多糖结构分析.docx

1、多糖结构分析实验八 多糖结构分析 多糖在生物学上的重要意义，尤其是在医药学上的重要意义决定了多糖研究的迅速发展，多糖构效关系的研究已成为多糖研究的热点。但由于多糖结构的复杂性和多样性，其结构测定远远落后于蛋白质和核酸,本实验选择天然多糖（半乳葡萄甘露聚糖）作为实验材料，对其一级结构做初步的分析。多糖一级结构的分析包括：纯度鉴定，分子量测定，单糖组成测定和糖链的序列测定。糖链的序列测定包括：单糖残基在糖链中的次序，单糖残基间连键的位置，链的分支情况等诸多方面。【实验目的】1.了解多糖结构分析的内容及方法。2.了解多糖一级结构分析的基本原理。3.掌握多糖一级结构分析的基本方法。一、糖含量测定【实验

2、原理】苯酚硫酸试剂与游离的或寡糖、多糖中的己糖、糖醛酸起显色反应，己糖在490nm处有最大吸收，吸收值与糖含量呈线性关系。【实验材料】1. 实验器材721型分光光度计。2. 实验试剂(1) 98 的浓硫酸。(2) 80 苯酚：80g苯酚加20ml水使之溶解，可置冰箱中避光长期贮存。 (3) 6 苯酚：临用前用80苯酚配制。(4) 标准葡萄糖溶液（0.1 mg/ml）：取100mg葡萄糖，用蒸馏水溶解，定容至1L。(5) 多糖样品：半乳葡萄甘露聚糖溶液（0.1 mg/ml）。【实验操作】 1. 制作标准曲线：取9支干燥试管，按下表操作管号012345678标准溶液（ml）00.40.60.81.

3、01.21.41.61.8蒸馏水（ml）2.01.61.41.21.00.80.60.40.26苯酚（ml）1.01.01.01.01.01.01.01.01.0浓硫酸（ml）555555555静止10分钟，摇匀，室温放置20分钟A490横坐标为多糖微克数，纵坐标为光密度值，绘制标准曲线。2. 样品含量测定：取样品液1.0ml，按上述步骤操作，测光密度。3计算: 糖含量(%)=C /（C0 V）100%C: 由标准曲线查得的糖微克数C0：样品溶液的浓度（0.1 mg/ml）V：测定时用的样品溶液体积（1.0ml）二、单糖组成分析【实验原理】多糖在浓硫酸中保温一定时间可完全水解为单糖，通过纸层析

4、分离，特定试剂显色后与已知糖的标准混合物作对比，可以鉴定多糖水解产物中单糖的组成。【实验材料】1. 实验器材水解管；滤纸；玻璃毛细管；层析缸；喷雾器。2. 实验试剂 标准糖溶液: 称取一定量的半乳糖、葡萄糖、甘露糖、阿拉伯糖，用蒸馏水溶解，得标准糖混合溶液(每种糖的点样量为20微克30微克)。 展层剂： 正丁醇：乙酸：水=4：l：5 (上层)。 显色剂：苯胺-邻苯二甲酸-正丁醇饱和水溶液(邻苯二甲酸1.6g溶于水饱和的正丁醇100 ml，加苯胺0.93g（相当于0.9 ml）。 BaCO3；1mol/L硫酸。【实验操作】l完全酸水解：称取20 mg多糖样品，加入1mol/L H2S04 2ml

5、；封管，l00水解8小时，然后加入BaC03中和，定量滤纸过滤，滤液留作分析。2纸层析：将层析滤纸剪成7cm40cm的纸条，距层析滤纸一端2cm处画一横线作为点样线,在点样线上画两个点分别作为标准糖溶液和多糖水解液的点样位置。用玻璃毛细管点样，斑点尽可能小，而且每点一滴，待点样点干燥后，在同一位置再点第二滴。然后将滤纸条悬挂于层析缸中进行层析，展层时间约为36小时。3显色：将滤纸取出，自然干燥，喷上苯胺-邻苯二甲酸-正丁醇饱和水溶液，100条件下15分钟即可显色。标准单糖混合物色斑在滤纸上由下而上的顺序是：半乳糖-葡萄糖-甘露糖-阿拉伯糖。与标准单糖混合物色斑比较，即可判断多糖样品的单糖组成。

6、三、糖链的序列测定（一）高碘酸氧化【实验原理】高碘酸可以选择性地氧化和断裂糖分子中连二羟基或连三羟基处，生成相应的多糖醛、甲醛或甲酸。反应定量地进行，每开裂个C-C键消耗一分子高碘酸。通过测定高碘酸消耗量及甲酸的释放量，可以判断多糖分子中糖苷键的位置、类型、多糖的分枝数目和取代情况等。【实验材料】1. 实验器材紫外分光光度计。2. 实验试剂(1) 溴甲酚蓝指示剂:0.1克溴甲酚蓝溶于250ml蒸馏水中（含1.43毫升0.1mol/L氢氧化钠）。(2) 30mmol/L高碘酸钠溶液。(3) 乙二醇。(4) 0.01mol/L氢氧化钠溶液。【实验操作】1. 制作标准曲线： 取6支干燥试管，按下表操

7、作管号012345高碘酸钠（ml）00.51.01.52.04.0蒸馏水（ml）4.03.53.02.52.00各取0.1 ml，定容至25 ml，在223nm处测定光密度A223横坐标为高碘酸钠毫摩尔数，纵坐标为光密度值，绘制标准曲线。2. 样品测定称取多糖样品25mg，用少量水溶解，加入30mmol/L高碘酸钠溶液,定容至25ml，置于暗处,室温下进行反应，于0，6，12，24，36，48小时间隔取样0.1 ml，蒸馏水稀释250倍后，使用紫外分光光度计在223nm处测定光密度。至光密度值达一稳定值时，加乙二醇破坏过量的高碘酸以终止反应。由此光密度值根据标准曲线计算出高碘酸的消耗量。取2m

8、l上述反应溶液，测定甲酸的生成量。剩余部分进行Smith降解。3. 甲酸测定：取2ml上述反应溶液，加一滴溴甲酚蓝作指示剂，用0.01mol/L氢氧化钠溶液滴定甲酸的释放量。【注意事项】为避免发生超氧化，反应溶液的pH值应控制在35，而且一定要在低温、避光处进行。（二）Smith降解【实验原理】Smith降解是将高碘酸氧化产物用硼氢化合物(如硼氢化钾或硼氢化钠)还原成稳定的多羟基化合物，然后进行适度的酸水解，用纸层析鉴定水解产物，由水解产物可以推断多糖各组分的连接方式及次序。以葡聚糖为例，其反应式如下图：以12位键合的糖基经高碘酸氧化，平均每个糖基仅消耗一分子高碘酸，并且无甲酸释放；Smith

9、降解后产生甘油。以14位键合的糖基经高碘酸氧化，平均每个糖基仅消耗一分子高碘酸，并且无甲酸释放；Smith降解后产生赤藓醇。以13位键和的糖基不被高碘酸氧化；Smith降解后仍为原来糖。以16位键和的糖基或非还原末端基（1）经高碘酸氧化，消耗二分子高碘酸，同时释放一分子甲酸；Smith降解后产生甘油。本实验通过纸层析检测终产物中的甘油、赤藓醇和糖。【实验材料】0.1molL醋酸。2molL硫酸。硼氢化钾。纸层析试剂及器材：除显色剂外，同实验二。硝酸银显色剂：A: 16%硝酸银水溶液: 丙酮为1：9（V/V）。B: 1%NaOH乙醇溶液(W/V)。C: 6molL氢氧化铵。【实验操作】1. 还原

10、和水解高碘酸氧化后经乙二醇处理的溶液对流水透析48小时，蒸馏水透析24小时，于40以下减压浓缩至10ml左右，加入70mg硼氢化钾于室温、暗处搅拌18小时24小时以还原多糖醛。用0.1 molL醋酸中和至pH67，对流水透析48小时，蒸馏水透析24小时，减压蒸干，加1 molL硫酸2ml，封管，100水解8小时，用碳酸钡粉末中和，定量滤纸过滤，滤液减压浓缩后，通过纸层析方法检测。2. 纸层析操作详见实验2（单糖组成分析）。展层后，将滤纸取出，自然干燥，喷上试剂A ，自然干燥，将滤纸浸入试剂B中，待斑点显出后，再浸入试剂C中以洗去滤纸上的氧化银，然后用水冲洗1小时左右，风干。以正丁醇：乙酸：水=

11、4：l：5为展层剂时，甘油的Rf为：0.48，赤藓醇的Rf为：0.35。【思考题】1. 多糖一级结构的分析包括哪些内容？2. 如果多糖经高碘酸氧化和Smith降解，发现产生的甘油量等于产生的甲酸量，说明该多糖主要含哪一种糖甘键，为什么？半乳葡萄甘露聚糖的一级结构通式：Experiment 20 Structure Analysis of PolysaccharideThe significance of polysaccharide in biology realm, particularly in Medical science and pharmaceutical realm，leads

12、to the quick development of research on polysaccharide. Research on the relationship between polysaccharide structure and function has become another hot point in polysaccharide research. Structure analysis of polysaccharide gets behind clearly the structure analysis of protein and nucleic acids due

13、 to the diversity of polysaccharide structure. Methods used currently for structure analysis of polysaccharide include chemical methods，zymological methods and instruments measure methods. We analyze essentially the primary structure of natural polysaccharide (Galactogluco-mannan) in this experiment

14、; the primary structure analysis of polysaccharide includes purity determination, molecular weight determination, and composition of monosaccharide determination and sequence of polysaccharide determination. The sequence of polysaccharide determination includes analysis of order of monosaccharide in

15、 polysaccharide molecule, the degree of branching of polysaccharide molecule, the position and nature of glycosidic bonds and so on.【Purpose】1. Understand the content and method on structure analysis of polysaccharide. 2. Understand the basic principle on the primary structure analysis of polysaccha

16、ride.3. Master the basic methods used for primary structure analysis of polysaccharide. Content Determination of Polysaccharide【Principle】Phenol- sulfuric acid reagents can react with free hexose, alduronic acid or hexose, alduronic acid of oligosaccharide and polysaccharide. After the reaction, the

17、 solution has the maximum absorption at 490nm (hexose), at which the relationship between absorbance and the content of polysaccharide takes on linearity in definite range.【Materials】1. Apparatus721 Spectrophotometer.2. Reagents(1) 98% concentrated sulfuric acid.(2) 80% phenol: Dissolve 80g of pheno

18、l into 20ml water, the solution can be stockpiled in the dark in refrigerator for a long time. (3) 6% phenol: dilute with 80% phenol on the day needed.(4) Standard glucose solution (0.1mg/ml): Dissolve 100mg of glucose, and dilute it to 1L with distilled water.(5) Polysaccharide solution: Galactoglu

19、co-mannan solution (0.1mg/ml).【Procedures】1. Draw standard curve:Prepare 9 dry test tubes and operate as the table belowTube No.012345678standard solution（ml）00.40.60.81.01.21.41.61.8Distilled solution（ml）21.61.41.21.00.80.60.40.26phenol（ml）1.01.01.01.01.01.01.01.01.0Concentrated sulfuric acid（ml）55

20、5555555 Place Statically for 10 minutes, shake evenly and place at room temperature for 20 minutes.A490We can draw the standard curve according to the result of determination.Abscissa: content of polysaccharide (g) Ordinate: the value of optical density2. Determination of saccharide content in sampl

21、e solution: Draw 1.0ml of polysaccharide sample solution, and perform as described above. The saccharide content in the sample can be looked up from the standard curve by the absorbency measured. 3. Calculation:Saccharide contentC /（C0 V）100 C ：saccharide content looked up from standard curve（g） C0：

22、sample concentration（0.1 mg/ml）V ：sample volume used for determination（1.0 ml）. Composition of Monosaccharide Determination【Principle】 Polysaccharide can be completely hydrolysed at definite temperature in concentrated sulfuric acid for enough time. Separate the hydrolysate by paper chromatography,

23、the separated monosaccharide can form colored compound with definite chromogenic reagent，Compare with standard saccharide color spots, composition of monosaccharide in polysaccharide hydrolysate can be determined.【Materials】1. ApparatusAmpoule, Filter paper, Glass capillaries, Chromatography tank, V

24、aporization.2. Reagents(1) Standard saccharide solution: Weight respectively certain amount of each saccharide sample below: galactose, glucose, mannose and arabinose. Then dissolve them in distilled water to prepare standard saccharide mixture. (20g 30g of each spotted on the filter paper)(2) Devel

25、op reagent: 1-butanol：acetic acid：water=4：l：5 ( upper phase).(3) Chromogenic reagent: Water saturation solution of anilin- phthalic acid-1-butanol. Dissolve 1.6g phthalic acid of in 100 ml Water saturation solution of 1-butanol，then add 0.93g of （equal to 0.9 ml）anilin to the solution.(4) BaCO3；1mol

26、/L H2S04.【Procedures】1. Complete acid hydrolysis: Weight 20mg of the polysaccharide sample, add 2ml of 1mol/L H2S04 in an ampoule and seal it, hydrolyze at 100C for 8h. Neutralized with BaC03, filter and collect the filtrate to analyze sugar analysis.2. Paper chromatography: Cut chromatography filte

27、r paper into 7 cm 40cm pieces. Draw a line as spotting end 2cm near the edge. Draw two spots on the line as spotting location of Standard saccharide solution and hydrolysate respectively. Spot the solution on the filter paper with glass capillary; the spot should be as small as possible. Dry the spo

28、t in the air and then spot the same solution again at the same location. Hang the filter paper inside chromatography tank and develop for 36h, pick out the paper and dry in the air. Spray Water saturation solution of anilin- phthalic - n-butanol on the filter paper. Dry 15min at 100 to make the colo

29、r spot emerges. The upward sequence of standard monosaccharide sample spot is: galactose-glucose-mannose-arabinose. Compare with standard saccharide color spots, sample composition can be determined. Sequence of Polysaccharide Determination1. Periodate Oxidation【Principle】Periodate oxidation involve

30、s the simultaneous oxidation and cleavage of carbon to carbon bonds that have adjacent free hydroxyl groups in saccharides, the products of periodate oxidation include homologous aldehyde of polysaccharide, formaldehyde or formate, the cleavage of one molecule of carbon to carbon bonds consumes one molecule of periodate in the reaction. Information about the structure of polysaccharides, such as the degree of branching, and the position and nature of glycosidic bonds and of non-carbohydrate residues in polysaccharides can be revealed by e