流动注射-抑制化学发光测定阿莫西林的方法研究

基于阿莫西林对Co2+催化的鲁米诺-H2O2化学发光体系的抑制作用,首次建立了阿莫西林的流动注射化学发光抑制分析法。发光信号的降低值ΔΙ与阿莫西林的质量浓度在8×10-9~4×10-5g/mL的范围内呈良好的线性关系,检出限为2.7×10-9g/mL,对1.0×10-5g/mL阿莫西林测定的相对标准偏差为1.89%(n=11)。本方法快捷、简便且具有很高的灵敏度,可用于阿莫西林胶囊中阿莫西林含量的测定,并与药典规定的方法进行对照,结果满意。     

流动注射-抑制化学发光法测定银杏叶中总黄酮含量

提出了流动注射-抑制化学发光测定银杏叶中的总黄酮含量的分析方法.它是基于银杏叶中物质黄酮类具有还原性,在碱性条件下还原H_2O_2,抑制鲁米诺-H_2O_2-KIO_4体系的化学发光,其抑制程度的大小与总黄酮的含量成线性关系.方法的线性范围为1.5～30μg/mL,检出限为0.03μg/mL,相对标准偏差(RSD)为1.2%,采样频率为240次/h,回收率为101%～104%.     

ClO--鲁米诺流动注射-抑制化学发光法测定银杏黄酮

基于在碱性条件下,银杏黄酮对ClO--鲁米诺体系有显著的抑制作用,结合反相流动注射技术,建立了流动注射-抑制化学发光测定银杏提取物中的银杏黄酮.银杏黄酮质量浓度在0.4～14.0 μg/mL范围与相对发光强度呈线性关系,检出限为0.06 μg/mL,采样频率为120次/h,对10.0 μg/mL的银杏黄酮平行测定11次,其RSD为1.2%,回收率为98.0%～104%.该方法可用于银杏黄酮制剂的测定.     

流动注射-抑制化学发光法测定葡萄籽提取物中原花青素

基于在碱性条件下,原花青素对H2O2-Luminol体系有显著的抑制作用,结合反相流动注射技术,首次建立了流动注射-抑制化学发光测定葡萄籽提取物中原花青素的含量;原花青素质量浓度在O．2～20．Omg／L范围与相对发光强度呈线性关系,检出限为O.lmg／L,采样频率为140次／h,对lO．Omg／L的原花青素平行测定11次,其RSD为1．2％,回收率为97％～103％;该方法用于原花青素的测定,结果令人满意。     

流动注射-抑制化学发光法测定司帕沙星

在碱性介质中,司帕沙星对鲁米诺-过氧化氢-铬(Ⅲ)化学发光有较强的抑制作用,据此,采用流动注射技术,建立了一种测定痕量司帕沙星药物的化学发光分析法。该法的线性范围为1.0×10-6~2.0×10-3mol/L,检出限为6.5×10-7mol/L。本方法简便、快速,用于片剂中司帕沙星的测定,回收率在90%~101%之间,结果满意。     

流动注射-抑制化学发光法测定克百威

基于在碱性介质中,克百威抑制鲁米诺-过氧化氢-叶绿素铜钠体系的化学发光,提出了流动注射-抑制化学发光法测定克百威含量的方法。试验结果表明:叶绿素铜钠对克百威荧光猝灭过程是静态猝灭过程,叶绿素铜钠与克百威结合形成物质的量比为1比1的稳定配合物,平衡常数(K0)为3.41×105L.mol-1(25℃),结合距离(r)为0.39 nm。克百威质量浓度在0.08～2.00 mg.L-1范围内与其发光强度呈线性关系,方法检出限(3σ/k)为0.03 mg.L-1。此法用于克百威杀虫剂样品的分析,测得方法的平均加标回收率为101.5%。     

流动注射-串联质谱法分析污水中11种毒品

建立了一种流动注射-串联质谱法(FIA-MS/MS)快速检测污水中11种常见毒品。样品经Oasis MCX固相萃取小柱净化和浓缩后,注入FIA-MS/MS系统进行分析。质谱采用正离子模式扫描,在多反应监测模式(MRM)下,同位素内标法定量。结果表明,11种毒品在0.16～200 ng/L范围内线性关系良好,相关系数r²≥0.9902,检出限(LOD)为0.05～0.53 ng/L,定量限(LOQ)为0.16～1.60 ng/L。11种毒品在低、中、高3种加标浓度下,方法回收率为74.5%～113.7%,日内相对标准偏差(RSD)在1.0%～6.9%之间,日间RSD在2.2%～11%之间。该方法适用于污水样品中毒品的高通量筛查和定量分析。     

H2O2-Luminol流动注射-抑制化学发光法测定银杏黄酮

基于在碱性条件下,银杏黄酮对H2O2 Luminol体系有显著的抑制作用,结合反相流动注射技术,首次建立了流动注射 抑制化学发光测定银杏提取物中的银杏黄酮。银杏黄酮质量浓度在0.2～20.0μg mL范围与相对发光强度呈线性关系,检出限为0.1μg mL,采样频率为140次 h,对10.0μg mL的银杏黄酮平行测定11次,其RSD为1.5%,回收率为100.0%～100.2%。该方法可用于银杏黄酮制剂的测定。     

流动注射-抑制化学发光法测定镱(Ⅲ)

在碱性介质中, Yb(Ⅲ)对Luminol-KMnO4体系化学发光强度具有抑制作用, 据此建立了一种测定Yb(Ⅲ)的化学发光新方法. 在优化的实验条件下, 化学发光强度与Yb(Ⅲ)的浓度在4.0×10-7～1.0×10-4 mol/L范围内呈现出良好的线性关系. 其检测限(3σ)为5.0×10-8 mol/L, 对8.0×10-5 mol/L的Yb(Ⅲ)溶液进行测定, 相对标准偏差为3.9% (n=11). 本法已应用于合成样品中的镱的测定.     

流动注射-化学发光法测定水果提取物对羟自由基的清除率

建立了Fe2+-H2O2-亚甲基蓝化学发光体系用于水果对羟自由基清除率的测定。实验发现,在酸性介质中,Fe2+和H2O2在线生成的羟自由基(.OH)与亚甲基蓝反应能产生较强的化学发光,加入水果水提取物可以清除部分羟自由基,使化学发光强度降低。据此,结合流动注射技术,建立了测定水果水提取物对.OH清除率的化学发光新方法。将该方法应用于9种水果水提取物对.OH清除能力的研究,结果满意。     

Electrochemical Activation of the Natural Catalytic Cycle of Cytochrome P450s in Human Liver Microsomes

The natural catalytic cycle of cytochrome (cyt) P450 enzymes in human liver microsome (HLM) films was activated electrochemically via the electron transfer sequence electrode→cyt P450 reductase (CPR)→cyt P450. Cyclic voltammograms for HLM films had midpoint potentials of ?0.50 V vs. SCE at pH 7.4 characteristic of CPR, not cyt P450s. HLM and CPR microsomes without cyt P450s did not electrocatalytically reduce H₂O₂, and did not shift midpoint potential when CO was added, also indicating that the peaks do not correspond to iron heme cyt P450 enzymes. Electrochemical activation of the natural cyt P450 cycle for substrate conversion via CPR in HLM films was confirmed by catalytic electrolysis in an electrochemical microfluidic array designed to generate and detect reactive metabolites by measuring their reactivity with DNA.     

Bacterial Cytochrome P450 Catalyzed Post-translational Macrocyclization of Ribosomal Peptides**

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating group of natural products that exhibit diverse structural features and bioactivities. P450-catalyzed RiPPs stand out as a unique but underexplored family. Herein, we introduce a rule-based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co-occurrence and co-conservation of precursors and P450s and interactions between them, successfully facilitating the identification of diverse P450-catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can catalyze the formation of Trp-Trp-Tyr (one C−C and two C−N bonds), Tyr-Trp (C−C bond), Trp-Trp (C−N bond), and His-His (ether bond) crosslinks, respectively, within three or four residues. KstB, ScnB, and MciB could accept non-native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s to expand the chemical diversity of strained macrocyclic peptides and the range of biocatalytic tools available for peptide macrocyclization.     

Rapid Screening of Olefin Polymerization Catalyst Libraries by Electrospray Ionization Tandem Mass Spectrometry

The simultaneous screening of catalysts according to their propensity for catalyzing the polymerization of ethylene (see scheme) can be achieved with the help of electrospray ionization tandem mass spectrometry. A small (eight catalysts) library of Pd^II complexes synthesized simultaneously in a one-pot reaction has demonstrated the efficiency of this new screening technique. This method could be widened to libraries of other olefin polymerization catalysts.     

高通量筛选策略在细胞色素P450单加氧酶定向进化中的应用

细胞色素P450单加氧酶具有催化活性混杂性的特点,可以催化多种氧化反应,因而在生物催化领域受到了极大的关注。然而P450单加氧酶往往存在催化活性低、稳定性差、区域和立体选择性不理想等问题,从而限制了其在生物催化领域的广泛运用。蛋白质定向进化的发展与运用为改善P450单加氧酶的催化性能提供了有效的途径,而一种高效的高通量筛选策略是保证酶蛋白定向进化成功实施的关键。本文综述了P450单加氧酶定向进化过程中高通量筛选策略的最新进展。     

A Compound I Mimic Reveals the Transient Active Species of a Cytochrome P450 Enzyme: Insight into the Stereoselectivity of P450-Catalysed Oxidations

Catching the structure of cytochrome P450 enzymes in flagrante is crucial for the development of P450 biocatalysts, as most structures collected are found trapped in a precatalytic conformation. At the heart of P450 catalysis lies Cpd I, a short-lived, highly reactive intermediate, whose recalcitrant nature has thwarted most attempts at capturing catalytically relevant poses of P450s. We report the crystal structure of P450BM3 mimicking the state in the precise moment preceding epoxidation, which is in perfect agreement with the experimentally observed stereoselectivity. This structure was attained by incorporation of the stable Cpd I mimic oxomolybdenum mesoporphyrin IX into P450BM3 in the presence of styrene. The orientation of styrene to the Mo-oxo species in the crystal structures sheds light onto the dynamics involved in the rotation of styrene to present its vinyl group to Cpd I. This method serves as a powerful tool for predicting and modelling the stereoselectivity of P450 reactions.     

Fast Screening of Chicken Egg Lysozyme in White Wine Products by Extractive Electrospray Ionization Mass Spectrometry

Fast detection of trace lysozyme,one of the most important food allergens in white wine samples,was achieved by extractive electrospray ionization mass spectrometry without sample pretreatment in this ...     

对氨基苯甲酸酯同系物与细胞色素P450代谢中间体络合物形成速率的定量构效关系

对氨基苯甲酸酯同系物与细胞色素Ｐ４５０代谢中间体络合物形成速率的定量构效关系傅旭春,刘志强,蒋惠娣（浙江医科大学药学系,杭州,３１０００６）关键词细胞色素Ｐ４５０,对氨基苯甲酸酯,代谢中间体络合物,定量构效关系,分子连接性细胞色素Ｐ４５０是混合功能氧...     

Electrochemistry of Cytochrome P450 2B6 on Electrodes Modified with Zirconium Dioxide Nanoparticles and Platin Components

The direct electrochemical and electrocatalytic behavior of the immobilized cytochrome P450 2B6 (CYP2B6) on zirconium dioxide nanoparticles (ZrO₂) was investigated. The film of nano‐structured ZrO₂ that incorporated cytochrome P450 2B6 (CYP2B6) with colloidal paltin, which was stabilized by poly‐lysine (Pt‐PLL), was prepared on glassy carbon electrodes. In anaerobic solutions, the immobilized CYP2B6 exhibited a reversible electron transfer between the heme electroactive center of CYP2B6 and electrodes with a formal potential of ?(0.449±0.004) V at pH 7.4. In air‐saturated solutions, an increased bioelectrocatalytic reduction current could be obtained with the CYP2B6‐modified electrode with the addition of anticancer drugs, such as lidocaine. This leads to the construction of disposable biosensors for drugs by utilizing the electrochemical activity and catalytic reactions of the immobilized CYP2B6.     

Novel Conformational Transitions of Human Cytochrome P450 2C8 during Thermal and Acid-induced Unfolding

Transitions among various heme coordination/spin states, heme environments and protein conformations of human cytochrome P450 2C8 were investigated under different denaturing conditions by means of electronic absorption and circular dichroism spectroscopies. It is the first report of it's kind. Our results indicated that the thermal and acid‐induced denaturation could convert P450 2C8 to various P420 forms. In the thermal unfolding process, the ferric P420 thermal form emerged with weakened Fe‐S (thiolate) bond. An absorption band at ca. 425 nm of the ferrous P420 2C8 thermal form was observed, suggesting that the axial Cys435 was protonated or displaced by other ligand. Moreover, the new coordination bond was stabilized when the temperature was cooled down. When binding with CO, the ferrous P420 2C8 thermal form had the protonated thiol of Cys435 as the axial ligand. X‐ray structure of P450 2C8 suggested that the specific structure of the β‐bulge where the axial cysteine ligand located might be the reason of the formation of these P420 2C8 thermal forms. In the acid‐induced unfolding studies, we found that at pH 3.0 the heme could be irreversibly released from the heme pocket of ferric and ferrous P450 2C8. Interestingly, the released heme could form a new coordination bond with an unidentified ligand at the surface of partially unfolded protein when binding with CO at reduced state.     

High-Throughput Screening of Ten Microcystins and Nodularins, Cyanobacterial Peptide Hepatotoxins, by Reversed-Phase Liquid Chromatography-Electrospray Ionisation Mass Spectrometry

Liquid chromatography-electrospray ionisation mass spectrometry was evaluated in the high-throughput analysis of microcystins and nodularins, cyanobacterial peptide hepatotoxins. Extracts originating from cyanobacterial strains and field material were separated on a 30 mm × 4 mm I.D. Merck Purospher STAR RP-18e column using a rapid gradient of aqueous formic acid and acetonitrile, ionised by electrospray technique and analysed on a Micromass Quattro II triple-quadrupole mass spectrometer operated in the selected ion recording (SIR) mode. The total analysis time per sample was 2.8 min corresponding to 514 samples a day. The system showed good robustness during a series of 320 repetitive injections of a field sample containing three major microcystins.