁饱和HTF、双铁HTF(HTF-2Fe3 )、单铁HTF(HTF-Fe3 )和脱铁HTF(apoHTF)的稳定性和裂解产物。以乙腈溶液作为洗脱相,发现铁饱和HTF在RP-HPLC分离纯化过程中产生裂解现象。铁饱和HTF和HTF-2Fe3 经乙腈处理后均能产生不同分子量的短肽裂解产物,指出HTF结构稳定性与络合铁离子数量有关。铁组分改善了HTF分子结构的稳定性。采用比对法,研究在乙腈作用下HTF裂解成为各种各样短肽的规律,初步阐明其裂解机理。在乙腈作用下,HTF可能通过蛋白质去折叠途径,形成不同多聚态HTF或多肽裂解产物。推测目前用于临床诊断先天性糖基化紊乱(CDG)和慢性酒精滥用(CAA)疾病低准确率的起因可能是受HTF裂解产物或多聚体的干扰。     

Stepwise Assembly of MII7 Clusters Revealed by Mass Spectrometry,EXAFS, and Crystallography

The square‐planar monomer NiL₂ ( Ni₁ ), L=2‐ethoxy‐6‐(N‐methyl‐iminomethyl)phenolate, reacts with M(H₂O)₆(ClO₄)₂, M=Ni or Co, to form heptanuclear disks [Co_xNi_7?x(OH)₆(L)₆](ClO₄)₂ ? 2 CH₃CN ( Co _x Ni_7?x , x=0–7) and the co‐crystal [Co_xNi_7?x(OH)₆L₆][NiL₂](ClO₄)₂ ? 2 CH₃CN ( Co _x Ni_7?x ‐Ni₁ ) under ambient conditions. It has proved possible to explore the bottom‐up assembly process of Co _x Ni_7?x and Co _x Ni_7?x ‐Ni₁ in real time. The final products have been characterized by thermogravimetric analysis, IR, elemental analysis, ICP‐MS, and single‐crystal X‐ray diffraction. Time‐dependent mass spectrometry (MS) revealed the following reaction steps: Ni₁→[M₂L₃]⁺→[M₄(OH)₂L₄]²⁺→[M₇(OH)₆L₆]²⁺. In contrast, the reaction of Ni₁ with Zn²⁺ only reaches halfway, and crystallographic evidence indicates a butterfly structure for [Zn₂Ni₂(OH)₂Cl₂] ( Zn₂Ni₂ ), an intermediate that is difficult to isolate in the above Ni‐Co series. A summation method has been used to analyze the MS of bimetallic clusters with very similar atomic masses, as is the case for Co and Ni. The results provide ample information on the distribution of Co and Ni within each cluster and their statistical distribution within selected crystals.     

知识发展、教材编排与学生学习的三序结合*——以“分子和原子”为例

“分子和原子”是九年级《化学》上册第三单元课题1的内容,是学生学习初中化学物质构成的启蒙。本文论述了“分子和原子”的知识研究发展顺序、教材编排顺序和学生学习顺序,针对该部分内容提出了在教学中三序结合的方式与建议。     

Isolation, Mapping, DNA Sequence and RFLPs Studies of Random Single-Copy DNA Segments on Human X Chromosome

Using the total human/mouse DNA as the probe, screening has been carried out three times with in situ plaque hybridization to obtain the single-copy DNA sequence from the human X chromosome genomic library. The effective rate of screening is 1. 45%. DNAs from clones containing single-copy inserts have been analyzed by a panel of hybrid cells with or without human X chromosome. Three segments, designated by DXFD52,73,75, are mapped to the X chromosome. DXFD52 has been precisely localized on Xq12-q13 with in situ chromosomal hybridization. DXFD52 has been partially sequenced. The results indicate that DXFD52 is a new isolated single-copy segment on the X chromosome. Great progress in the RFLPs study with DXFD52 has been achieved in the population of Chongqing, Sichuan Province. The results show that the DXFD52 can be used to detect the RFLP with Hind Ⅲ, Bgl Ⅱ, and Hinf Ⅰ. DXFD52 will be a potential "landmark" for the construction of the complete linkage map of human genome and the analysis of genomic s     

Determination of Arbutin,Niacinamide, and Adenosine in Functional Cosmetic Products by High-Performance Liquid Chromatography

A reversed-phase high-performance liquid chromatography method using a diode array detector at 260 nm was developed and validated for the determination of arbutin, niacinamide, and adenosine in cosmetics. The analytes were extracted using methanol and deionized water. The separations were performed on a C₁₈ column with gradient elution. At the optimized conditions, the limits of detection for arbutin, niacinamide, and adenosine were 0.75, 0.12, and 0.01 µg/mL, respectively. The average recoveries were between 98.1% and 107.5%. The method was used to analyze thirty cosmetics.     

Volumetric, Electric, and Magnetic Properties of Thioxanthen-9-one in Aprotic Solvents as Revealed by High-Precision Densitometry, High-Accuracy Refractometry and Magnetic Susceptibility Measurements and by DFT Calculations

High-precision densitometry measurements of solutions of thioxanten-9-one (TX) in 1,4-dioxane, DMSO, toluene, and benzene have been obtained at 293.15, 303.15, 313.15, 323.15, 333.15, and 343.15K. The partial molar volumes of TX ( ) and the corresponding values at infinite dilution ( ) were determined. The partial molar expansibility ( ) of TX at infinite dilution in each solvent is temperature independent. Dynamic electronic polarizabilities of TX in each aprotic solvent were determined by the Singer–Garito approach. These values are in excellent concordance with the theoretical value for TX of 2.611×10⁻²³cm³ estimated here using DFT/B3LYP/6-311++G(d,p). The partial molar volumes of TX at infinite dilution were calculated and interpreted in terms of the Scale Particle Theory (SPT). The solvent influence on the partial molar volume of TX was found to be due mainly to cavity formation and intermolecular dispersion forces.     

Catalysis of Hydrogen Evolution by Polylysine,Polyarginine and Polyhistidine at Mercury Electrodes

Protein catalyzed hydrogen evolution reaction at mercury‐containing electrodes controlled by constant‐current chronopotentiometric stripping (CPS) is representing a new tool useful in protein research. The resulting CPS peak H is sensitive to changes in the protein structure and its amino acid composition. Besides CPS, cyclic voltammetry appears to be useful for study of poly(amino acids) as an intermediate model system between peptides and macromolecular proteins. Here we show that similarly as arginine in polyarginine and lysine in polylysine also histidine residues in polyhistidine contribute to the catalysis of hydrogen evolution under the given conditions. Peak potentials of individual poly(amino acids) are different and depend on the type of amino acid residues.     

毛细管区带电泳法测定冬虫夏草中的腺苷、腺嘌呤和尿嘧啶

建立了毛细管区带电泳法测定天然和人工冬虫夏草中腺苷、腺嘌呤和尿嘧啶含量的分析方法。采用15mmol/L硼砂＋14mmol/L磷酸二氢钠＋5％（V／V）甲醇（pH＝9.5）作为缓冲体系，在电压为18kV和检测波长为254nm的条件下，冬虫夏草提取液中的腺苷、腺嘌呤和尿嘧啶实现了基线分离。定量分析表明，3种成分的校正峰面积与质量浓度呈较好的线性关系(r≥0.9991）。考察了缓冲溶液的pH值、浓度及有机改性剂对腺苷、腺嘌呤和尿嘧啶迁移行为的影响。     

电感耦合等离子体原子发射光谱(ICP-AES)法测定人发中铜、锌、钙、镁、铁

样品经硝酸-高氯酸消化溶解,高氯酸冒烟,盐酸溶解盐类后,在盐酸(5%)介质中,在选定的测定条件下,用电感耦合等离子体原子发射光谱(ICP-AES)法测定人发中微量元素铜、锌、铁、镁、钙。选择Cu 327.3、Zn 206.2、Fe 238.2、Mg 279.5、Ca 315.8nm分别作为铜、锌、铁、镁、钙的分析线与混合标准溶液同时测定;方法加标回收率为98.6%～101%,铜、锌、铁、镁、钙的精密度(RSD,n=8)为0.37%～2%,准确度(RE)为-3.4%～1.15%,检出限分别为0.002 3、0.001 6、0.004 6、0.003 0、0.001 4μg/mL。方法克服了分光光度法和原子吸收光谱法操作繁琐、周期长、成本高、灵敏度低等缺点。用于测定人发样品中的铜、锌、铁、镁、钙元素,测定结果与原子吸收光谱法测定值基本一致。经GBWO7061标准物质和自制标样分析验证,测定值与标准值吻合,结果准确可靠。     

Binding Studies of AICAR and Human Serum Albumin by Spectroscopic,Theoretical, and Computational Methodologies

The interactions of small molecule drugs with plasma serum albumin are important because of the influence of such interactions on the pharmacokinetics of these therapeutic agents. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) is one such drug candidate that has recently gained attention for its promising clinical applications as an anti-cancer agent. This study sheds light upon key aspects of AICAR’s pharmacokinetics, which are not well understood. We performed in-depth experimental and computational binding analyses of AICAR with human serum albumin (HSA) under simulated biochemical conditions, using ligand-dependent fluorescence sensitivity of HSA. This allowed us to characterize the strength and modes of binding, mechanism of fluorescence quenching, validation of FRET, and intermolecular interactions for the AICAR–HSA complexes. We determined that AICAR and HSA form two stable low-energy complexes, leading to conformational changes and quenching of protein fluorescence. Stern–Volmer analysis of the fluorescence data also revealed a collision-independent static mechanism for fluorescence quenching upon formation of the AICAR–HSA complex. Ligand-competitive displacement experiments, using known site-specific ligands for HSA’s binding sites (I, II, and III) suggest that AICAR is capable of binding to both HSA site I (warfarin binding site, subdomain IIA) and site II (flufenamic acid binding site, subdomain IIIA). Computational molecular docking experiments corroborated these site-competitive experiments, revealing key hydrogen bonding interactions involved in stabilization of both AICAR–HSA complexes, reaffirming that AICAR binds to both site I and site II.     

Molecular Imaging of Growth,Metabolism, and Antibiotic Inhibition in Bacterial Colonies by Laser Ablation Electrospray Ionization Mass Spectrometry

Metabolism in microbial colonies responds to competing species, rapidly evolving genetic makeup, and sometimes dramatic environmental changes. Conventional characterization of the existing and emerging microbial strains and their interactions with antimicrobial agents, e.g., the Kirby–Bauer susceptibility test, relies on time consuming methods with limited ability to discern the molecular mechanism and the minimum inhibitory concentration. Assessing the metabolic adaptation of microbial colonies requires their non‐targeted molecular imaging in a native environment. Laser ablation electrospray ionization (LAESI) is an ambient ionization technique that in combination with mass spectrometry (MS) enables the analysis and imaging of numerous metabolites and lipids. In this contribution, we report on the application of LAESI‐MS imaging to gain deeper molecular insight into microbe–antibiotic interactions, and enhance the quantitative nature of antibiotic susceptibility testing while significantly reducing the required incubation time.     

Molecular Imaging of Growth,Metabolism, and Antibiotic Inhibition in Bacterial Colonies by Laser Ablation Electrospray Ionization Mass Spectrometry

Metabolism in microbial colonies responds to competing species, rapidly evolving genetic makeup, and sometimes dramatic environmental changes. Conventional characterization of the existing and emerging microbial strains and their interactions with antimicrobial agents, e.g., the Kirby–Bauer susceptibility test, relies on time consuming methods with limited ability to discern the molecular mechanism and the minimum inhibitory concentration. Assessing the metabolic adaptation of microbial colonies requires their non‐targeted molecular imaging in a native environment. Laser ablation electrospray ionization (LAESI) is an ambient ionization technique that in combination with mass spectrometry (MS) enables the analysis and imaging of numerous metabolites and lipids. In this contribution, we report on the application of LAESI‐MS imaging to gain deeper molecular insight into microbe–antibiotic interactions, and enhance the quantitative nature of antibiotic susceptibility testing while significantly reducing the required incubation time.     

Simultaneous Determination of Buprenorphine, Norbuprenorphine and Naloxone in Human Plasma by LC-MS-MS

A novel sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method simultaneously determined buprenorphine (BUP) and its active metabolite, norbuprenorphine (NBUP), and a coformulant, naloxone was developed, validated and applied successfully in humans. Buprenorphine-d ₄ and norbuprenorphine-d ₃ were used as the internal standard. The analysis was performed on a silica column, and the mobile phase was isocratic and composed of acetonitrile:2 mM ammonium formate in H2O (82:18, v/v). Mass spectrometry employed multiple reaction monitoring modes with transitions of m/z 468.1?C55.2 for BUP, 414.2?C101.2 for NBUP, 328.3?C310.3 for naloxone, 472.1?C59.2 for buprenorphine-d ₄ and 417.2?C101.2 for norbuprenorphine-d ₃. Lower limit of quantification (LLOQ) of the analytical method was 0.05 ng mL^?1 for BUP, 0.1 ng mL^?1 for NBUP and 0.025 ng mL^?1 for naloxone, respectively. The standard calibration curves of BUP, NBUP and naloxone were linear over the concentration range of 0.05?C20 ng mL^?1, 0.1?C20 ng mL^?1 and 0.025?C20 ng mL^?1, respectively. The precisions (RSD) and accuracies (RE) of LLOQ and other QC samples were in acceptable range, with RSD < 20% and RE ± 20% for LLOQ and RSD < 15% and RE within ±15% for QC samples. The method was accurate, precise and specific, and was applied to the pharmacokinetic study of buprenorphine in healthy volunteers.     

Determination of Piribedil in Human Serum, Urine and Pharmaceutical Dosage Form by LC-DAD

A rapid, selective and sensitive reversed-phase liquid chromatographic (LC) method was developed for the determination of piribedil in human serum, urine and pharmaceutical dosage form. LC analysis was carried out using reversed-phase isocratic elution with a C₁₈ column and a mobile phase of 0.01 M phosphate buffer-acetonitrile (50:50, v/v). The chromatograms showed good resolution and sensitivity with no interference of human serum and urine. Piribedil concentrations were determined using diode array detection at 240 nm. Sildenafil citrate was used as internal standard. The limit of quantification (LOQ) and limit of detection (LOD) concentrations were 107.2 and 321.6 pg mL^?1, 96.6 and 290.4 pg mL^?1, 161.7 and 53.9 pg mL^?1 for urine, serum and pharmaceutical dosage forms, respectively. The method was validated for its linearity, precision and accuracy and applied to the tablets, urine and human serum. In addition, the results were compared to those obtained from UV-spectrophotometry.     

Quantitation of Tamoxifen, 4-Hydroxytamoxifen,and N-Desmethyltamoxifen in Human Plasma by High Performance Liquid Chromatography

Abstract

A reverse phase high performance liquid chromatography assay used for quantitative analysis of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen extracted from human plasma was developed. Plasma samples were spiked with an internal standard, nafoxidine, extracted with 2% butanol in hexane, and activated to fluorescence by exposure to high intensity short-wave ultraviolet (254 nm) light. Aliquots of extracted plasma were then injected onto a C18 reverse phase column and eluted isocratically with a mobile phase of water and triethylamine in methanol. Fluorescence of activated tamoxifen and its metabolites was measured at 266 nm. Peak height ratios were used to quantitate tamoxifen and its metabolites from extracted human plasma specimens.     

Determination of Ofloxacin,A New Oxazine Derivative in Human Serum,Urine, and Bile by High-Performance Liquid Chromatography

Abstract

A simple and precise high-performance liquid procedure has been developed for the determination of Ofloxacin (DL-8280), a new oxazine derivative in human serum, urine and bile using Norfloxacin as internal standard. The work-up procedure involves a chemical extraction step followed by isocratic chromatography on a anion-exchange analytical column, with ultraviolet detection. The run time for the assay was 11.5 minutes.     

Simultaneous Determination of Pyridoxine,Meclizine and Buclizine in Dosage Formulations and Human Serum by RP-LC

Rapid liquid chromatographic procedures for analytical quality control of pharmaceutical preparations and human serum containing antihistamine drugs, meclizine and buclizine alone or in combination with pyridoxine are proposed, using acetonitrile:water (80:20) as a mobile phase (pH adjusted to 2.6), methylparaben as internal standard and UV detection was made at 230 nm. The results obtained showed a good agreement with the declared content. The method shows good linearity in the range of 30–10,000 ng mL⁻¹ for pyridoxine and 25–10,000 ng mL⁻¹ for meclizine and buclizine serum concentrations with a correlation coefficient 0.9999 (inter- and intra-day CV < 3.91%). The recovery was >97.8%. The proposed method may be used for the quantitative analysis of meclizine and buclizine alone or in combination with pyridoxine from raw materials, in bulk drugs, dosage formulations and in serum.