Experiment No. 10 DIGESTION In order for the nutrients in food to be absorbed, they must first be broken down into particles that are small enough to be transported through carrier proteins into the epithelial cells that form the mucosal lining of the digestive tract. The process of breaking down food is termed digestion. It occurs through two different processes: physical digestion, where large chunks of food are ground into tiny particles, and chemical digestion, whereby through the use of enzymes released into the digestive tract large polymeric biomolecules are broken into individual monomers or oligomers (e.g. dimers or trimers). Digestion occurs primarily within three particular segments of the digestive tract: the mouth, the stomach, and the small intestine. Digestion in the Mouth The digestion of food starts in the mouth where it is masticated, broken into small pieces and mixed with saliva before entering the digestive tract. Carbohydrates are the first type of biomolecule to be chemically digested in the digestive tract, as chemical digestion begins in the oral cavity through a salivary enzyme called salivary amylase or ptyalin. Salivary amylase hydrolyzes starch to maltose and dextrins. It acts on starch by randomly cleaving -1,4 linkages at any interior part of the polysaccharide chain. Starch is progressively hydrolyzed into simpler oligosaccharides that give different colors when reacted with iodine: amylodextrin – purple; erythrodextrin – red; achroodextrin – colorless; maltose – colorless. Salivary amylase is most active at a temperature of 40°C and the activity of the enzyme is enhanced by the presence of chloride ions. Carbohydrate digestion effectively stops when the food is swallowed and transferred to the stomach. Only when the masticated food or chyme passes into the small intestine will carbohydrate digestion resume. Digestion in the Stomach The chyme is further digested by gastric juice when it enters the stomach. This is also where the chemical digestion of protein begins. The lining of the stomach produces a mixture of fluids called gastric juice in response to neural stimulation (induced by smell, sight and taste of food), by distension of the stomach as food enters, and by pH changes induced as the more neutral pH food enters the acidic stomach. Normal gastric juice is a light colored, thin fluid of high acidity containing 0.2-0.5% HCl, 97-99% water with organic acids (lactic acid), mucin, inorganic salts, and digestive enzymes (pepsin, rennin, lipase) comprising the remaining 1-3%.
The acidity of pure gastric juice is mainly due to HCl that is produced by the parietal cells of the gastric glands. The normal stomach pH is 1.6-1.8 due to the mixing of HCl with food and the secretions of chief and mucous cells. Gastric secretions are evaluated in terms of Free and Total Acidities using suitable indicators. Free acidity is the amount of free HCl present in the gastric juice and total acidity is the total amount of organic acids, acid salts and free HCl present in the gastric juice. By titrating gastric juice with 0.1M alkali solution, gastric acidities can be expressed as mL of 0.1N alkali required to neutralize 100 mL of gastric juice. Normal gastric juice contents have a total acidity of about 60-90 and a free acidity of 40-50. The presence of the organic acid, lactic acid, in gastric juice is determined by a color reaction with very dilute FeCl 3 solution. Pepsin is first secreted by the chief cells in the gastric mucosa as pepsinogen, a zymogen. A zymogen or proenzyme is an inactive precursor that is converted into an active enzyme by action of an acid or another enzyme or by other means. Pepsinogen is converted to its active form pepsin, both by the acidity of the gastric juice and pepsin itself. pepsin Pepsinogen + H+
Pepsin
Pepsin hydrolyzes native and denatured proteins into shorter polypeptides. Like other enzymes, pepsin requires a rather definite pH for its maximum activity. Its optimum pH lies between 1.0 and 2.0. Another proteolytic enzyme found in the gastric juice of some animals is rennin. It is secreted first as its zymogen, prorennin, that is converted to rennin by addition of HCl. Rennin acts on casein, the chief protein in milk which occurs mainly as calcium caseinate, by causing its partial hydrolysis to soluble paracasein. Rennin acts best at a pH of 6.0 to 6.5. Digestion in the Small Intestine From the stomach, the food is transported to the small intestine for further digestion. Three types of juices enter the intestines: pancreatic juice from the pancreatic gland, intestinal juice from the duodenal wall, and bile from the gall bladder. These juices mix with the partially digested food and digestive enzymes from the stomach. Pancreatic Juice In the small intestine the chyme is exposed to pancreatic amylase that hydrolyzes starch, glycogen and other carbohydrates except cellulose into disaccharides and trisaccharides. The functional conformation of pancreatic amylase is stabilized by calcium and chloride ions. Pancreatic juice contains pancreatic nucleases such as ribonuclease and deoxyribonuclease that hydrolyzes RNA and DNA, respectively. Cholesterol esterase
that catalyzes the hydrolysis of cholesterol esters and pancreatic lipases that hydrolyze fats into glycerol and fatty acids are also found in the pancreatic juice. (Although there is a gastric lipase secreted in the stomach that causes a small amount of fat digestion, and infants produce a salivary lipase, almost all of the digestion of fat takes place in the small intestine.) The proteases trypsin, chymotrypsin, and carboxypeptidase are secreted in the pancreatic juice as the inactive proenzymes trypsinogen, chymotrypsinogen and pro-carboxypeptidase. Enzymes in the brush-border cells activate these zymogens, which ultimately digest the polypeptides into a combination of free amino acids, dipeptides, and tripeptides that are absorbed by the intestinal epithelium. Complete protein digestion is achieved through the activity of both pancreatic and intestinal brush border proteases. Bile Bile contains water, mucin, pigments, cholesterol, esterified and unesterified fatty acids, inorganic salts and bile acids. Bile salts can emulsify fats and fat digestion products. Prior to lipase chemical digestion, the fats are emulsified to reduce them into fine colloidal droplets to colloidal dimension. Bile salts indirectly enhance lipase activity because of their capacity to emulsify acids making them more available for the enzyme. Intestinal Secretion The final digestion in the small intestine is carried by the intestinal secretion. Enteropeptidase is found in the intestinal juice; it is an enzyme which converts trypsinogen into active trypsin. Nucleases further hydrolyze nucleic acids. Certain brush-border enzymes such as lactase, sucrase, maltase and isomaltase break down specific oligosaccharides and disaccharides into monosaccharides that are absorbed by the intestinal epithelium. The final products of carbohydrate digestion can be analyzed using Lugol’s solution by noting the colors produced when the hydrolysate is reacted with iodine. Benedict’s reagent can likewise be used to test the products of carbohydrate digestion. Objectives Upon completion of the experiment, the student should be able to: 1. understand the process of digestion of different molecules. 2. distinguish the enzymes responsible for the digestion of carbohydrtaes, proteins and lipids. 3. calculate the free and total acidties of gastric juice samples. 4. examine the extent of digestion of food samples under different conditions.
A. Carbohydrate Digestion Tub 1% H2O 0.2% e starch amylase solution solution* 1 5.0 ml 3 ml 2 5.0 ml 3 ml 3 5.0 ml 3 ml
1.0 M HCl
1.0 M H3PO4
1.0 M NaCl
Work-up
-
-
-
10 drops -
-
Heat in boiling H2O bath, 5 mins. -
4 5
5.0 ml 5.0 ml
-
3 ml 3 ml
10 drops -
6
5.0 ml
-
3 ml
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10 drops *If no stock amylase is available, use 5 mL salivary secretion per tube. Incubate all tubes in a 37 °C water bath for 1.5 hours. Divide the contents of each tube evenly into 2 test tubes after incubation. Add 2-3 drops I2 in KI solution to one set of tubes. Note any color change. To the other set of tubes, add 5 ml Benedict’;s reagent. Place a marble at the mouth of each tube and heat in a boiling water bath for 5 mins. Record observations. B. Protein Digestion Tub H2O 2% neutral e pepsin solution 1 10 5 ml drops 2 5 ml 3 4 5
5 ml -
5 ml 5 ml
1.0 M HCl 10 drops
1.0 M NaOH -
Work-up
Place in ice bath, 5 mins.
10 drops Place a piece of (10-min) boiled egg white (1cm x 1 cm x 1 cm) in each tube. Incubate all tubes in a 37 °C water bath for 1.5 hours. Observe any difference in the size of the egg white. Place 5 drops of the solutions from test tubes 1-5 into separate test tubes. Add 5 drops Biuret reagent to each test tube. Shake well. Observe color changes, if there are any. C. Fat Digestion Tub H2O 2% neutral e pancreatin solution 1 5 ml 2 5 ml
10 drops 10 drops -
Bile salt
1.0 M CaCl2
0.1 g -
-
1.0 M Na2CO3 -
3 5 ml 0.1 g 4 5 ml 0.1 g 10 drops 5 5 ml 0.1 g 10 drops Add 3 ml cream to each tube. Shake gently until emulsification is complete. Take the pH of each tube before incubation in a 37 °C water bath for 1.5 hours. Take pH readings at 20, 40, 60 mins during the incubation process and after the 1.5 hour-period. Ensure that the pH electrode is washed with detergent solution & thoroughly rinsed with water after each pH reading. Use wash bottles for both detergent and water. D. Gastric Analysis 1. Titration of free acidity Pipette 10 ml of strained gastric contents into an Erlenmeyer flask, add 2 drops of Topfer’s reagent and titrate with 0.1N NaOH until the initial red color is replaced by salmon pink. Record the buret reading and calculate free acidity as ml of 0.1N alkali for 100 ml of gastric contents. 2. Titration of total acidity To the same solution which you have just titrated for free acidity add 2- 3 drops phenolphthalein and continue the titration until the yellow color of the solution (due to Topfer’s reagent) changes to the definite pink of phenolphthalein. Record the buret reading and calculate the total acidity as ml of 0.1N alkali required per 100 ml of gastric contents. 3. Test for lactic acid Measure 1 ml. dilute FeCl 3 solution (faintly yellow) into two test tubes. Add 1 ml gastric juice to one of the tubes. Compare the color obtained with that of the untreated dilute FeCl3 solution. WASTE DISPOSAL 1. Collect all solid wastes in a garbage bag and dispose in the trash bin. 2. Dispose neutral pepsin and neutral pancreatin down the drain but flush with ample amounts of water. 3. Discard mixtures from Part D-3 into the heavy metal waste jar. 4. Dispose all other solutions into the sink. Flush with ample amounts of running water. References
Biochemistry Laboratory Manual, Quezon City: UP Diliman, 2007. http://chestbooks.com/health/nutriton Robyt, J.F. and B.J. White. Biochemical Techniques: Theory and Practice. Michigan, USA: Waveland Press, 1987.
NAME SECTION INSTRUCTOR PRE-LAB SHEET FOR EXPERIMENT 10 DIGESTION 1. Create a flowchart or concise outline of the procedure.
DATE
2. Use the protocol to fill up the ff. table of materials, equipment & glassware needed for each part of the experiment (Parts A-D).
3. Give a brief overview of the digestion process. Include all enzymes needed in each step.
4. What is the difference between free and total acidities? Which one is used in evaluating whether a person is hypo- or hyperacidic?
5. Provide a material safety data sheet (MSDS) for two hazardous reagents/ chemicals involved in this experiment. Refer to the internet for the appropriate MSDS facts and use the back page(s) of your prelab sheet for
this requirement.
Formal Report due ___________________________________ Include answers to the following questions in your formal report: A. Show sample calculations for free and total acidities. B. Postlab Questions: 1. What are the end products of the action of amylase on starch? Of pepsin on a protein?of pancreatic enzymes on cream? 2. Whay was hard-boiled egg and not raw egg white used in this experiment? 3. How can you make use of the Biuret reagent to follow the progress of the tryptic digestion of a solid protein under optimum conditions? 4. Show the action of the pancreatic enymes on the polypeptide V-K-
M-R-T-D-C 5. What is the role of CaCl2 in the action of pancreatic lipase? 6. What results show the substrate specificity of enzymes? 7. In the tabulated form, trace the complete digestion of a meal consisting of rice, red egg with sliced tomatoes and fried pork chop. Use the format below. Site
Secretion
Enzyme
Substrate
End Product
Optimum Conditions
