利用乙醇系统RP－HPLC分析PTC－氨基酸

首次报道了用乙醇作梯度洗脱有机相分离ＰＴＣ－氨基酸的新方法同乙腈相比,乙醇毒害性小,价廉、易得。在优化的色谱条件下,乙醇洗脱分辨率,灵敏度,准确度均佳,可成为ＰＴＣ－氨基酸分析的一种极有前途的新方法。     

几种氨基酸─铜（Ⅱ）配合物的超氧化物歧化酶活性

用四氮唑蓝光化学还原法对所合成的ＫＣｕ（ＩＤＡ）（Ｓｅｒ）·２Ｈ２Ｏ、ＫＣｕ（ＩＤＡ）（Ａｌａ）·Ｈ２Ｏ、Ｃｕ（ＩＤＡ）（ｅｎ）、ＫＣｕ（ＩＤＡ）（Ｇｌｙ）·Ｈ２Ｏ和Ｃｕ（ＩＤＡ）·２Ｈ２Ｏ（ＩＤＡ＝Ｎ（羧基甲基）－甘氨酸,Ｓｅｒ＝丝氨酸,Ａｌａ＝丙氨酸,ｅｎ＝乙二胺,Ｇｌｙ＝甘氨酸）等５种氨基酸─铜（Ⅱ）配合物进行了活性测定,发现它们均具有天然超氧化物歧化酶活性,其活性依次为０．３４、０．４５、０．５０、０．５４、０．７２Ｃｕμｍｏｌ·Ｌ－１。     

PICO-TAG方法测定氨基酸的研究

详细讨论了用异硫氰酸苯酯（ＰＩＴＣ）柱前衍生,反相色谱法测定氨基酸（即ＰＩＣＯ－ＴＡＧ法）的最佳条件。研究了流动相配制时溶液ｐＨ值、乙腈含量等对色谱分析的影响,并且对色谱柱的退化与再生进行了说明。氨基酸含量在１．２５～１５００ｐｍｏｌ范围内线性良好,相关系数大于０．９９９,且大多数氨基酸保留时间的变异系数小于０．５％。氨基酸采用峰面积定量,测定变异系数为０．１～１．５％。     

反相高效液相色谱法测定血清中γ—氨基本酸

建立一种高灵敏度的氯甲酸芴甲酯柱前衍生荧光检测反相高效相色谱法测定血清中γ－氨基丁酸的方法,内标为己氨酸,固定相为Ｓｈｉｍ－ＰａｃｋＣＬＣ－ＯＤＳ（Ｍ）,４．６ｍｍ＊１５０ｍｍ,填充料５μｍ;流动相采用二元梯度洗脱,     

基因工程菌发酵合成L－苏氨酸－N~（15）

采用含有稳定同位素１５Ｎ－硫酸铵为主要氮源的专用发酵培养基配方和提取精制条件,在国内、外首次采用基因工程菌ＡＡ７（ｐＴＨ２）（ＡＨＶｒＡＥＣｒ,Ｔｈｒ－Ｎ－,Ｈｏｍｒ,Ａｐｒ）直接发酵方法研制Ｌ－苏氨酸－Ｎ１５高丰度精制产品。每ｍｏｌ１５Ｎ－硫酸铵实际得到０．６３８ｍｏｌＬ－苏氨酸－Ｎ１５,产品Ｎ１５丰度达９９．０９％,仅比原料１５Ｎ－硫酸铵丰度下降０．４２％,提取精制得率高达９２．８３％。     

DL－氨基酸拆分条件的优化

本文利用高效液相色谱,应用两种不同类型的方法拆分Ｄ、Ｌ－氨基酸,对拆分条件进行了优化。当利用脲衍生型手性色谱柱时,流动相组成为：正已烷／二氯乙烷／乙醇＝７５：１８：７拆分迅速、有效;当利用柱前衍生反相色谱拆分时,在甲醇／ＮａＡｃ缓冲溶液流动相中加入适量ＴＨＦ得到很好的分离效果。     

自装高效液相色谱柱分析氨基酸

本文尝试用国产填料自装色谱柱进行氨基酸组成分析及氨基酸顺序测定，实验证明，自装柱不仅成本低廉，而且能将ＰＴＣ和ＰＴＨ氨基酸很地分离，完全能替代进口及国产商品柱。     

水稻巯基蛋白酶抑制剂的纯化及其性质研究

水稻的糠皮和胚经生理盐水浸取、离心后的上清液加热至８０℃处理１０ｍｉｎ,离心获得的上清液调ｐＨ至８．０,得到沉淀。沉淀溶解于０．０１ｍｏｌ／ＬＨＣｌ,经透析冷冻干燥得水稻巯基蛋白酶抑制剂（ＣＰＩ）粗品;粗品再经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱线性离子梯度洗脱和ＳｅｐｈａｄｅｘＧ－１００柱分子筛层析,即可获得在ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ上均为单一蛋白带的ＣＰＩ样品。经上述步骤,ＣＰＩ可被纯化５８倍。经ＳｅｐｈａｄｅｘＧ－１００和ＳＤＳ－ＰＡＧＥ测定其分子量均为１２０００,Ｎ末端氨基酸为Ｐｒｏ,等电点５．６．水稻ＣＰＩ经１００℃处理１０ｍｉｎ后,其抑制活性无任何变化,在ｐＨ２．０～９．０之间,活性也不发生改变,但ｐＨ在９．０以上,其活性逐渐下降,水稻ＣＰＩ对木瓜蛋白酶是一种高亲和性的抑制剂,它对木瓜蛋白酶和无花果蛋白酶有强抑制作用,对菠萝蛋白酶仅有弱抑制作用,但对胰蛋白酶则全无抑制作用;其抑制类型属竞争性抑制剂类型,Ｋ＿ｉ值约３．５×１０￣（－８）ｍｏｌ／Ｌ对木瓜蛋白酶的抑制摩尔比约为１：１。     

蚯蚓体内一种纤溶酶原激活剂(e-PA)对ATEE的降解

赤子爱胜蚓（Ｅｉｓｅｎｉａｆｅｔｉｄａ）体内的一种纤溶酶原激活剂（ｅ－ＰＡ）能够降解人工合成底物Ｎ－乙酰－Ｌ－酪氨酸乙酯（ＡＴＥＥ）,该降解反应的最适ｐＨ为８．５,而且在０．２ｍｏｌ／ＬＮａ２ＨＰＯ４中的活性要强于在０．０５ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．５）中．分别测定了ｅ－ＰＡ的大小亚基及全酶在０．２ｍｏｌ／ＬＮａ２ＨＰＯ４与０．０５ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．５）两种体系中的Ｋｍ和Ｋｃａｔ．结果表明,在０．２ｍｏｌ／ＬＮａ２ＨＰＯ４中,全酶的ＡＴＥＥ活性远远高于大小亚基单独的ＡＴＥＥ活性,而在０．０５ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．５）中则没有这种现象．从蛋白质结构的角度对这一结果作了解释．用不同抑制剂和ｅ－ＰＡ作用,结果表明,ｐｅｐｓｔａｔｉｎ,Ｅ－６４和ＥＤＴＡ对ｅ－ＰＡ的ＡＴＥＥ活性都有不同程度的抑制,这一点与ｅ－ＰＡ的ＢＡＥＥ活性不同．     

蜈蚣碱性蛋白SSmp-d的分离纯化及其部分理化性质的鉴定

少棘巨蜈蚣（ＳｃｏｌｏｐｅｎｄｒａｓｕｂｓｐｉｎｉｐｅｓｍｕｔｉｌａｎｓＬ．Ｋｏｃｈ）经９５％乙醇脱脂后,再经４℃水冷渗,水提液低温旋转浓缩,冻干,得到的冻干粉先后经过ＳｅｐｈａｄｅｘＧ－２５柱,等电聚焦制备电泳,再经ＳｅｐｈａｄｅｘＧ－１５０柱,ＳｅｐｈａｄｅｘＧ－１００柱,最后经ＨＰＬＣ制备得到一个纯的碱性蛋白,命名为ＳＳｍｐ－ｄ．该蛋白经ＨＰＬＣ、超薄等电聚焦电泳检验是均一的．采用ＨＰＬＣ和Ｐｒｏｔｅｉｎ－ＰａｋＴＭ１２５柱测定其分子量为２４．６４ｋＤ．ＩＥＦ－ＨＰＣＥ显示其等电点为９．２７．氨基酸分析表明ＳＳｍｐ－ｄ含较多的Ａｒｇ、Ｌｙｓ等碱性氨基酸,另外还含有较多的Ａｌａ、Ｌｅｕ．使用蛋白质自动序列分析仪测定了ＳＳｍｐ－ｄＮ端的１１个氨基酸,序列为ＮＨ３＋－Ａｓｐ－Ｖａｌ－Ａｓｎ－Ｐｈｅ－Ａｒｇ－Ｌｅｕ－Ｓｅｒ－Ｇｌｙ－Ａｌａ－Ａｓｐ－Ｐｒｏ．     

柱前衍生反相高效液相色谱法测定绞股蓝茶叶中17种游离氨基酸含量

建立2,4-二硝基氟苯柱前衍生化-反相高效液相色谱法测定绞股蓝茶叶中17种游离氨基酸的含量。以Phenomenex Gemini NX C18(4.6mm×250mm,5μm)为分析柱,采用梯度洗脱,流动相A为0.05mol·L-1乙酸钠(pH=6.4,含0.1%N,N-二甲基甲酰胺),流动相B为乙腈-水(1∶1,v/v),检测波长为360nm,柱温35℃;经方法学考察,该方法具有良好的稳定性和重现性。测定结果表明,绞股蓝茶叶中17种游离氨基酸总量为39.79mg·g-1,其中人体必需氨基酸占游离氨基酸总量的36.57%。从氨基酸含量考虑,绞股蓝茶叶具备一定的开发利用价值。     

2,4-2硝基氟苯柱前衍生法测定氨基酸的改进

本文报道了用2,4-二硝基氟苯柱前衍生反相高效液相色谱法测定氨基酸,15种常见的氨基酸可得到很好的分离。衍生物很稳定,在室温下保存两个月无明显变化,检测极限可达到5pmol。本法可应用于食品、药品等样品的氨基酸测定。     

两种柱前衍生反相高效液相色谱法测定血液和尿液中游离氨基酸

建立以PITC法和AQC法为柱前衍生试剂测定血液和尿液中游离氨基酸含量的测定方法。采用Waters-e2695操作系统,色谱柱为Shim-vp,ODS(250mm×4.6mm,5μm)(日本,岛津公司),以甲醇/乙腈/水和醋酸钠溶液(pH 6.5)为流动相,梯度洗脱。分别采用紫外和荧光检测器对血液和尿液中游离氨基酸进行含量测定。结果显示,两种衍生化方法灵敏度好、分离度高,具有良好的线性范围(r>0.990 0),准确度高(平均回收率为75.1%～127.0%),进样精密度好(RSD为0.12%～3.42%)。PITC法在尿液中游离氨基酸含量测定中显示了良好的测试准确性;而AQC法测定尿液中组氨酸、苏氨酸、脯氨酸超出线性范围,需要对尿样的前处理进行深入研究。     

反相高效液相色谱法测定烟叶中的游离氨基酸

用不同浓度的乙醇溶液提取烟样中的游离氨基酸 ,结果显示 ,存在最佳的乙醇溶液浓度 ,使烟样中被提取的游离氨基酸总量最大 ;对比了活性炭、乙醚、5 %磺基水杨酸、阳离子交换柱的纯化效果 ,发现阳离子交换柱的纯化效果较其它三种试剂要好。在提取和纯化之后 ,采用OPA、FMOC联合在线衍生反相高效液相色谱法测定了烟样中的游离氨基酸 ,该方法使烟样中的氨基酸和亚氨基酸能被同时测定 ,并且分析方法的重现性和回收率均令人满意。最后用该方法对云南B2 F98(上部、橘黄、二等烟叶 ,98年产 )烟叶中的游离氨基酸进行了测定 ,有 15种氨基酸被测出 ,其中Pro含量最高 ,约占总量的 2 5 % ,Thr含量最低 ,约占总量的 1%。     

A simplified method for the estimation of individual amino acid radioactivity in plasma samples

A method is presented for the quantitative estimation of the individual amino acid radioactivity in biological samples. The material is deproteinized with cold acetone, and, after acetone evaporation, is passed through a column containing 1 g of Amberlite XAD-2, then eluted with 10% ethanol. The samples are derivatized with Sanger's reagent (alkaline 1-fluoro-2,4-dinitrobenzene) and passed again through the Amberlite XAD-2 column; the 10% ethanol eluate is now discarded and the DNP-amino acids eluted with acetone. Aliquots are used for TLC chromatography on Silicagel plates; the spots are identified, cut away and their radioactivity estimated. The actual recovery of radioactivity in the spots is about 86-92% of the initial radioactivity. No contamination with radioactive glucose, lactate, pyruvate or glycerol has been observed.     

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.     

利用乙醇系统RP－HPLC分析PTC－氨基酸

首次报道了用乙醇作梯度洗脱有机相分离PTC－氨基酸的新方法.同乙腈相比,乙醇毒害性小、价廉、易得.在优化的色谱条件下,乙醇洗脱分辨率、灵敏度、准确度均佳,可成为PTC－氨基酸分析的一种极有前途的新方法.     

Quantitative determination of phenyl isothiocyanate-derivatized amino sugars and amino sugar alcohols by high-performance liquid chromatography

Simple and rapid methods for the preparation of phenylthiocarbamyl (PTC) derivatives of amino sugars and amino sugar alcohols and their quantitative determination with high sensitivity (less than 10 pmol) by C18 reversed-phase high-performance liquid chromatography are described. Rapid sample preparation of the phenyl isothiocyanate (PITC)-derivatized amino sugars and amino sugar alcohols was achieved by a simple extraction of the reaction mixture with chloroform to remove the excess PITC and its adducts. Baseline separation of the PTC derivatives of amino sugars and amino sugar alcohols was obtained within 30 min, using a simple solvent system consisting of 0.2% each of n-butylamine, phosphoric acid, and tetrahydrofuran. The mobile phase containing n-butylamine, in conjunction with a C18 stationary phase, mimics the conditions for the separation of carbohydrates on an amino-bonded column. GlcNH2 and GalNH2 derived from the initial protein-sugar linkages were also separated from the amino acids for quantitative estimation of sugar chains in glycoproteins. Amino sugar alcohols gave single reaction products with PITC while the reaction with amino sugars was accompanied by the formation of secondary products. Apparently the secondary products were formed in an acid-catalyzed intramolecular cyclization of the PTC-hexosamines involving the aldehyde functional group. Conditions were developed to stop the transformations and maintain the stability of PTC derivatives for their convenient determination by HPLC.