高效液相色谱法直接测定芳香族氨基酸

芳香族氨基酸包括苯丙氨酸(Phcnylalanine,Phe)、酪氨酸(Tyrosine,Tyr)、色氨酸(Tryptophan,Trp)等.定量分析芳香族氨基酸在蛋白质化学和临床诊断与研究中具有重要意义~([1]).高效液相色谱法(HPLC)~([2,3])是分析氨基酸的常用方法.文献~([3])采用梯度洗脱在不同波长处分别测定Tyr、Phe和Trp,在7 min内完成测试.本研究采用高效液相色谱法(HPLC)-紫外检测,检测波长为257nm,同时测定Tyr、Phe和Trp的含量.     

芳香氨基酸光敏化瞬态产物的光谱学及动力学表征

运用KrF激光闪光光解瞬态吸收光谱 ,以丙酮为光敏剂 ,研究了水溶液中芳香氨基酸的光化学反应 .通过动力学分析和猝灭实验 ,鉴别了光化学反应过程中的瞬态产物 ,获取了激发三重态的瞬态吸收光谱及动力学参数 .在丙酮存在下 ,色氨酸(Trp)和酪氨酸 (Tyr)的水溶液光解 ,分别观察到Trp激发三重态、N中心色氨酸自由基 (Trp/N·)和酪氨酸的酚氧自由基 (Tyr/O·) ,阐述了二者是丙酮三重态与Trp ,Tyr分别通过三重态 三重态 (T T)激发能转移和电子转移生成 ;苯丙氨酸 (Phe)不能与丙酮三重态进行激发能转移和电子转移 .进一步 ,在色氨酰酪氨酸 (Trp Tyr)敏化光解过程中 ,观察到分子内的电子转移 ,即Trp/N· Tyr→Trp Tyr/O·自由基的生成过程 .     

非衍生化毛细管区带电泳直接紫外检测法测定茶叶中的4种氨基酸

采用非衍生化毛细管区带电泳直接紫外检测法同时分离测定精氨酸(Arg)、色氨酸(Trp)、苯丙氨酸(Phe)和酪氨酸(Tyr)4种氨基酸,并应用于不同发酵过程的茶叶样品的测定。在分离电压为20 kV、柱温为25 ℃、检测波长为190 nm条件下,以25 mmol/L硼酸-硼砂缓冲溶液(pH 10.0)为运行缓冲液,4种组分在8 min内达到基线分离,Arg、Trp、Phe、Tyr的检出限分别为5.0,1.0,0.3和0.5 mg/L。7次平行测定中,4种组分迁移时间的相对标准偏差(RSD)均小于2.8%,峰电流的RSD均小于4.0%。将所建立的方法用于11种实际茶叶样品中Arg、Trp、Phe和Tyr含量的测定,结果令人满意。该方法可以为茶叶的质量评估提供借鉴。     

LC/MS/MS方法筛查新生儿苯丙酮尿症

苯丙酮尿症 [1～ 4 ] ( PKU )发病原因是患者基因缺陷使肝脏不能合成苯丙氨酸羟化酶而导致体内苯丙氨酸 ( Phe)不能正常代谢为酪氨酸 ( Tyr) ,前者在体内大量堆积并氧化为对人体有害的苯丙酮酸 . PKU是目前筛查范围最广的氨基酸代谢遗传疾病 ,在全世界每年 [5]约有一千万婴儿接受 PKU筛查 ;在我国 ,PKU也是卫生部要求重点筛查的病种 .Chace[5]等在 1 993年报道了 MS/ MS方法筛查新生儿PKU:直接使用 MS/ MS的中性碎片丢失扫描方式检测 Phe和 Tyr,通过氘代内标与待测氨基酸的质谱峰高比来定量 .MS/ MS方法速度快、准确性好、可以…     

GC—CI—MS—SIM方法论断新生儿苯丙酮尿症

苯丙酮尿症（PKU）的发病原因是患者苯丙氨酸羟化酶的缺陷,从而导致体内苯丙氨酸（Phe）不能正常代谢为]酷氨酸（Tyr),前者在体内大量堆积并氧化为有害的七人酮酸,PKU是目前筛查范围最广的氨基酸代谢遗传疾病,全世界每年约有一千万儿接受PKU调查,在中国PKU也是卫生部要求重点筛查的病种^[1],目前常规筛查方法是细菌抑制法（BIA),它的特点是用低、速率慢、空易产生假阳性;Chace等^[2,3]报道了MS－MS）京都是新生儿PKU,曲峻等^[4]发展了Chace的方法,采用HPLC与MS－MS联用方法（LCD－MS－MS）筛查新生儿PKU^[4],利用HPLC富集,MS－MS作为HPLC的检测器定量血样中的苯丙氨酸和酷氨酸,MS－MS和LC－MS－MS方法速度快,准确性好,由于是通过测定Phe与Tyr的比值来筛查PKU,克服了假阳性的产生^[2~4],但是LC－MS－MS和MS－MS价格都很昂贵,不适合国内普及推广,本文将血样的Phe与Tyr进行化学修饰后,采用化学电离源选择离子的方式用气相色谱质谱（Gas chromatography-chemical ionizatino -mass spectrograph-selective ion measurement简称GC－CI－MS－SIM）进行了分析,测定了Phe和Tyr相应衍生物的准分子离子峰面积,用外标法计算出血样中的Phe与Tyr摩尔浓度,与MS－MS、LC－MS－MS一样取得同样满意的效果,由于GC－MS价格较低,适合国内普及推广。     

荧光光谱法研究亚甲蓝与三种芳香族氨基酸的相互作用

本文运用荧光光谱法研究亚甲蓝(MB)分别与色氨酸(Trp)、酪氨酸(Tyr)和苯丙氨酸(Phe)三种芳香族氨基酸的相互作用。在pH 7.4 Tris-HCl缓冲溶液中,MB引起上述三种氨基酸明显的荧光猝灭现象,最大荧光猝灭波长分别位于346、303和282nm。其荧光猝灭值(F0-F)在一定范围内与MB成正比,用于测定MB具有高灵敏度。通过Stern-Volmer作图及温度的影响,表明MB与三种芳香族氨基酸之间以摩尔比1∶1形成基态复合物,均产生显著的静态猝灭,相互作用力较强,其中MB主要通过疏水作用与Trp结合,与Tyr和Phe之间的结合过程以静电作用力为主。     

Pd(Ⅱ)与色氨酸、酪氨酸及苯丙氨酸相互作用的荧光光谱

当Pd(Ⅱ)与色氨酸(Trp)、酪氨酸(Tyr)及苯丙氨酸(Phe)等芳香族氨基酸相互作用时,能观察到3种氨基酸的荧光均发生猝灭. 从吸收光谱的变化,温度对猝灭作用的影响以及猝灭常数Ksv,可以判定荧光猝灭作用是由于Pd(Ⅱ)与上述氨基酸形成基态配合物而导致的静态猝灭过程. 并认为在一定浓度的Cl-存在下,Pd(Ⅱ)与氨基酸分别以N, N配位和N, O配位形成以下混配型三元配合物Pd(HR)Cl2 (Trp和Phe体系)和Pd(H2R)Cl2(Tyr体系),并推测了配合物相应的结构. 该荧光猝灭体系不仅可用于研究钯(Ⅱ)与上述芳香族氨基酸的相互作用,也可成为以氨基酸(特别是Trp)作探针高灵敏荧光猝灭法测定钯的基础.     

高效液相色谱-同位素稀释质谱法测定血清中的苯丙氨酸

苯丙酮尿症(PKU)是一种世界性分布的遗传疾病,病人体内缺少苯丙氨酸羟化酶(PAH),导致苯丙氨酸(Phe)不能正常代谢为酪氨酸,引起Phe代谢产物在人体中过量蓄积,造成患者中枢神经系统受到不可逆的损害而影响智力.     

在饮水中典型溶解性有机氮酪氨酸氯化生成氯仿的机理分析

饮用水氯化消毒可以有效杀灭细菌, 但同时会产生危害人体健康的消毒副产物(DBP). DBP生成机理研究是有效控制DBP的前提. 溶解性有机氮(DON)是DBP的重要前体物, 选取典型DON-丙氨酸(Ala)、苯丙氨酸(Phe)和酪氨酸(Tyr)作为氯仿(CF)等DBP的前体物, 研究三种氨基酸(AA)的耗氯量和CF产率; 同时考察了Tyr氯化中间产物2,4,6-TCP的氯化特性和CF产率; 采用GC/MS扫描和前线轨道理论验证, 探讨了CF的主要生成路径. 研究发现, 在同等氯化反应条件下, 由于侧链基团的不同, Tyr的耗氯量以及CF产率都明显高于Ala和Phe, 从而说明Tyr确实是一种重要的CF前体物质. CF的主要生成路径为Tyr→ 4-MCP → 2,4-DCP → 2,4,6-TCP → CF. 氯胺消毒工艺可有效控制CF的生成, 并能减少2,4,6-TCP的产生, 但不能确保饮用水的生物安全性. 氯化消毒之前将Tyr等重要前体物去除可能是控制CF等DBP更加有效的措施.     

GC-CI-MS-SIM方法诊断新生儿苯丙酮尿症

苯丙酮尿症 (PKU)的发病原因是患者苯丙氨酸羟化酶的缺陷 ,从而导致体内苯丙氨酸 (Phe)不能正常代谢为酪氨酸 (Tyr) ,前者在体内大量堆积并氧化为有害的苯丙酮酸 .PKU是目前筛查范围最广的氨基酸代谢遗传疾病 ,全世界每年约有一千万婴儿接受 PKU筛查 ;在中国 PKU也是卫生部要求重点筛查的病种[1] .目前常规筛查方法是细菌抑制法 (BIA) ,它的特点是费用低、速度慢、容易产生假阳性 ;Chace等 [2 ,3] 报道了 MS- MS方法筛查新生儿 PKU.曲峻等 [4 ] 发展了 Chace的方法 ,采用 HPLC与MS- MS联用方法 (LC- MS- MS)筛查新生儿 PK…     

Fast Diagnosis of Neonatal Phenylketonuria by Gas Chromatography-Mass Spectrometry Following Microwave-Assisted Silylation

Phenylketonuria (PKU) is a fairly common autosomal recessive disease. Phenylalanine (Phe) and tyrosine (Tyr) are the biomarkers of PKU, and it can be diagnosed by the measurement of Phe and Tyr in neonatal blood samples. A fast diagnostic procedure for neonatal PKU has been developed using microwave-assisted silylation followed by gas chromatography-mass spectrometry. Amino acids extracted from neonatal blood samples are rapidly derivatized with N, O-bis(trimethylsilyl)-trifluoroacetamide under microwave irradiation; the derivatives are then analyzed by GC-MS. The silylation conditions have been optimized, and the method validated for linear range, detection limit, precision and recovery. The proposed method is linear from 20 to 800 µM, detection limit is less than 0.35 µM, relative standard deviation (RSD) is less than 9.0%, and recoveries of 87% and 83% were obtained for Phe and Tyr. The proposed method was tested by the determination of Phe and Tyr in blood spots from eight PKU-positive neonates and twelve control neonates. Microwave irradiation considerably accelerates the derivatization reaction of amino acid with BSFTA, and shortens the whole analysis time. Microwave-assisted silylation coupled to GC-MS is a powerful tool for fast screening of neonatal PKU.     

Solid phase synthesis and opioid receptor binding activities of [D-Ala2, D-Leu5]enkephalin analogs containing a fluorinated aromatic amino acid

Five [D-Ala2, D-Leu55]enkephalin (DADLE) analogs containing fluorinated Tyr1 or Phe4 residue, that is, [Phe(2F)4] (I), [Phe(3F)4] (II), [Phe(4F)4] (III), [Tyr(3F)1] (IV) and [Tyr(2F)1] (V), were synthesized by the solid phase method and their opioid receptor affinities were examined. Affinity profiles of five derivatives for the mu- and delta-receptor were similar to those of DADLE, and the affinity for kappa-receptor was zero or negligible.     

Determination of phenylalanine and tyrosine by liquid chromatography/mass spectrometry

Phenylketonuria is a common metabolic disorder disease. Those affected appear normal at birth, but without treatment they develop severe psychomotor retardation. Throughout life, they must control their blood levels of phenylalanine (Phe) and consume a diet containing adequate amounts of Phe and tyrosine (Tyr). We have developed a liquid chromatographic/mass spectrometric (LC/MS) method for the quantitative evaluation of Phe and Tyr in food samples. This method takes advantage of the good separation of LC and the selective and reliable quantification provided by MS in the electrospray ionization mode. The LC/MS method is very suitable for the determination of selected amino acids in various matrixes. It is sensitive to levels as low as about 0.30 ppm for Tyr and 0.70 ppm for Phe and robust. Nearly 100 nondietary food samples were analyzed by the developed method.     

Posttranslational hydroxylation of human phenylalanine hydroxylase is a novel example of enzyme self-repair within the second coordination sphere of catalytic iron

Phenylalanine hydroxylase, a mononuclear non-heme iron enzyme, catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and reduced pterin cofactor. X-ray structural studies have established the coordination around the iron metal center and point to significant interactions within the second coordination sphere. One such interaction involves Tyr325 in human phenylalanine hydroxylase (hPAH), which forms a hydrogen-bonding network with an aqua ligand on iron and the pterin cofactor. The full-length tetramer (1-452) and truncated dimer (117-424) Tyr325Phe hPAH mutant enzymes showed similar kinetics, thermal stabilities, and oligomerization profiles as their corresponding wild-type proteins. The possibility of in vivo posttranslational hydroxylation that would restore the activity of hPAH was examined by mass spectrometry on the trypsin digested full-length (1-452) hPAH Tyr325Phe point mutant. The amino acid tags obtained by ESI-MS/MS confirmed the presence of a Phe325 in the peptide corresponding to the doubly charged precursor ion at m/z 916.4 (L A T I F W F T V E F G L C K), and its hydroxylated counterpart in the peptide corresponding to the m/z 924.4 (L A T I F-OH W F T V E F G L C K) byproduct ion series comprising the fragments y(5)-y(12). Furthermore, the point mutation Tyr325Ala resulted in an enzyme that was totally inactive and did not display any evidence of hydroxylation. These results demonstrate the importance of Tyr325 for proper conformation of the active site, substrate binding, and catalysis. The rescue of the Tyr325Phe mutant in hPAH via self-hydroxylation presents a novel example of oxidative repair on the molecular level.     

Aromatic ring-flipping in supercooled water: implications for NMR-based structural biology of proteins.

We have characterized, for the first time, motional modes of a protein dissolved in supercooled water: the flipping kinetics of phenylalanyl and tyrosinyl rings of the 6 kDa protein BPTI have been investigated by NMR at temperatures between -3 and -16.5 degrees C. At T = -15 degrees C, the ring-flipping rate constants of Tyr 23, Tyr 35, and Phe 45 are smaller than 2 s(-1), i.e., flip-broadening of aromatic NMR lines is reduced beyond detection and averaging of NOEs through ring-flipping is abolished. This allows neat detection of distinct NOE sets for the individual aromatic (1)H spins. In contrast, the rings of Phe 4, Tyr 10, Tyr 21, Phe 22, and Phe 33 are flipping rapidly on the chemical shift time scale with rate constants being in the range from approximately 10(2) to 10(5) s(-1) even at T = -15 degrees C. Line width measurements in 2D [(1)H,(1)H]-NOESY showed that flipping of the Phe 4 and Phe 33 rings is, however, slowed to an extent that the onset of associated line broadening in the fast exchange limit is registered. The reduced ring-flipping rate constant of Phe 45 in supercooled water allowed very precise determination of Eyring activation enthalpy and entropy from cross relaxation suppressed 2D [(1)H,(1)H]-exchange spectroscopy. This yielded DeltaH = 14 +/- 0.5 kcal.mol(-1) and DeltaS = -4 +/- 1 cal.mol(-1).K(-1), i.e., values close to those previously derived by Wagner and Wüthrich for the temperature range from 4 to 72 degrees C (DeltaH = 16 +/- 1 kcal.mol(-1) and DeltaS = 6 +/- 2 cal.mol(-1).K(-1)). The preservation of the so far uniquely low value for DeltaS indicates that the distribution of internal motional modes associated with the ring flip of Phe 45 is hardly affected by lowering T well below 0 degrees C. Hence, if a globular protein does not cold denature, aromatic flipping rates, and thus likely also the rates of other conformational and/or chemical exchange processes occurring in supercooled water, can be expected to be well estimated from activation parameters obtained at ambient T. This is of keen interest to predict the impact of supercooling for future studies of biological macromolecules, and shows that our approach enables one to conduct NMR-based structural biology at below 0 degrees C in an unperturbed aqueous environment. A search of the BioMagResBank indicated that the overwhelming majority of the Phe and Tyr rings (>95%) are flipping rapidly on the chemical shift time scale at ambient T, while our data for BPTI and activation parameters available for ring-flipping in Iso-2-cytochrome c reveal that in these smaller proteins a total of six out of seventeen rings ( approximately 35%) are "frozen in" at T = -15 degrees C. This suggests that a large fraction of Tyr and Phe rings in globular proteins that are flipping rapidly on the chemical shift time scale at ambient T can be effectively slowed in supercooled water. The present investigation demonstrates that supercooling of protein solutions appears to be an effective means to (i) harvest potential benefits of stalled ring-flipping for refining NMR solution structures, (ii) recruit additional aromatic rings for investigating protein dynamics, and (iii) use multiple slowly flipping rings to probe cold denaturation. The implications for NMR-based structural biology in supercooled water are addressed.     

高效液相色谱-荧光检测法测定血清中的犬尿氨酸

建立了高效液相色谱-荧光检测(HPLC-FLD)测定血清中犬尿氨酸(kynurenine,Kyn)含量的方法。采用Hypersil C8色谱柱(300 mm×6.0 mm,10 μm), 流动相为0.25 mol/L醋酸锌及50 mmol/L醋酸溶液(含3%乙腈),流速为1.5 mL/min, 荧光检测激发波长和发射波长分别为365 nm和480 nm。血清标本经5%(v/v)高氯酸溶液去除蛋白质后取上层清液直接进样, Kyn经流动相等度洗脱分离后,用FLD进行测定。研究结果表明Kyn保留时间约为8.3 min,线性范围为0.098～19.6 μmol/L,最低检出浓度为0.04 μmol/L,回收率为90.8%～96.2%,日内、日间测定的相对标准偏差均小于5%,苯丙氨酸、酪氨酸、色氨酸、5-羟色胺和犬尿喹啉酸等物质对犬尿氨酸的测定均无干扰。建立的方法简便、快速、灵敏、特异,适用于临床和科研应用。     

A New Sensor for Simultaneous Determination of Tyrosine and Dopamine Using Iron(III) Doped Zeolite Modified Carbon Paste Electrode

A new chemically modified electrode is constructed based on iron(III) doped zeolite modified carbon paste electrode (Fe³⁺Y/ZMCPE). The new sensor could be used for the simultaneous determination of the biologically important compounds dopamine (DA) and tyrosine (Tyr). The measurements are carried out using differential pulse voltammetric (DPV) method. The prepared modified electrode shows voltammetric responses of high sensitivity, selectivity and stability for DA and Tyr under optimal conditions, which makes it a suitable sensor for simultaneous trace detection of DA and Tyr in solution. Application of the DPV method demonstrates that in the Briton Robinson buffer solutions (pH=5) containing 50 µmol/L Tyr, there is a linear relationship between the oxidation peaks and the concentrations of DA over the range of 0.1–200 µmol/L, with a detection limit of 0.05 µmol/L (S/N=3). For Tyr a linear correlation between oxidation peak current and concentration of Tyr over the range of 0.5–200 µmol/L (containing 50 µmol/L DA), with a detection limit of 0.08 µmol/L is obtained. The analytical performances of this sensor are evaluated for the detection of DA and Tyr in human serum and a medicine.