毛细管电泳分离寡糖衍生物及其电泳行为研究

将葡聚糖部分酸水解成寡糖混合物,经ＡＮＴＳ胺化还原衍生,在ｐＨ２．５、５０ｍｍｏｌ／Ｌ磷酸缓冲液（含或不含１０ｍｍｏｌ／Ｌ ＴＥＡ）中,以及在ｐＨ９．３、１００ｍｍｏｌ／Ｌ硼砂缓冲液中用毛细管电泳分离衍生物,分别得到１至２１和１至１８个聚合度的衍生物电泳梯度图谱。作出了准确定位的毛细管电泳双向电泳图。对电泳行为研究发现,在低ｐＨ磷酸电泳缓冲液中,衍生物相对电迁移率（μｅｐ）ｒ与（Ｍｒ＾－２／３）、     

荧光标记糖电泳在新琼寡糖定性和定量分析中的应用

目的:建立荧光辅助糖电泳(FACE)对系列新琼寡糖进行定性、定量分析的方法。方法:将系列新琼寡糖用7-氨基-1,3-萘胺二磺酸钾(AGA)氨化还原衍生后,在浓度梯度为18%-25%的聚丙烯酰胺凝胶中电泳分离,于波长264nm直接检测寡糖衍生物,得到聚合度为4-14的新琼寡糖衍生物电泳分析图谱。结果:寡糖衍生物的相对电迁移率与其分子量的负三分之二次方成线性关系;运用图像分析软件对电泳图谱进行数字化转换,发现寡糖衍生物的灰度积分面积与样品浓度呈线形关系。结论:建立了快速、精确的新琼寡糖微量定性、定量分析的方法,为新琼寡糖的质量分析提供了技术支撑。     

用于荧光辅助糖电泳寡糖标尺的构建

荧光辅助糖电泳(FACE)是一种简洁廉价的分离糖类方法。寡糖首先与8-氨基萘基-1,3,6-三磺酸(ANTS)反应标记,然后,ANTS标记的寡糖通过在32％丙烯酰胺-2．4％双丙烯酰胺组成的分离胶上电泳从而得以相互分离。结果表明,电泳图谱能准确反映寡糖的聚合度梯度,因而,一种具有连续聚合度的淀粉水解液的荧光标记电泳图谱可以作为荧光辅助糖电泳的分子量标尺。     

地衣芽孢杆菌β－甘露聚糖酶的纯化及酶学性质

地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＮＫ－２７菌株发酵产生的β－甘露聚糖酶（β－ｍａｎｎａｎａｓｅ）经硫酸铵盐析沉淀,两次ＤＥＡＥ纤维素和ＳｅｐｈａｄｅｘＧ－１００离子交换柱层析以及制备ＰＡＧＥ筹步骤,获得了凝胶电泳均一的样品。用ＳＤＳ－凝胶电泳测得纯化后的β－甘露聚糖酶分子量为２６ｋＤ,用凝胶聚焦电泳测得等电点ＰＩ为５．０。酶反应的最适ｐＨ为９．０,最后温度为６０℃,稳定ｐＨ为６．０—９．０,稳定温度为４０℃。金属离子中Ｍｇ￣（２＋）、Ｃａ￣（２＋）、Ｆｅ￣（２＋）、Ｎｉ￣（２＋）对该酶有一定的激活作用;而Ｓｎ￣（２＋）、Ｚｎ￣（２＋）、Ａｌ￣（３＋）、Ａｇ￣＋和Ｈｇ￣（２＋）对该酶有强烈的抑制作用。ＮＫ－２７菌株的β－甘露聚糖酶对魔芋葡萄甘露聚糖和角豆胶半乳甘露聚糖的Ｋｍ值分别为７．１４和５．５６ｍｇ·ｍｌ￣（－１）;Ｖ＿（ｍａｘ）分别为２００．５３和１５７．４５μｍｏｌ·ｍｇ￣（－１）·ｍｉｎ￣（－１）。     

盐生杜氏藻生油—3—磷酸脱氢酶的分离化及其特性的研究

利用ＰＥＧ分级,ＤＥＡＥ离子交换层析,Ｂｈｕｅ Ｓｅｐｈａｒｏｓｅ拟亲和层析,ＭｏｎｏＱ离子交换层析等手段,分离纯化直二氏藻甘油三磷酸（Ｇ－３－Ｐ）脱氢酶（ＥＣ１．１．１．８）得到比活为１２．６ｕ／ｍｇ的电泳纯的酶,并对此酶的生化特性进行了研究。４－２０％非变性聚丙烯酰胺梯度凝胶电泳测得全酶分子量约为２７０ｋＤ,ＳＤＳ－ＰＡＧＥ表明该酶只有一种分子量约为６５ｋＤ的亚基,据此推测该酶应为同四聚体。酶     

毛细管电泳分析常见单糖的2—氨基吖啶酮衍生物

建立毛细管电泳同时分析了１１种常见还原单糖的２－氨基吖啶酮胺化还原衍生物的方法。衍生反应在４５℃进行５ｈ,用毛细管电泳分离,于波长２６４ｎｍ在线检测单糖衍生物,单糖的化学衍生极限为１０μｍｏｌ／Ｌ,单糖的浓度和质量检测极限分别为０．６μｍｏｌ／Ｌ和１７ｆｍｏｌ。     

枯草芽孢杆菌中性内切β-甘露聚糖酶的纯化及性质

三草芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＢＭ９６０２产生的中性内切β－甘露聚糖酶（ｅｎｄｏ－β－１,４－Ｄ－ｍａｎｎａｎ ｍａｎｎａｎｏｈｙｄｒｏｌａｅｓ,ＥＣ,３．２．１．７８）经硫酸铵分级沉淀、ＤＥＡＥ－纤维素（ＤＥ２２）离子交换柱层析,得到电泳纯的样品,提纯了４５．５倍,收率为５．９％。用ＳＤＳ－ＰＡＧＥ测得该酶的分子量为３５ｋＤ。用ＰＡＧＥＩＥＦ测得其等电点ｐⅠ为４．５。酶反应的     

对百日咳脂寡糖特异的保护性和非保护性单抗的分析

对百日咳脂寡糖特异的保护性和非保护性单抗的分析百日咳脂寡糖（ＬＯＳ）经电泳出现ＬＯＳＡ和ＬＯＳＢ两条带,人和鼠抗ＬＯＳＡ中多糖分子的单克隆抗体对百日咳菌具有高度的特异性,在体外具有杀菌活性。本文作者将鼠抗ＬＯＳＡ的单克隆抗体ＢＬ－１、ＢＬ－６、ＢＬ－...     

人参培养细胞中活性六糖的分离及结构鉴定

将用酸水解人参细胞方法得到的寡糖素,是一组聚合度由３－１２单糖组成的水溶性混合物,经活性碳柱,ｏｗｅｘ５０（Ｈ＾＋）柱,Ｂｉｏ－Ｇｅｌ Ｐ－２凝胶柱后由半制备型ＯＤＳ柱在ＨＰＬＣ分离到一个六糖,通过ＧＣ,ＧＣ－ＭＳ,ＦＡＢ－ＭＳ和＾１３４Ｃ－ＮＭＲ测定,推测出了这个     

地衣芽孢杆菌β—甘露聚糖酶的纯化及其性质的研究

地衣芽孢杆菌１Ｂａｃｉｕｕｓ Ｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＢＬ－３０６产生的胞外β－甘露聚糖酶经硫酸铵分级盐析,ＤＥＡＥ－纤维素柱层析。Ｓｅｐｈａｄｅｘ－Ｇ１００柱凝胶过滤和ＤＥＡＥ－纤维素柱再层析分离纯化,得到ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）均一样品。用ＳＤＳ－ＰＡＧＥ测得纯化后β－甘露聚糖酶分子量为２６０００道尔顿。用凝胶等电聚焦电泳（ＰＡＧＥＩＥＦ）测得等电点ＰＩ为５．０。该酶     

Recovery of free oligosaccharides from derivatives labeled by reductive amination

This study examined chemical regeneration of free oligosaccharides from their fluorescent derivatives prepared by reductive amination with various aromatic amines. Maltose derivatives of ethyl 4-aminobenzoate (p-ABEE), 2-aminobenzonitrile (o-ABN), 4-aminobenzonitrile (p-ABN), 7-amino-4-methylcoumarin (AMC), 2-aminobenzoic acid (o-ABA), 2-aminobenzamide (o-ABAD), 2-aminopyridine (AP), and 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) were incubated at 30 degrees C with an aqueous solution of hydrogen peroxide/acetic acid. Recoveries of maltose from p-ABEE, p-ABN, and AMC derivatives were fairly good and gave approximately 90% of maltose. Recoveries of maltose from its o-substituted aniline (o-ABA, o-ABAD, and o-ABN) derivatives were 5-40%, but maltose was unrecoverable from AP and ANTS derivatives. Nevertheless, prior treatment of an AP derivative with cyanogen bromide enabled the regeneration of maltose in high yields. As an application, p-ABEE-labeled N-glycans from some glycoproteins separated on an amide column were identified by converting peak components to their AP derivatives via free saccharides and following mapping by reversed-phase chromatography.     

Electrophoretic resolution and fluorescence detection of N-linked glycoprotein oligosaccharides after reductive amination with 8-aminonaphthalene-1,3,6-trisulphonic acid.

The relative mobilities of various N-linked oligosaccharides reductively aminated to the charged fluorophore 8-amino-naphthalene-1,3,6-trisulphonic acid (ANTS) were determined by electrophoresis on high-density polyacrylamide slab gels. Each ANTS-derivatized oligosaccharide was assigned a relative migration index (RMI) expressed in terms of glucose equivalents, which was conveniently estimated by reference to a homologous series of ANTS--maltooligosaccharides run on each gel as oligosaccharide size standards. High-mannose-, complex- and hybrid-type structures were generally well resolved and easily visualized at picomole levels by simple UV light excitation. Application of these methods for the qualitative analysis of the oligosaccharides released from bovine fetuin and bovine asialofetuin by peptide-N-glycosidase F illustrates the usefulness of these techniques as fast, simple, and inexpensive tools for the characterization of N-linked oligosaccharides attached to glycoproteins.     

A detailed analysis of neutral and acidic carbohydrates in human milk.

Reverse- and normal-phase chromatography have been used to separate a number of standard human milk oligosaccharides derivatized via a reductive amination reaction with 2-aminoacridone (2-AMAC). Analytes were detected by spectrofluorimetry and injected simultaneously with a hydrolyzed dextran ladder derivatized with methyl-4-aminobenzoate. The latter probe does not fluoresce at the wavelength of emission by the 2-AMAC derivatives, and the derivatized, hydrolyzed dextran components were visualized by their ultraviolet absorbance. This procedure gave precise measurements of the "size" of 2-AMAC oligosaccharides in terms of their glucose equivalent values. Analytical amounts of 2-AMAC oligosaccharide standards were also isolated for further characterization by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry. MS-MS was also used to provide information on oligosaccharide sequences. This methodology was used successfully to characterize mixtures of neutral and acidic oligosaccharides from samples of human milk. This approach could be usefully applied to the study of glycoforms from a variety of samples such as those released from glycoproteins/glycolipids; these have been reported to be altered in a number of diseases, for example, cancer, cystic fibrosis, and autoimmune disease such as rheumatoid arthritis.     

Analysis of the oligosaccharides in ovalbumin by high-performance capillary electrophoresis

The oligosaccharides in ovalbumin as a glycoprotein model were released with anhydrous hydrazine, and reductively pyridylaminated after re-N-acetylation. The derivatives were analyzed by capillary zone electrophoresis (CZE) with on-column fluorometric detection. Direct CZE could separate the derivatives on the basis of the degree of polymerization, giving five peaks of hepta-, octa-, nona-, deca-, and undecasaccharides. Coelectrophoresis with the standard mixture of isomaltooligosaccharide derivatives was effective for peak assignment. CZE as borate complexes allowed separation on the basis of structural difference, especially in the peripheral monosaccharide residues. Peaks were tentatively assigned to the derivatives of reported oligosaccharides by comparing their relative mobilities with those of the chromatographic fractions obtained by using the ODS and Dowex 50W x 2 columns. These two modes gave excellent separation and were complementary to each other. Although the actual amount analyzed in the capillary tube was quite small (ca. 5 ng as carbohydrates), a larger amount (ca. 25 micrograms as carbohydrates) was required to make sample concentration sufficiently high to be detected by a modification of a commercial fluoromonitor for HPLC.     

Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins.

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.     

Microscale synthesis of dextran-based multivalent N-linked oligosaccharide probes

We developed a convenient method for the synthesis of dextran-based multivalent probes containing N-linked oligosaccharides which is efficient even in a small scale. Oligosaccharides were derivatized with succinic dihydrazide and dimethylamine borane under a mild acidic condition. The derivatized oligosaccharides were then conjugated in a good yield to periodate-oxidized dextran (500 kDa). Thus, the conjugates containing 120 to 140 oligosaccharide chains per dextran molecule were successfully synthesized. Their practical advantage was shown by the example that the asialofetuin oligosaccharide-dextran conjugate has much higher affinity to Ricinus communis agglutinin (RCA-I) than asialofetuin oligosaccharide itself or asialofetuin. The conjugates were further labeled with fluorescent reagent or biotinylation reagent containing a hydrazino group by the use of the unreacted aldehyde groups of the oxidized dextran, yielding probes with similar densities of fluorophores or biotin groups. Direct binding of the biotinylated asialofetuin oligosaccharide-dextran probe to RCA-I coated on the titer plate at a concentration of 50 ng/50 microl was easily detected using 50 fmol (as oligosaccharides) of the probe. The method for the synthesis of dextran-based oligosaccharide probes will facilitate the investigation of carbohydrate-mediated molecular interactions based on the native oligosaccharide structures.     

荧光标记电泳对透明质酸寡糖片段鉴定的方法建立

近年来,透明质酸寡糖片段（hyaluronan oligosaccharides, o-HA）的生物学活性引起国外学者的重视,因为o-HA具有一定的生物学活性,如参与免疫调节、刺激新生血管形成等.本研究建立一种经济、简便的ANTS（8-氨基奈-1,3,6-三磺酸）荧光标记电泳对透明质酸寡糖片段大小鉴定的新实验方法.实验原理为,ANTS能与糖分子发生还原反应,在反应时提供3个电子和1个荧光基团,通过高浓度PAGE分离,在特定波长下呈现颜色反应.采用酶消化法得到不同分子量大小的o-HA片段,测得不同片段大小的o-HA聚合度,分别与高效液相色谱（high-performance liquid chromatography, HPLC）和静电喷雾电离质谱（electrospray ionization mass spectrometry, ESI-MS）进行比较,结果吻合.研究提示,用荧光标记电泳法分析寡糖分子量,操作简单、设备低廉、灵敏度较高且检测速度快,是一种检测鉴定寡糖分子的较好方法.     

Enhanced efficiency due to the use of achiral additives in the optical resolution of 1-phenylethylamine by its glutaric acid derivative

Racemic 1-phenylethylamine was optically resolved by its own derivative formed with glutaric acid namely (+)-(R)-N-(1-phenylethyl)glutaramic acid. The amide acid resolving agent was synthesized from (+)-(R)-1-phenylethylamine by N-derivatization. The glutaric acid derivative was the next in a homologous series of dicarboxilic acid derivatized resolving agents of racemic 1-phenylethylamine. Resolution results obtained with the oxalic, malonic, and succinic acid derivatives were previously discussed(1). Each of the above derivative resolving agents could be successfully applied as resolving agents of 1-phenylethylamine. The efficiency of the present optical resolution using (+)-(R)-N-(1-phenylethyl)glutaramic acid resolving agent was remarkably inferior to the results obtained by its shorter chained homologues(1). Use of achiral additives, like urea, thiourea, N-methylurea, and N,N'-dimethylurea caused large increase in the efficiency of the resolution by (+)-(R)-N-(1-phenylethyl)glutaramic acid resolving agent. Precipitated salts obtained in the resolutions performed in the presence of the additives were investigated by thermoanalysis, X-ray powder diffraction, and optical microscopy. Based on the analytical data, the improvement of the resolution results was attributed to the influence of the additives on the crystal nucleation processes of the diasteromeric salts.     

Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones--vasopressin and oxytocin, and pancreatic hormone--human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3-6.0 micromol dm(-3) and in the PDA detector 1.6-3.1 micromol dm(-3). The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm(-3).