老年性痴呆患者血浆中氨基酸类神经递质的HPLC荧光检测

采用邻苯二甲醛(OPA)柱前衍生反相高效液相色谱法测定了老年痴呆患者(AD)血浆中4种氨基酸类神经递质(天门冬氨酸、谷氨酸、γ-氨基丁酸、甘氨酸)的含量.用Hanbon反相柱分离,荧光检测器检测;流动相A为四氢呋喃-甲醇-0.05 mol/L磷酸二氢钾(pH 6.0)(体积比2 : 35 : 63);流动相B为90%甲醇;采用二元梯度洗脱.天门冬氨酸、谷氨酸、γ-氨基丁酸、甘氨酸的检出限分别为0.21、0.01、0.05、0.02 pmol/L,在对应的浓度范围内线性关系良好,相关系数为0.997 5 ～0.999 1,峰面积的相对标准偏差分别为1.08%、1.24%、1.12%和1.35%,回收率为98% ～99%.AD患者血浆中天门冬氨酸和谷氨酸的浓度显著高于对照组(P＜0.05),甘氨酸与对照组无显著差异.     

示波极谱法同时测定两种氨基酸

在蛋白质中的谷氨酸和天门冬氨酸的电分析方法,目前只有单个测定方法,未见有连续测定方法的报道.本文研究了在浓度为0.020g/L谷氨酸和0.010 g/L天门冬氨酸,pH为3.5～4.5的硼酸体系中同时出现两灵敏度和分辨率均比较高的极谱波,峰电位分别为0.30V和0.60V,检出限分别为0.0050g/L和0.0025g/L,当其浓度分别为0.0100～0.1000g/L和0.005～0.05g/L与峰电流呈线性关系.浓度为0.500g/L胱氨酸、酪氨酸、亮氨酸、脯氨酸等中性氨基酸对测定无大的影响.回收率分别为95.0～100.1%和96.5～101.6%,未发现有系统误差存在.经3次对人发水解液分析,并与氨基酸分析仪测量结果对照,平均值分别为9.50%和9.55%,相对误差为1.50%和1.80%,均在国家规定的范围内.本法简便快速,灵敏度高,稳定性好,可用于各种蛋白质中的谷氨酸和天门冬氨酸的测定.     

米根霉胞内氨基酸的高效液相色谱测定及其代谢途径分析

建立了米根霉胞内代谢物提取方法定量评价体系,用100％甲醇作为提取剂获得对米根霉胞内能荷物质最佳的提取效果.用HPLC法建立了米根霉产富马酸体系胞内18种氨基酸的含量测定方法.采用异硫氰酸苯酯( PITC)作为柱前衍生化试剂,所有氨基酸在28 min内洗脱完毕.以Sepax AA柱(250 nn×4.6 mm,5μm)为分析柱,流速1.0 mL/min,检测波长254 nm,以0.1 mol/L醋酸钠(pH 6.5)-乙腈(93∶7,V/V)为流动相A,乙腈-水( 80∶ 20,V/V)为流动相B,梯度洗脱.18种氨基酸在1.87～45.3 mg/L范围内线性关系良好,相关系数大于0.991;加标回收率在96.3％～102.7％之间;相对标准偏差在1.6％～2.9％之间.运用本方法测定不同发酵时间米根霉胞内氨基酸的含量,得到其在发酵过程中的变化趋势,并结合氨基酸代谢途径分析了该发酵条件下富马酸产量偏低的原因.     

柱前衍生高效液相色谱测定小儿复方氨基酸注射液中氨基酸

建立了高效液相色谱(HPLC)测定小儿复方氨基酸注射液中各种氨基酸含量的方法.采用Kromasil C18(250×4.6 mm,5 μm)色谱柱,以2,4-二硝基氟苯为衍生试剂,梯度洗脱.流动相A为乙腈∶水(50∶50,V/V),流动相B为0.04 mol/L的磷酸二氢钾溶液(pH=6.8);流速为1.0 mL/min;检测波长为360 nm.所测氨基酸的线性范围为0.008～0.2213 mg/mL,平均回收率在97.85%～102.86%之间.     

高效液相色谱法直接测定谷丙甘氨酸胶囊中3种氨基酸的含量北大核心CSCD

建立HPLC法直接测定谷丙甘氨酸胶囊中3种氨基酸含量的方法。采用Sharpsil氨基柱(250 mm×250 mm,5μm),通过优化流动相中盐相种类、pH、有机相比例等参数,得到最佳流动相:溶液A(20 mmol/L KH_(2)PO_(4)溶液(KH_(2)PO_(4)2.72 g,加水1 L溶解后,加浓氨水5 mL,用H_(3)PO_(4)调节pH值至5.0))∶乙腈=45∶55(V/V),流速为1 mL/min,检测波长为210 nm。样品用溶液A溶解后再加乙腈稀释。3种氨基酸线性关系良好,加样回收率在98.58%~100.32%之间,相对标准偏差(RSD)均小于0.6%。该方法操作简单,具有良好的精密度、重复性和准确度,可用于谷丙甘氨酸胶囊中3种氨基酸含量的测定。     

3-(4-酮基苯甲酸)-2-喹啉甲醛衍生氨基酸的高效毛细管电泳-激光诱导荧光检测分析

1引言在众多的神经递质中，兴奋性和抑制性氨基酸一直是神经科学中所关注的生化指标。兴奋性氨基酸如谷氨酸（Glu）、天门冬氨酸（Asp）和抑制性氨基酸γ-氨基丁酸（GABA）、甘氨酸（Gly）等在体内具有广泛的生理意义。牛磺酸（Tau）不仅是神经递质，而且具有调节机体的作用。氨基酸在血和脑脊液中的含量较低，大部分氨基酸由于无紫外吸收，在分析过程中常用柱前和柱后衍生氨基酸的方法使之具有紫外或荧光性，3－（4-酮基苯甲酸）－2－喹啉甲醛（CBQCA，3－（4-carboxybenzoyl）－2-quinolinecarbox-aldehyde）为新的柱前氨基酸类衍生…     

OPA-FMOC在线柱前衍生化HPLC法测定甘露聚糖肽中氨基酸组成及含量

建立了OPA-FMOC在线柱前衍生化高效液相色谱法测定甘露聚糖肽中氨基酸组成及含量的方法,采用色谱柱为Agilent ZORBAX Eclipse XDB-C18柱(250 mm×4.6 mm,5μm),流动相A为40 mmol/L Na_2HPO_4缓冲溶液(pH 7.8);流动相B为甲醇:乙腈:水(45∶45∶10,V/V/V);梯度洗脱;流速:1.5 m L/min;柱温:40℃;检测波长:338,262 nm。20种氨基酸在53 min内得到较好地分离,各氨基酸在5~500μg/m L范围内线性关系良好,相关系数在0.9941~0.9999之间;平均加标回收率在85.6%~102.6%之间;检出限在0.56~8.33 ng(进样量1μL)范围内。甘露聚糖肽样品中含有16种氨基酸,氨基酸总量平均为3.56%(n=3)。     

毛细管电泳-激光诱导荧光检测法分离检测谷胱甘肽及其构成氨基酸

以4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD-Cl)为柱前衍生试剂,建立了一种毛细管电泳-激光诱导荧光直接检测氧化型和还原型谷胱甘肽及其构成氨基酸(谷氨酸、半胱氨酸和甘氨酸)的新方法。经过实验条件的优化,采用25 mmol/L硼砂-20 mmol/L聚氧乙烯月桂醚(Brij-35)-5%乙腈(pH 9.5)的缓冲体系,在柱温为25°C、分离电压为20 kV的条件下,压力进样3447.5 Pa(0.5 psi)×3 s,五种物质在11 min内实现高效基线分离。在该方法下,还原型谷胱甘肽、氧化型谷胱甘肽、谷氨酸、半胱氨酸和甘氨酸的线性范围分别为:1～50μg/mL,1～50μg/mL,0.5～25μg/mL,1～50μg/mL,0.1～20μg/mL;检测限分别为:0.1μg/mL,0.1μg/mL,0.005μg/mL,0.1μg/mL,0.001μg/mL。以还原型谷胱甘肽钠粉针剂为样品,方法的加标回收率为99.5%～110.7%,相对标准偏差为0.26%～3.272%(n=3)。该方法准确、快速、灵敏、检测限低,有望用于样品中氧化型和还原型谷胱甘肽及其构成氨基酸的含量分析。     

嗜碱微生物细胞内氨基酸的高效液相色谱测定及代谢途径分析

建立了嗜碱微生物细胞内17种氨基酸的高效液相色谱分析方法。采用Sinochrom ODS-BP色谱柱,以乙腈∶水=1∶1(溶液A),0.06mol/L乙酸钠溶液 0.1%N,N-二甲基甲酰胺 10%乙腈,用5%醋酸溶液调至pH=6.4(溶液B)为流动相进行梯度洗脱,流速0.8mL/min,柱温35°C,检测波长360nm。在此色谱条件下,各组分在32min内均得到较好分离,回收率符合含量测定要求。运用本方法测定嗜碱微生物发酵不同时间细胞内氨基酸含量,得到嗜碱微生物细胞内氨基酸在发酵过程中的变化趋势,分析了相关代谢途径。     

场增强样品堆积-毛细管电泳法测定山楂粉中的氨基酸

采用场增强样品堆积-毛细管电泳法建立了在线富集氨基酸的分析方法,用于中药山楂中水解氨基酸的检测,并进行了回收率实验. 采用富集电压为-20 kV,进样压力为3 psi,进样时间为50 s,紫外检测波长为214 nm,运行缓冲溶液为270 mmol/L乙酸-270 mmol/L乙酸钠（pH=4.15）-6%（V/V）乙腈溶液,分离电压为17 kV,组氨酸、精氨酸、色氨酸、酪氨酸、缬氨酸、苯丙氨酸、亮氨酸、苏氨酸、丙氨酸、甘氨酸、谷氨酸和门冬氨酸等12种氨基酸在50 min内达到分离,检出限在0.000 3~0.08 μg/mL之间.     

Pattern Recognition of Amino Acids in Wines

New synthesized fatty acid amides (N‐(2‐(piperidin‐1‐yl)ethyl)oleamide, and N‐(2‐(pyrrolidin‐1‐yl)ethyl)oleamide) were used for the design of stochastic sensors based on nanographene paste. The stochastic sensors were used for pattern recognition of four amino acids: L‐histidine, L‐tyrosine, L‐ornithine, and L‐lysine in wines. The pattern recognition was performed based on the signatures recorded for each of the amino acids. The limits of determination allow the assay of amino acids in wine at very low concentrations faster, reliable, and more cost effective than other methods proposed to date.     

A Facile Synthesis of N-Phosphoryl Amino Acids Containing Hydroxy Group

three kinds of N-(diisopropyloxyphosphoryl) amino acids containing hydroxyl group were prepared in high yield by using diisopropyl phosphite as the phosphorylating agent, sodium hypochlorite as the chlorinating agent and tetrabutyl ammonium bromide as the phase transfer catalyst in basic aqueous media.     

Applications of Unnatural Amino Acids in Protease Probes

Since proteases are involved in a wide range of physiological and disease states, the development of novel tools for imaging proteolytic enzyme activity is attracting increasing interest from scientists. Peptide substrates containing proteinogenic amino acids are often the first line of defining enzyme specificity. This Minireview outlines examples of major recent advances in probing proteases using unnatural amino acid residues, which greatly expands the possibilities for designing substrate probes and inhibitory activity‐based probes. This approach already yielded innovative probes that selectively target only one active protease within the group of enzymes exhibiting similar specificity both in cellular assays and in bioimaging research.     

蛋白质功能化新策略:嵌入非天然氨基酸

天然蛋白质由20种天然氨基酸组成,这些蛋白质的构筑基元包含功能基团:羧基、氨基、巯基、硫醚、羟基、碱性胺、烷基和芳基。然而,这些有限的功能基团却不足以完成生物体内所有的生物学功能。为了更好地让生命的体现者--蛋白质完成更加精确和多样的生物学功能,自然界会对蛋白质进行翻译后的修饰,包括:磷酸化,甲基化,乙酰化或者羟基化,甚至在某些情况下,进化出一种新型的翻译机制以便插入硒代半胱氨酸或者吡咯霉素。受此启发,生物化学家发展出各种生物或化学方法来改变或插入新的蛋白质构筑基元,使天然蛋白质完成其相应的生物学功能或者使其具有某些特殊的性质,甚至是创造一种新酶。该文将简单介绍这些蛋白质修饰策略以及该领域的最新进展。     

固定化D-氨基酸氧化酶催化DL-氨基酸去消旋化制备非天然L-氨基酸

在过氧化氢酶和氧气存在下,固定化D-氨基酸氧化酶(D-AAO)对映选择性催化DL-氨基酸中的D-对映体氧化脱氨为相应酮酸,L-对映体保留.研究了D-AAO的底物特异性并对反应条件进行了优化.结果表明:D-AAO具有较宽的底物谱,能够催化疏水性α-氨基酸的D-对映体氧化脱氨.在最优反应条件下,D-AAO催化DL-2-氨基丁酸、DL-2-氨基戊酸去消旋化,L-2-氨基丁酸、L-2-氨基戊酸的收率分别为48%和47%,ee分别为99.5%和99.8%.进一步地利用Pd-C/HCOONH4催化氧化脱氨过程中产生的亚氨基酸原位还原,有效提高了L-2-氨基丁酸、L-2-氨基戊酸的收率并保持高的光学纯度.     

Catalysts Based on Amino Acids for Asymmetric Reactions in Water

Wet and wild : Water is a powerful force in nature, as evident in the painting “ES” by Zdzislaw Beksinski (1985, oil, 92 cm×90 cm; reproduced with permission from Piotr Dmochowski), as well as a promising medium for asymmetric reactions. As catalysts, natural amino acids are ideal candidates. This Minireview summarizes recent advances in asymmetric catalysis in water, with amino acids and their derivatives as effective catalysts or essential components.

     

柱前衍生法在氨基酸分析测定中的应用

介绍了目前柱前衍生法分析氨基酸常用的几类衍生试剂及相应的衍生方法，并且比较了各类柱前衍生试剂的优缺点,对近期氨基酸分离分析中的色谱检测方法做了系统综述。     

The Determination of Protonation Constants of Some Amino Acids and Their Esters by Potentiometry in Different Media

In this study, stoichiometric protonation constants of L-tyrosine, L-cysteine, L-tryptophane, L-lysine, and L-histidine, and their methyl and ethyl esters in water and ethanol–water mixtures of 30, 50, and 70% ethanol (v/v), were determined potentiometrically using a combined pH electrode system calibrated as the concentration of hydrogen ion. Titrations were performed at 25^∘C and the ionic strength of the medium was maintained at 0.10 mol⋅L⁻¹ using sodium chloride. Protonation constants were calculated by using the BEST computer program. The effect of solvent composition on the protonation constants is discussed. The log₁₀ K₂ values of esters generally decreased with increasing ethanol content. However, the log₁₀ K₁ values of the esters of L-tyrosine, L-cysteine, and L-tryptophane were found to increase with increasing ethanol content in contrast those of L-lysine and L-histidine esters.     

Ionic Liquid Crystals Derived from Amino Acids

Novel chiral amino acid derived ionic liquid crystals with amine and amide moieties as spacers between the imidazolium head group and the alkyl chain were synthesised. The key step in the synthesis utilised the relatively uncommon SO₃ leaving group in a microwave‐assisted reaction. The mesomorphic properties of the mesogens were determined by differential scanning calorimetry (DSC), polarising optical microscopy (POM) and X‐ray diffraction. All liquid crystalline salts exhibit a smectic A mesophase geometry with strongly interdigitated bilayer structures. An increase of the steric bulk of the stereogenic centre hindered the formation of mesophases. In case of phenylalanine‐derived derivatives a mesomorphic behaviour was observed for shorter alkyl chains as compared to other amino acid derivatives indicating an additional stabilising effect by the phenyl moiety.     

Microwave-Assisted Esterification of Diverse Carboxylic Acids and Chiral Amino Acids

A facile and efficient synthetic method of esters from their corresponding carboxylic acids and amino acids is described. The esterification reaction of carboxylic acids and amino acids could be greatly accelerated under microwave irradiation because the reactions described in this article took place in only 5 min with almost quantitative yields, and distinct acidity of catalytic acids was well tolerated. Unlike the racemation problem in microwave-assisted N-acylation reactions, the esters of chiral amino acids could be achieved with retention of configuration under this condition.