Digestion and Enzyme Activity

Background:

The digestive system is made up of the digestive tract – a series of hollow organs joined in a long, twisting tube from the mouth to the anus- and other organs that help the body break down and absorb food. Organs that make up the digestive tract are the mouth, esophagus, stomach, small intestine, large intestive – also called the colon – rectum, and anus. Two accessory digestive organs, the liver and pancreas, produce digestive juices that reach the intestines through small tubes called ducts. The gallbladder stores the liver’s digestive juices until they are needed in the intestine.

The digestive glands that act first are in the mouth – the salivary glands. Salivaproducedbythese glands contains an enzyme called amylasethatbeginstodigestthestarchfromfoodintosmaller molecules. An enzyme isasubstancethatspeedsupchemicalreactionsinthebody.

Thenextsetofdigestiveglandsisinthestomach lining. Theseproducehydrochloricacidand an enzyme called pepsin thatdigestsprotein. Athick mucuslayercoats the mucosaandhelpskeepthe acidic digestive juice from dissolving the tissue of the stomachitself. After the stomach emptiesthefood andjuicemixtureintothesmallintestine,thejuicesof two other digestive organs mix with thefood. One of theseorgans,thepancreas,producesajuicethat containsawidearrayofenzymessuchasamylaseto breakdownthecarbohydrate,lipase tobreak down fat, and trypsin to break down protein.

Thesecondorgan,theliver,producesyetanotherdigestivejuice—bile.Bileisstoredbetweenmeals in the gallbladder. At mealtime, it is squeezed out of the gallbladder, through the bile ducts, and into the intestine to mix with the fat infood. The bile acids dissolve fatinto the watery contents of the intestine,muchlikedetergentsthat dissolve grease from a frying pan.

Purpose: The goal of this lab will be to observe how digestive enzymes are able to break down macromolecules and how environmental changes affect their functioning.

Digestion and Enzyme Activity

Materials:

Spot Plate / Lipase / Albumin
10 Stirring Sticks / Dishsoap / HCl
11 pH Strips / Starch Solution / Pepsin
Water / Amylase / Biuret
Corn Oil / Potassium Iodine / Boiled Amylase

Procedure:

+++ All data tables are on Pg 3+++

PART A: Fat Digestion

The corn oil represenst the lipid polymer, and lipase is the enzyme responsible for breaking the lipid down. A change in pH will demonstrate that the enzyme is active. The soap works similarly to bile, and will break up the oil so lipase can work more easily.

  1. 2. Add10dropsofwatertowells1-3.
  2. Add 3 drops of Corn oil to wells 1-3.
  3. Use separate stirring sticks to mix each well thoroughly.
  4. Use the forceps to dip a separate pH strip intoeachofthewells1-3. Comparethecolor change of each strip to the pH strip chart to determinethepHofthemixtureineachwell. RecordyourresultsinDataTable1.
  5. Add 5 drops of lipase to well 2 and 3.
  6. Add2dropsofliquidsoaptowell3.
  7. Re-stir the mixture in each well, and allow them to sit for 20 minutes.
  8. Retest the pH and record your results inDataTable1.
  9. Recordyourobservations of each of the mixtures in DataTable1.
  10. Rinseoutyourspotplateand dry it off with a paper towel.

PART B: Carbohydrate Digestion

InpartBstarchisapolysaccharide,andamylaseistheenzymeresponsibleforbreakingstarchdown intomonosaccharides. Sincethepotassiumiodinetests for starch, you will be looking for a negative test to determine whether amylaseactuallybrokedownthestarch.

  1. Add 15 drops of 1% Starch solution to wells 1-3.
  2. Add 5 drops of 2% Amylase solution to well 2.
  3. Add 5 drops of 2% Boiled Amylase solution to well 3
  4. Use separate stirring sticks to mix each well, and allow them to sit for 5-7 minutes.
  5. Add1dropofPotassiumiodinetowells1-3. A positive test for starch will turn dark blue- black. If a well is positive for starch, put a+in the DataTable2. If the testis negative for starch put a–.
  6. Recordyourobservations of each of the mixtures in DataTable2.
  7. Rinseoutyourspotplateand dry it off with a paper towel.

PART C: Protein Digestion

InpartCthealbuminrepresents apolypeptideprotein. Pepsinistheenzymeneededtobreakdown the albumin into amino acids, but can only work withinacertainpHrange. TheBiuretwillturnpink with smallerproteinchainsandwillturnpurpleinthepresenceoflargeproteins.

  1. Place5dropsof2%Albumininwells1-4.
  2. Add 5 drops of 1% HCl to wells 2 and 4
  3. Add 5 drops of 3% Pepsin to wells 3 and 4.
  4. Use separate stirring sticks to mix each well, and allow them to sit for 5-7 minutes.
  5. Use the forceps to dip a separate pH strip intoeachofthewells1-4. Comparethecolor change of each strip to the pH strip chart to determinethepHofthemixtureineachwell. RecordyourresultsinDataTable1.I will provide you with the data for HCl
  6. Add 2 drops of Biuret to wells 1-4. A positive test for protein breakdownwillturnpink. Ifthe wellispositive,puta+in Data Table 1. If the well is negative put a -.
  7. Recordyourobservations of each of the mixtures in DataTable1.
  8. Rinseoutyourspotplateand dry it off with a paper towel.

++++ Clean up your area and complete the analysis questions for all parts ++++

Data Tables:

TABLE 1

Well / Contents / InitialpH / FinalpH / Observations
1 / Oil + Water
2 / Oil + Water + Lipase
3 / Oil + Water + Lipase +Soap

TABLE 2

Well / Contents / StarchTest / Observations
1 / Starch
2 / Starch+Amylase
3 / Starch + B. Amylase

TABLE 3

Well / Contents / pH / ProteinTest / Observations
1 / Albumin
2 / Albumin + HCl
3 / Albumin+ Pepsin
4 / Albumin+ Pepsin+HCl
5 / HCl

Analysis:

Answer on a separate sheet of paper

PART A:

  1. HowdoesthepHofeachsolutionshowthatfatdigestionhasoccurred?
  2. Whichwellshowedthegreatest degreeoffatdigestion? Explain.
  3. Whateffectwouldfatdigestionhaveonthe pH of the surrounding solution? Explain.
  4. What do you predict would happen to the pH of a corn oil solution if we added Pepsin. Explain.

PART B:

  1. Whatarethesubunitsthat make up carbohydrates?
  2. How could you tell that carbohydratedigestionhadtakenplace?
  3. What do you think may happen if HCl was added to the starch + amylase mixture? Explain your answer.
  4. What do you predict would happen to a starch solution if we added lipase? (i.e.What would be the results of an iodine test?)

PART C:

  1. Whatarethesubunitsthat make up a protein?
  2. How could you tell that proteindigestiontookplace?
  3. Which of the wells showed the greatestdegreeofproteindigestion? Why?
  4. DoestheeffectivenessofpepsindependonpH? Explain.
  5. WhatcanyouconcludeabouttheoptimalpHforpepsin?
  6. What do you predict would happen to an albumin solution if we added amylase? (i.e. What would be the results of a Biuret test?)

Conclusion:

Explain, in detail and with examples, how this experiment demonstrates that enzymes are highly specific to both substrates and environmental conditions.