生化實驗 | 肝糖原的提取、鑑定與定量(八年制)

(這是Hmaide的第344篇推送)

關注和分享

----感謝 17級八年制某位同學 的投稿

（均分95，複製過來格式有些不對應，特別是空格有些沒有，閱讀一般也能看得出）

格式要求：正文請統一用：小四號，宋體，1.5倍行距；數字、英文用Times New Roman；標題用：四號，黑體，加粗。需強調的地方請用藍顏色標出。不得出現多行、多頁空白現象。

Extraction, Identificationand Quantification of Hepatic Glycogen

1.Purpose

1.1 To master the preparationmethod of tissue samples, understand the matters which need to attention.

1.2 To understand the principle and precautions of glycogen extraction, glycogen and glucose identification andanthrone colorimetric determination of glycogen content, and to master itsoperation method. 

1.3 Correctly operate and use the measuringpipette and adjustable micro liquid extractor. 

1.4 Skillfully use various methodsof solution mixing. 

1.5 Correctly grasp the solution transfer operation. 

1.6 Correctly operates pectrophotometer.

 

2.Principle

2.1 Extraction of hepatic glycogen

Glycogen stored in cells, grinding homogenate and other methods can makecells broken, low concentration of trichloroacetic acid can make proteindenaturation, destroy the enzymes in liver tissue and precipitate protein,while glycogen is still stable in the supernatant. As a result, glycogen isseparated from other components such as protein, and glycogen is insoluble inethanol and soluble in hot water, so 95% ethanol is used to precipitateglycogen in the filtrate and then dissolve it in hot water.

2.2Identification of hepatic glycogen

Glycogen aqueous solution is milky luster, iodine isreddish brown. This is the color of iodine molecules adsorbed by intermoleculargravity in the helix formed by the long chain of glucose in glycogen. Glycogencan also be hydrolyzed to glucose by acid. The presence of glycogen in livertissue can be determined by color reaction and glucose reduction. The colorreactions are as follows:

2.3Quantitative analysis of hepatic glycogen

Glycogen can behydrolyzed into glucose in concentrated acid, and concentrated sulfuric acidcan further dehydrate glucose to furfural derivative-5-hydroxymethylfuranal,which then condenses with anthrone to form blue compound. The substance has themaximum absorption at 620nm. When the sugar content is in the range of 10 ~ 100μ g, the color of the solution is proportional to the soluble sugar content.Using this reaction to compare the color of the standard solution with theknown glucose content treated with the same treatment, the content of glycogenin the sample can be calculated by the standard control method. Glycogen is verystable in concentrated alkali solution, so before color development, livertissue is heated in concentrated alkali to destroy other components and retainhepatic glycogen.

 

1.Materials and Method：

3.1 Experimentalmaterials (including experimental samples, main reagents, main instruments andequipment)

3.2 Experimental steps (including experimentalprocedures, operating steps, and considerations)

     3.2.1Experimental process

(1) Extraction and identification of hepatic glycogen

3.2.2 Matters needing attention

(1)extraction of hepatic glycogen: one step of adding ethanol to the supernatant,be sure to mix the solution. Because the supernatant is water, the density ishigher than ethanol, the solution is stratified. The volume of supernatant isabout 4ml. After adding ethanol, the volume of liquid increases, whichincreases the difficulty of mixing. Therefore, the dumping mixing or blowingand suction mixing operation with sample gun is adopted at this time.

(2)Identification of hepatic glycogen: the color of iodine adsorbed by glucosehelix chain in glycogen is related to the number of glucose residues. Glucoseresidues below 20 will make iodine red, 20-30 will make iodine purple, and morethan 60 will make iodine blue. The branched chain in starch is longer, so it isblue, while the glucose residue in the branch of hepatic glycogen is less than20, which is reddish brown after adsorbing iodine.

(3) theidentification of glucose in glycogen hydrolysate: the overtime reagent shouldnot be excessive. Otherwise, the reaction solution was black and turbid, and theexpected experimental results could not be observed.

(4)quantitative determination of liver glycogen: liver tissue must be dissolved inboiling water, otherwise it will affect colorimetry.

(5) useof spectrophotometers: reagents shall not be transferred at the interface ofthe instrument so as not to corrode the spectrophotometer. The liquid containedin the colorimetric cup shall not exceed 2/3 of the colorimetric cup to preventthe solution from overflowing and corroding the colorimetric cup holder.

1.Results and Discussion: (①Results: experimental data, phenomena, maps;②Discussion: results-based logical inferences, anddraw conclusions.)

4.1 Results

4.1.1 Experimental Phenomena

    (1) Extraction and Identificationof Hepatic Glycogen

After the firstcentrifugation, the liver homogenate was divided into two layers, the upperlayer was supernatant, incomplete clarification, and the lower layer was lightbrown precipitate.

After adding ethanol,mixing and standing, the solution is layered, the upper layer is supernatantand the lower layer has white flocculent precipitate.

After the second centrifugation, the solution was divided into 2 layers, the upper layer wassupernatant and there was a small amount of white precipitate at the bottom ofthe tube.

After adding distilled water and boiling water bath, thewhite precipitate dissolves.

On the white porcelain plate, the glycogen solution isreddish brown after being added with iodine reagent, which is obviouslydifferent from the yellow of iodine itself.

The solution is blue (the Benedict's reagent itself is blue). After adding the Benedict'sreagent to the glycogen hydrolysate. After boiling water bath, the solutionturns brick red.

(2) Quantitative Determinationof Hepatic Glycogen

After adding KOH tochicken liver and boiling water bath, the solution turns red brown.

Before the water bath, the blank tube is yellow, the standard tube isgreen, and the sample tube is yellow (lighter in color than the blanktube).  After the water bath, the blanktube has no obvious change, the color of the standard tube is slightly darker,and the sample tube changes from yellow to dark green (the color is darker thanthe standard tube).

Results:The content of hepatic glycogen is 2.565g/100g of liver tissue.

Therefore,the conclusion of this experiment is that the chicken liver sample containsliver glycogen, and the content of liver glycogen is 2.565g/100g of livertissue.

4.2Discussion

(1) Analysis of experimental data:

Liver glycogen content measured in this experiment is 2.565g/100gof liver tissue. The content of glycogen in chicken liver of a well-fed chickenis 2~3g/100g of liver tissue. The results of this experiment are relatively normal,which indicates that liver tissue contains normal level of liver glycogen, thatmeans the chicken liver used in the experiment may come from chickens with adequatenutrition.

(2) Analysis of color contrast results:

The reaction between glycogen solution and iodinereagent is reddish brown, which is obviously different from the yellow ofiodine reagent itself, indicating that glycogen content is pointing to an expectedresult.

(3) Analysis of reaction results between glycogenhydrolysate and Bancroft reagent:

The reaction mainly indirectly identifies theexistence of glycogen in the solution by detecting the reductive sugar reactionbetween glucose and Bancroft reagent. Glycogen solution is hydrolyzed into glucose by concentratedhydrochloric acid, aldehyde groups in glucose can be oxidized to carboxylgroups by Cu(OH) 2, namely glucose is oxidized togluconic acid, and Cu2+ is reduced to generate Cu2O brick red precipitate. Therefore,after the water bath, the solution changed from blue to brick red, and theexperimental phenomenon was obvious.

(4) Precautions:

1) Anthrone reagent isprepared from concentrated sulfuric acid and should be used carefully.  In case of contact with skin, rinseimmediately with plenty of clean water.

2) After centrifugation,the tip of the pipette should be prevented from touching the precipitate whenabsorbing the supernatant with the pipette, otherwise the precipitate willfloat upward, resulting in turbidity of the supernatant.  Do not push the button until the tip of thegun reaches below the liquid level, otherwise the bubbles emitted from the tipof the gun will also stir up sediment.

3) Be careful of watervapor when opening the cover of the room temperature to 100℃ thermostatic water bathto avoid scalding.

4) Pay attention tobalancing when using the centrifuge, otherwise the centrifuge will be damaged.

5) When using thespectrophotometer, the reagent shall not be transferred on the instrumentinterface to avoid corrosion of the spectrophotometer.

6) During liver glycogenextraction, add 95% ethanol to the supernatant and mix well. The total amountof the solution is large, and the mixing operation is not easy. It can beuniformly mixed by pouring or by stirring with a glass rod.

7) Liver tissue must becompletely dissolved in boiling water bath, otherwise the colorimetric effectwill be affected.

8) Anthrone reagent mustbe 2 times as much as the tested solution.

(5) Conclusion

This experiment consists oftwo parts: extraction, identification and quantitative determination of hepaticglycogen. In the qualitative identification, our color reaction is totallyobvious, so it is inferred that the concentration of hepatic glycogen may in anormal level. Further quantitative experiments showed that the concentration ofhepatic glycogen is 2.565%. Confirms our conjecture. Therefore, this experimentis generally successful.