Lab 16: Digestion Lab

Carbohydrate Digestion

• enzymes used to break down starch (polysaccharides) via hydrolysis are:

  1. saliva amylase

     • salivary amylase: enzyme found in salvia, breaks down starch into short chains of polysaccharides and maltose

  2. pancreatic amylase

     • pancreatic amylase: takes starch and produces short-chain polysaccharides AND maltose (disaccharides)

       • any starch that doesn’t get absorbed by salivary amylase will get digested by pancreatic amylase

     • break down occurs in the small intestine

  3. sucrase: a brush boarder enzyme that digest sucrose (disaccharides) to glucose and fructose (monosaccharides)

  4. maltase: a brush boarder enzyme that digest maltose (disaccharides) to glucose (monosaccharides)

  5. lactase: a brush boarder enzyme that digest lactose (disaccharides) to glucose and galactose (monosaccharides)

• monosaccharides are absorbed in duodenum and jejunum

  • glucose and galactose (monosaccharides) are absorbed via secondary active transport

  • fructose (monosaccharides) are absorbed via facilitated diffusion

Protein Digestion

• Enzymes used to break down Proteins into amino acids, dipeptides, or tripeptides via hydrolysis:

  1. stomach low pH denatures proteins allowing access to peptide bonds for peptidase enzymes (aminopeptidase)

  2. pepsin: stomach enzyme that catalyzes digestion of ingested proteins to polypeptide chains

     • Chief cells: produce pepsinogen

       • pepsinogen gets transformed to active form pepsin by HCl cleaving portions of the enzyme

  3. typsin: breaks polypeptides into smaller polypeptides chains in the duodenum and jejunum

  4. chymotrypsin: further breaks down smaller polypeptides into dipeptide chains in the duodenum and jejunum

  5. Carboxypeptidase: breaks down even further into individual amino acids in the duodenum and jejunum

  6. aminopeptidase: brush border enzyme and type of peptidase enzyme that breaks down even further into individual amino acids in the duodenum and jejunum

     • final GI lumen digestion products are amino acids, dipeptides, and tripeptides

       • amino acids, dipeptides, and tripeptides absorbed via secondary active transport

         • Dipeptides and tripeptides are further broken down into amino acids in epithelial cells

           • intestinal lining causes active transport of amino acids into blood stream

Lipid Digestion

• Enzymes that break down Triglycerides into fatty acids, or monoglycerides via hydrolysis:

  1. bile: produced in the liver; contains bile salt micelles that are secreted into the duodenum to and breaks down fats into tiny emulsification droplets of triglycerides

     • Emulsification: process of breaking down fat into smaller particles

  2. Pancreatic lipase: pancreatic enzyme that removes 2 of 3 fatty acids from each triglyceride and forms free fatty acids and a monoglyceride

     • fatty acids and a monoglyceride (lipids) can enter epithelial cell without carriers or active transport

       • once in the epithelial cell, they are regenerated into triglycerides, cholesterol, and phospholipids

         • those are combine with lipoproteins to form chylomicrons

         • chylomicrons: article of lipids and protein secreted by the intestinal epithelial cells into the lymph and transported by the lymphatic system to the blood.

           • chylomicrons are secreted by exocytosis into lacteals

             • then transported into lymphatic system to the thoracic duct

               • then returned to the blood at the left subclavian artery

Experiment:

• Digestion of Carbohydrates by salivary amylase:

  • starch (polysaccharide) and water combine forms maltose and small polysaccharides

    • starch + water $\rightarrow$ maltose + small polysaccharides

  • enzyme: salivary amylase

  • substrate: starch

  • substrate test (assay): Lugol’s reagent

    • test for presence of starch

    • contains iodine

      • positive test indicated by purplish black color

  • Product: maltose

  • Product test (assay): Benedict’s test

    • tests for presence of maltose, a reducing sugar

    • contains cupric ions (Cu2+), which in presence of maltose are reduced to cuprous ions (Cu+)

      • positive test indicated by yellow color precipitate (Cu2O)

  • Factors necessary and inhibitory for enzymatic action in lab and GI tract

    • Test Tube 1: contained water and starch solution

      • Starch test: No digestion of starch because water does not digest starch

        • should have highest concentration of starch as starch remained undigested

      • Maltose test: no maltose produced, indicating that without an enzyme, starch cannot be converted to maltose

    • Test Tube 2: contained saliva and starch solution

      • Starch Test: complete digestion occurred because salivary amylase was present

      • Maltose test: some maltose present, confirming that salivary amylase effectively catalyzed the breakdown of starch into simpler sugars.

    • Test Tube 3: contained pepsin and starch solution

      • Starch Test: No digestion occurred because wrong enzyme present

      • Maltose test: No digestion of maltose because enzyme was wrong to break down, making breakdown of starch into maltose impossible

    • Test Tube 4: contained saliva and starch solution that was boiled

      • Starch Test: No digestion occurred because it was boiled, causing denaturation of enzymes

      • Maltose test: no maltose present due salivary amylase being denatured and unable to breakdown starch.

    • Test Tube 5: contained saliva, starch solution, and 10 drops HCl

      • Starch Test: No digestion occurred because pH was too acidic for salivary amylase to function

      • Maltose test: no maltose present due to wrong pH causing inactivity of salivary amylase breakdown down starch into maltose

• Digestion of Proteins (photographic film made from collagen protein) by pepsin:

  • enzyme: pepsin

  • substrate: collagen on film

  • substrate test (assay): observe color

    • dark film indicates no protein digestion

    • opaque film indicates partial protein digestion

    • clear film indicates complete protein digestion

  • Product: amino acid

  • Factors necessary and inhibitory for enzymatic action in lab and GI tract

    • Test Tube 1 and 5: contains pepsin and HCl

      • pH: 2

      • amount of film coating: none

      • pepsin activity: complete digestion of proteins due to presence of enzyme pepsin and ideal pH levels

    • Test Tube 2: contains boiled pepsin and HCl

      • pH: 2

      • amount of film coating: some

      • pepsin activity: no digestion of proteins due to enzymes being denatured in boiled solution

    • Test Tube 3: contains pepsin and NaOH

      • pH: 8

      • amount of film coating: some

      • pepsin activity: no digestion of proteins due to NaOH creating basic pH levels

        • pepsin needs acidic environments to be active

    • Test Tube 4: contains pepsin and water

      • pH: 5.5

      • amount of film coating: some

      • pepsin activity: no digestion of proteins due to water creating basic pH levels

    • Test Tube 6: contains pepsin and HCl

      • pH: 2

      • amount of film coating: some

      • pepsin activity: no digestion of proteins due to no enzyme present

    • Test Tube 7: contains salivary amylase and HCl

      • pH: 0

      • amount of film coating: some

      • pepsin activity: no digestion of proteins due to wrong enzyme being present

    • Test Tube 8: contains pepsin and HCl in cold ice bath:

      • pH: 1

      • amount of film coating: some

      • pepsin activity: no digestion of proteins due to temperature being too low

Specificity

• digestive enzymes are specific

  • specificity: every functional protein will bind with only one ligand to the functional site of the enzyme and perform only one function

    • in context of enzymes, every enzyme will bind only one substrate and participate in only one chemical reaction

Factors necessary and inhibitory for enzymatic action in lab and GI tract

• Factors necessary for enzymatic action in lab and GI tract:

  • Test tubes were incubated for 1 hour in 37°C water bath for carbohydrate digestion lab: replicating temperature conditions

  • HCl was added to protein digestion lab: mimicking acidity of the stomach

• Factors inhibitory for enzymatic action in lab and GI tract:

  • NaOH was added to protein digestion lab: denaturing enzymes, demonstration effect of non-optimal pH

  • in both carbohydrate and protein lab, some solutions were boiled: denaturing enzyme

  • omission of enzyme: acts as control

    • control test tubes were the ones that did not have enzyme present

  • different enzymes: tests for specificity

    • substrates bind with specificity to their enzymes

      • if substrates and enzymes do not match, reaction will not occur

Purpose of Control

• purpose of control is to ensure results of experiment are due to experimental factors and not due to chance

  • in context of experiment, control determines if the substrate will spontaneously be broken down into products under conditions of experiment, without the enzyme OR require action of enzyme to break down products

Distinguish between Enzymatic Digestion and Emulsification

• digestion: chemical breakdown of macromolecules into their monomers or subinits, catalyzed by enzymes

• Emulsification: production of fine suspension of lipid droplets

  • no chemical change to the lipid

Distinguish between Chylomicrons, HDLs, and LDLs

• Chylomicrons: particles of lipid and proteins secreted by the intestinal epithelium into lymphatic vessels, then transported by lymphatic vessels to the blood.

• HDLs (high density lipoprotein): particles of lipid and proteins that transport cholesterols away from arteries and to the liver

  • offer protection from atherosclerosis.

• LDLs (low density lipoprotein): lipids and proteins that transport cholesterol to the arteries

  • may contribute to arteriosclerosis.