22__糖、氨基酸、蛋白质的性质-EN

The Properties of Carbohydrates, Amino Acids, and Proteins Objective: To learn and understand the chemical properties of carbohydrates, amino acids, and proteins. To distinguish monosaccharides, disaccharides, and polysaccharides using simple chemical methods. To learn methods to characterize amino acids and protein. Key points: To distinguish monosaccharides, disaccharides, and polysaccharides using simple chemical methods. To learn methods to characterize amino acids and proteins. Teaching methods: Comparing and Summarizing 【Lecture】The experiment is to study the properties of carbohydrates, amino acids, and proteins. Through the experiment, students will learn how to distinguish monosaccharides, disaccharides, and polysaccharides using simple chemical methods, and how to characterize amino acids and proteins. 【Procedure】 1. The properties of carbohydrates (1) Molisch’s test[1]: To a solution of 1 ml 5% glucose in a test tube, add 2 drops of 10% α-naphthol in 95% ethanol solution (Molisch’s reagent). Tilt the test tube to 45°. Slowly add 1 ml of concentrated sulfuric acid down the sides of the sloping test tube without mixing. Observe the reaction. Heat the reaction in hot water bath if no color change. Samples: 5% glucose, fructose, maltose and sucrose. (2) 1,3-Benzenediol test[2]: Add 2 mL of 1,3-benzenediol and 1 ml 5% glucose to a test tube. Heat the reaction for 1-2 min with boiling water bath. Observe the reaction. Keep heating for 20 min, check the reaction and explain the observations. Samples: 5% glucose, fructose, maltose, and sucrose. (3) Fehling’s test and Tollen’s test on reducing sugars: A) Fehling’s test: Take 3 ml of Fehling’s reagent A and Fehling’s reagent B each. Mix thoroughly. Divide the mixture into 6 portions and put into 6 test tubes. Label each test tube. Heat the mixture to reflux then add 0.5 ml samples respectively. Observe and compare the results. Samples: 5% glucose, fructose, maltose, sucrose, lactose and starch. B) Tollen’s test: Add 1.5 ml Tollen’s reagent in 5 test tubes. Add 0.5 ml samples respectively. Heat the reaction with 60-80 °C hot water bath. Observe the reaction. Keep heating for 20 min, check the reaction and explain the observations. Samples: 5% glucose, fructose, maltose, sucrose and starch. (4) Osazone formation: Add 2 ml of phenylhydrazine in 2 test tubes each. Add 5% glucose and lactose respectively. Heat the reaction in boiling water bath. Observe the formation of crystalline (take a drop of the suspension on a microslide and check the crystalline under microscope). Record the time needed. [3] (5) Hydrolysis of carbohydrates: A) Hydrolysis of sucrose: Add 2 drops of conc. HCl into 8 ml 5% sucrose solution in a test tube. Boil the mixture for 3-5 min. After cooling, neutralize the mixture with 10% NaOH. Use the resulting solution for a Fehling’s test. B) Hydrolysis of starch and the iodine test: Add 9 ml water in 1 ml starch. Mix thoroughly. Add several drops of iodine-KI solution. Diluted the mixture to very light blue. Heat the mixture and observe. Cool the mixture and observe the color change. Explain the results. [4] 2. The properties of amino acids and proteins (1) Precipitate reactions of proteins: A) Precipitate of protein using heavy metal salts: [5]Add 1 ml protein solution into 2 test tubes each. Add 2-3 drops saturated CuSO4 solution and basic Pb(OAc)2 respectively. Observe the reactions. B) Reaction of proteins with alkaloid: [6] Add 0.5 ml protein solution into a test tube. Acidify the solution with 5% HOAc. Add saturated picric acid solution till precipitate forms. [4] (2) Protein reactions with color change: A) Ninhydrin test: [7] Add 1 ml of glycine solution and 1 ml of protein solution into 2 test tubes respectively. Add 2-3 drops of ninhydrin solution. Heat the reaction with boiling water bath for 10-15 min. Observe the reaction. B) Biuret test: [8] Add 1-2 ml 20% NaOH solution in a test tube. Add 1 drop of CuSO4 solution and 1 ml protein solution. Heat the mixture and observe. Do the same reaction with 1 ml 1% glycine solution. Compare the results. 【Note】 1. In Molisch reaction, carbohydrates were dehydrated by conc. H2SO4 to form furfural derivatives, which condense with α-naphthol to form a purple colored product. 2. Ketose reacts with 1,3-benzenediol to from a bright red precipitate. The product is soluble in ethanol giving a bright red solution. Maltose and sucrose hydrolyze to give glucose or fructose under acidic conditions. High concentration glucose can partially convert to fructose in the presence of acid. So glucose, maltose and sucrose have positive 1,3-benzenediol test if heated for long time. 3. It takes 5 min for glucose to precipitate. Lactose takes 20 min. Lactose osazone is soluble in hot water, so participation forms after cooling. 4. Starch turns to blue with iodine. It is because the size of the straight chain starch helix and that of iodine molecule match so they form blue colored host-guest complex. 5. Heavy metal forms precipitate with protein even at very low concentration. They form water soluble, salt like compounds. So protein is used as an antidote for heavy metal poisoning. Both protein precipitate by heavy metal salt and heating is irreversible. 6. Many alkaloid precipitate reagents are heavy metal salts, macromolecule acid, and iodo complex salt with high molecular weight. Alkaloids can form precipitate with protein. 7. See text book p184-185 for the structure of ninhydrin, preparation of ninhydrin solution, and the color change principle for the reactions with amino acids and proteins. 8. All proteins and their hydrolysis intermediates have positive biuret test results because proteins and the hydrolysis intermediates contain peptide bonds. In the hydrolysis intermediates of proteins, the reaction color is related to the numbers of peptide bonds. In general, those with one peptide bond turn to blue; those with two peptide bonds turn to purple; compounds with three peptide bonds turn to red. Proteins form complex with copper ion so the color turns to purple. Do not add too much copper salt. Otherwise, the formation of too much Cu(OH)2 may affect the observation of purple or red color.