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.     

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。     

小鼠肠道对壳寡糖的吸收率及吸收成分的影响

目的通过分析壳寡糖在小鼠肠道内的吸收率以及吸收成分,了解肠道对壳寡糖代谢的影响,初步判断壳寡糖的有效生物成分,为壳寡糖生物活性的进一步研究提供必要的实验材料和线索。方法首先用蜗牛酶将壳聚糖水解成聚合度不同的壳寡糖,并通过聚丙烯酰胺凝胶层析柱（Bio-Gel P4凝胶）将不同聚合度的壳寡糖分开,分别收集聚合度为1-3,8-11的壳寡糖,并用异硫氰酸荧光素（FITC）标记,再通过聚丙烯酰胺凝胶层析柱（Bio-Gel P-2/P4）将游离的FITC除去,随后对两组禁食24 h的小鼠分别用FITC标记的大分子量和小分子量的壳寡糖灌胃。1 h后取血清和小肠,经分离水溶成分后,通过荧光分光光度计检测血清样品和肠溶物样品中荧光的强度,进而确定壳寡糖的吸收率和吸收成分。结果通过改变酶解时间,蜗牛酶可以将壳聚糖水解成不同聚合度的壳寡糖。利用Bio-Gel PA聚丙烯酰胺凝胶层析柱可以将不同聚合度的壳寡糖分离成具有一定聚合度范围的壳寡糖。用F1TC标记的大、小分子量的壳寡糖给小鼠灌胃,从血清和肠溶物中均检测到荧光强度,两者比值平均值分别为5.68 ： 1和9.84 ： 1。结论壳寡糖在肠道的吸收率随分子量的减小而增大,除小分子壳寡糖外,吸收成分也包括部分大分子量壳寡糖。     

糖的有色试剂标记微量测定法

本文报告用有色试剂4-N,N-二甲氨基-4′-氨基偶氮苯标记还原糖。标记产物可在聚酰胺薄膜上经双向层析分离鉴定。用盐酸熏后,标记产物由黄绿色转为蓝紫色。灵敏度达10~(-9)～10~(-10)克分子糖。本法可鉴定寡糖的组成成分和聚合度;结合酶解可鉴定寡糖的还原末端。     

寡糖分离和结构分析进展

寡糖,尤其是糖缀合物中的寡糖链,在生命过程的细胞识别、信号传导和受体调节现象中扮演着重要的角色.高效液相色谱、毛细管电泳、质谱、核磁共振、荧光标记糖电泳和试剂阵列分析方法等新技术的应用使得寡糖的分离和结构鉴定变得更为快速、简便和准确;同时多种有力工具的联合运用将更深刻地揭示极微量寡糖的结构-功能关系成为可能.     

糖的有色试剂标记微量测定法

本文报告用有色试剂4-N,N-二甲氨基-4'-氨基偶氮苯标记还原糖。标记产物可在聚酰胺薄膜上经双向层析分离鉴定。用盐酸熏后,标记产物由黄绿色转为蓝紫色。灵敏度达10~(-9)～10~(-10)克分子糖。本法可鉴定寡糖的组成成分和聚合度;结合酶解可鉴定寡糖的还原末端。     

荧光标记IgHV1抗原九肽及其分离纯化

目的 :建立IgHV₁抗原九肽荧光标记及分离纯化的方法。方法 :利用硫代磷酸化的原理以荧光试剂标记IgHV₁抗原九肽 ,用葡聚糖凝胶 (Sephadex)G 1 5层析柱及聚丙烯酰胺凝胶电泳(PAGE)对荧光标记的IgHV₁抗原九肽进行分离纯化并以毛细管电泳技术进行鉴定。结果 :用毛细管电泳技术对纯化后样品进行鉴定 ,其电泳图谱只出现单一峰。结论 :初步建立IgHV₁抗原九肽荧光标记及分离纯化的方法     

寡糖的荧光标记高效液相色谱分析

 利用氨化还原的方法把高量子产率的荧光标记物——α-萘胺,接到异麦芽糖寡糖的还原端。用硅胶薄层色谱、荧光光谱及快原子轰击质谱证实反应物的完全性及其结构。高效液相色谱用Micropak si5硅胶柱,以乙腈-水(含0.05％三乙胺)为梯度洗脱液可使含有二到九个糖残基的异麦芽糖寡糖的α-萘胺衍生物全部分离。本法对异麦芽糖二糖的荧光检测灵敏测度为2.35Pmol。     

灵芝β-葡寡糖的分离纯化和体外抗炎活性

为阐释抗炎活性与灵芝葡寡糖聚合度之间的关系,将灵芝β-葡寡糖组分GLPW-A(DP2–14)通过Bio-Gel P-2凝胶色谱柱分离,分别从洗脱液、流速以及每管接样量3个方面优化分离条件。通过阴离子色谱和基质辅助激光解析串联飞行时间质谱(MALDI-TOF-MS)分析测定其聚合度,并进行体外抗炎活性评价。当洗脱液为0.1 mol/L的NH₄HCO₃溶液,流速为0.1 mL/min,接样量为2 mL时分离效果最佳,根据出峰时间先后收集得到了8个组分(F1–F8)。聚合度分析表明,F8和F7分别为DP2和DP3的葡寡糖组分,其余6个组分F6–F1均为主要含有2–3种聚合度的葡寡糖。体外抗炎活性表明,主要含有DP4–DP6的F5和F6组分在浓度为0.5–10μg/mL的范围内无明显抗炎活性,而其余各组分均在一定浓度下表现出不同程度的抗炎作用,且活性优于GLPW-A组分,说明灵芝β-葡寡糖的抗炎活性与寡糖片段的聚合度有关。     

凝胶色谱法制备寡糖

采用已知结构的多糖,控制其水解条件,使之产生所需寡糖片断。用聚丙烯酰胺凝胶色谱(BiO-6el P-4)分离,对中性糖可以分离到含十一个糖残基的寡糖。对氨基糖可分离到含七个糖残基的寡糖。用薄层色谱和快原子轰击质谱鉴定了它们的纯度。     

Interactions of carbohydrates and proteins by fluorophore-assisted carbohydrate electrophoresis

A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions is demonstrated by fluorophore-assisted carbohydrate electrophoresis (FACE). The procedure is simple and the cost is low. The advantage of this method is that carbohydrate-protein interactions can be easily displayed by FACE, and the carbohydrates do not need to be purified.     

Comparison of high-performance liquid chromatography and fluorophore-assisted carbohydrate electrophoresis methods for analyzing peptidoglycan composition of Escherichia coli

Currently, reversed-phase high-performance liquid chromatography (HPLC) is the method of choice for determining the types and amounts of muropeptide subunits comprising bacterial peptidoglycan. Although effective and sensitive, the technique does not lend itself to high throughput screening, and its complexity and equipment requirements may dissuade some investigators from pursuing certain types of cell wall experiments. Previously, we showed that muropeptides can be labeled with a fluorescent dye and separated by fluorophore-assisted carbohydrate electrophoresis (FACE), a simple and rapid gel procedure that might serve as a prelude to more intense analysis by HPLC. To validate the utility of FACE, we used both techniques to perform a side-by-side analysis of the peptidoglycan of eight mutants and their Escherichia coli parent strain. FACE and HPLC both detected the seven major muropeptides, which represent more than 95% of the total muropeptides present in this organism. In addition, FACE returned the same relative and quantitative results in 92% of 72 measurements, indicating that the procedure gives an accurate overview of peptidoglycan composition. The results also suggest a possible biochemical activity for the AmpC and AmpH proteins of E. coli, and the use of FACE as an in vitro enzyme assay detected possible substrate preferences for the endopeptidase penicillin binding protein 4.     

Optimization of fluorophore-assisted carbohydrate electrophoresis

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a simple and inexpensive method for separating saccharides. Oligosaccharides were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and the reductive amination reactions were essentially complete after approximately 16 h under the given experimental conditions. Saccharide-ANTS adducts were then separated by electrophoresis on 32% C(ACR)/2.4% C(BIS) polyacrylamide gel at alkaline pH. This technique doesn't require sophisticated instrumentation and highly trained personnel.     

Quantitative, small-scale, fluorophore-assisted carbohydrate electrophoresis implemented on a capillary electrophoresis-based DNA sequence analyzer

Fluorophore-assisted carbohydrate electrophoresis (FACE) is an analytical method for characterizing carbohydrate chain length that has been applied to neutral, charged, and N-linked oligosaccharides and that has been implemented using diverse separation platforms, including polyacrylamide gel electrophoresis and capillary electrophoresis. In this article, we describe three substantial improvements to FACE: (i) reducing the amount of starch and APTS required in labeling reactions and systematically analyzing the effect of altering the starch and 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) concentrations on the reproducibility of the FACE peak area distributions; (ii) implementing FACE on a multiple capillary DNA sequencer (an ABI 3130xl), enabling higher throughput than is possible on other separation platforms; and (iii) developing a protocol for producing quantitative output of peak heights and areas using genetic marker analysis software. The results of a designed experiment to determine the effect of decreasing both the starch and fluorophore concentrations on the sensitivity and reproducibility of FACE electrophoregrams are presented. Analysis of the peak area distributions of the FACE electrophoregrams identified the labeling reaction conditions that resulted in the smallest variances in the peak area distributions while retaining strong fluorescence signals from the capillary-based DNA sequencer.     

High-performance size-exclusion chromatography (HPSEC) and fluorophore-assisted carbohydrate electrophoresis (FACE) to describe the chain-length distribution of debranched starch

Chain-length (CL) distribution is an important feature of the "fine structure" of starch molecules, which are comprised of amylose and amylopectin. The objective of the present work was to combine data for two methods to achieve a more comprehensive data set that would allow a fuller comparison of the CL distribution for different starches. Both high-performance size-exclusion chromatography (HPSEC) and fluorophore-assisted carbohydrate electrophoresis (FACE) were carried out on endosperm starch isolated from five maize genotypes. For the CL distribution in the range DP50, data in the HPSEC chromatogram were transformed to the form of a FACE electrophoregram, in which the x-axis is DP and the y-axis is the number of chains. The two sets of data in this region were shown to be similar. We conclude that the data sets from HPSEC and FACE may be considered together to describe the CL distribution more completely than for either method alone. We further note that for DP 6-50, data from HPSEC may be transformed to allow a similar presentation as for that obtained by FACE, such that FACE analysis might not be required for comparison of CL distribution of different starches.     

Analyses of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues by fluorophore-assisted carbohydrate electrophoresis

Lipid-linked oligosaccharides (LLOs) are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I congenital disorders of glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, simple and sensitive nonradioactive methods for LLO analysis are lacking. Thus, almost all studies of LLO synthesis have relied on metabolic labeling of the oligosaccharides with radioactive sugar precursors. We report that LLOs in cell cultures and tissues can be easily detected and quantified with a sensitivity of 1-2 pmol by fluorophore-assisted carbohydrate electrophoresis (FACE). These analyses required efficient removal of contaminants, most likely trace quantities of glycogen breakdown products, that interfered with FACE. Studies with CHO-K1 cells showed that LLOs detected by FACE and by metabolic labeling had similar turnover rates. Glc(3)Man(9)GlcNAc(2)-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc(0-2)Man(5-9)GlcNAc(2)-P-P-dolichol intermediates in tissues, such as liver and kidney, were unexpectedly greater than for cultured cells. IV injection of D-mannose, raising the circulatory concentration by three- to fourfold, did not affect LLO composition. Thus, the relative accumulation of LLO intermediates in mouse liver and kidney is not likely due to inadequate D-mannose in the circulation. In summary, FACE is a facile, accurate, and sensitive method for LLO analysis, permitting investigations not feasible by metabolic labeling.     

开放式空气CO2浓度增高对水稻冠层微气候的影响

利用位于江苏省无锡市安镇的我国唯一的农田开放式空气CO2 浓度增高 (FACE)系统平台 ,于2 0 0 1年 8月 2 6日至 10月 13日 (水稻抽穗至成熟期 )进行水稻作物冠层微气候连续观测 ,以研究FACE对水稻冠层微气候特征的影响 .结果表明 ,FACE降低了水稻叶片的气孔导度 ,FACE与对照水稻叶片气孔导度的差异上层叶片大于下层叶片 ,生长前期大于生长后期 .FACE使白天水稻冠层和叶片温度升高 ,这种差异生长前期大于生长后期 ;但FACE对夜间水稻冠层温度的影响不明显 .在水稻旺盛生长的抽穗开花期 ,晴天正午前后FACE水稻冠层温度比对照高 1.2℃ ;从开花至成熟期 ,FACE水稻冠层白天平均温度比对照高 0 .4 3℃ .FACE对冠层空气温度也有影响 ,白天水稻冠层空气温度FACE高于对照 ,这种差异随太阳辐射增强而增大且冠层中部大于冠层顶部 ;冠层中部空气温度FACE与对照的差异 (Tface-Tambient)日最大值在 0 .4 7～ 1.2℃之间 ,而冠层顶部的Tface-Tambient日最大值在 0 .37～ 0 .8℃之间 .夜间水稻冠层空气温度FACE与对照差别不大 ,变化在± 0 .3℃之内 .而FACE对水稻冠层空气湿度无显著影响 ,表明FACE使水稻叶片气孔导度降低 ,从而削弱了植株的蒸腾降温作用 ,导致水稻冠层温度和冠层空气温度升高 ,改变了整个水稻冠层的温度环     

Structure of N-glycosidic carbohydrates of secretory proteins of Tetrahymena thermophila

Glycoproteins secreted by Tetrahymena into the culture medium were isolated and the N-glycosidic oligosaccharides analyzed using lectin blots and fluorophore-assisted carbohydrate gel electrophoresis (FACE). Lectin blots showed that the glycoproteins secreted by Tetrahymena contain only N-glycosidic structures of the high mannose type. Further analysis using the FACE technology revealed the presence of four different N-glycosidic structures differing only in the number of mannose residues attached to the core chitobiose unit.     

Adaptation of FACE methodology for microanalysis of total hyaluronan and chondroitin sulfate composition from cartilage

Protocols for analyzing the fine structure of hyaluronan and chondroitin sulfate using fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone-derivatized hyaluronidase/chondroitinase digestion products were adapted for direct analysis of previously characterized cartilage-derived samples. The chondroitin sulfate disaccharide compositions for fetal and 68 year human aggrecan from FACE analyses were DeltaDi4S (50%), DeltaDi6S (43%), and DeltaDi0S (7%); and DeltaDi4S (3%), DeltaDi6S (96%), and DeltaDi0S (1%), respectively. The nonreducing terminal structures included predominantly 4S-galNAc with minor amounts of 6S-galNAc and Di6S for the fetal aggrecan sample and, in addition, included 4,6S-galNAc in the 68 year aggrecan sample. FACE analysis of a proteinase K digest of rat chondrosarcoma tissue gave an internal disaccharide composition for its chondroitin sulfate chains of DeltaDi0S (7%) and DeltaDi4S (93%) with no DeltaDi6S and DeltaDi4, 6S detected, while DeltaDiHA from hyaluronan was 5% of the total. Analysis of nonreducing terminal structures indicated the presence of 4S-galNAc (51%), galNAc (27%), and Di4S (22%) with no 4,6S-galNAc or Di6S detected. Unexpectedly, FACE analysis detected putative linkage oligosaccharide structures from the chondroitin sulfate chains including both unsulfated (85%) and 4-sulfated (15%) linkage oligosaccharides. Finally, the number averaged chain length estimated from the ratio of the molar fluorescence of the Deltadisaccharides to that of the nonreducing termini or the linkage oligosaccharide structures was calculated as approximately 16 kDa. A tissue glucose concentration of 0.72 g/l was also measured. These results for both samples as determined by FACE analysis were similar to results previously reported, using more labor and time intensive procedures, validating the FACE protocols.     

Effect of ammonia on the glycosylation of human recombinant erythropoietin in culture

Recombinant human erythropoietin (EPO) was produced by a stable transfected CHO-K1 cell clone (EPO-81) grown in serum-free medium. Our previous work showed that there was a significant increase in the heterogeneity of the glycoforms of EPO and a reduction of the sialylation at 20 mM NH(4)Cl. In the work presented here, the effects of ammonia on EPO N-linked oligosaccharides were analyzed. EPO was purified from culture supernatants by immunoaffinity chromatography. The N-linked oligosaccharides were released enzymatically and analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and HPLC. The FACE N-linked oligosaccharide profile showed that the sialylated glycans contain one prominent band at a position corresponding to eight glucose units. The density of the major band was greatly diminished and the width was significantly increased in cultures containing added ammonia. The proportion of tetraantennary structures was reduced by 60%, while the tri- and biantennary structures were increased proportionally in the presence of ammonia. Glycan analysis by HPLC using a weak anion exchange column showed that the most significant characteristic effect of ammonia was a reduction of the proportion of glycans with four sialic acids from 46% in control cultures to 29% in ammonia-treated cultures. Analysis of the desialylated glycans by normal phase chromatography indicated a distribution of tetra-, tri-, and biantennary structures similar to that shown by FACE. The N-linked glycan sequence was determined by sequential exoglycosidase digestion followed by FACE. The results indicated a typical N-linked complex oligosaccharide structure. Glycans from ammonia-containing cultures showed the same sequence pattern. In conclusion, we showed that ammonia in the culture medium affected EPO glycosylation, which was observed as a reduction of the tetraantennary and tetrasialylated oligosaccharide structures. However, the presence of ammonia in the cultures did not change the oligosaccharide sequence.