study of enzymes we consume

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Part A

The enzyme tested in part A was pancreatic lipase. The proper substrate for pancreatic lipase is fat droplets. The products formed are glycerol, fatty acids, and monoglycerides. All of this takes place in the lumen of the small intestine and the optimum pH range is 7.1 to 8.2. (Campbell et al. 2008)

Phenol Red was used as a pH indicator in this test and it would become yellow to indicate acid, orange to indicate neutral, red to indicate alkaline, and a pink color indicated that the enzyme has denatured and is above 10.

Triglyceride Digestion with Pancreatic Lipase

Observations (Color)

Results (pH condition) indicated by Phenol Red

Tube 1

Orange - red

More Acidic than neutral

Tube 2

Red

Alkaline

Tube 3

Pink

Denatured (pH above 10)

Results for Tube 1 indicate that the enzyme is more acidic than neutral because bile salts were added to the enzyme solution. Tube 2 indicates that it is alkaline, with distilled water, the pancreatic solution and 0.8% NaOH. Tube 3 is denatured because bile salts were added and it was boiled.

Part B

The enzyme tested in part B were pancreatic proteases, which include Trypsin, Chymotrypsin, and Carboxypeptidase. The proper substrates for this part are polypeptides. The products formed are smaller polypeptides and amino acids. All of this takes place in the lumen of the small intestine and the optimum pH range is 7.1 to 8.2. (Campbell et al. 2008)

A coagulation test for proteins was preformed which was done by boiling the tubes for 5 minutes after incubation, and observing any precipitate. Flocculation indicates protein digestion, and large, chunky pieces indicated that the protein was not digested.

Biuret is used to indicate a positive or negative reaction. A positive reaction is indicated by a violet color and would also indicate the presence of proteins for a dark violet color, and the presence of polypeptides is indicated by a light violet color. A negative reaction is indicated by a blue color.

Protein Digestion with Pancreatic Proteases - Test for COAGULATION

Observations (Type of precipitate)

Results (Digestion or not)

Tube 4

None

No digestion

Tube 5

Chunky

No digestion

Tube 6

Flocculation

Digestion

Tube 4 shows no precipitate, which indicates no digestion. This happened because distilled water was used instead of albumin solution in the tube. This was our negative control. Tube 5 shows chunky precipitate which indicates no digestion. This happened because the pH was 4, which is not the proper range for this enzyme to work properly. Tube 6 shows flocculation which indicates digestion. This happened because the pH and substrate where both correct for digestion.

Protein Digestion with Pancreatic Proteases - Biuret Test

Observations (Color)

Results (Positive or Negative)

Tube 4'

Blue

Negative

Tube 5'

Dark Violet

Positive for proteins

Tube 6'

Light Violet

Positive for polypeptides

Tube 4' remained blue after addition of the Biuret, which indicates a negative result, which again happened distilled water was used instead of the albumin solution. This was our negative control. Tube 5' turns dark violet, indicating that it is positive for proteins. The results shown are a result of the improper pH of 4 being used in this tube and the enzyme being inactive. Tube 6' turns light violet, indicating that it is positive for polypeptides. This happened because all of the conditions were proper for the enzyme and substrate to work, including pH.

Part C

The enzyme tested in part C was pancreatic amylase. The proper substrates are polysaccharides. The products formed are maltose and other disaccharides. All of this takes place in the lumen of the small intestine and the optimum pH range is 7.1 to 8.2. (Campbell et al. 2008)

Lugol's test is used to indicate starch. A positive result would show a color of blue/black. Any other color would indicate a negative reaction.

Benedict's test is used to indicate reducing sugars. A positive reaction is indicated by a green, red or brick reddish/brownish color. A negative reaction would be a blue color.

Starch Digestion with Pancreatic Amylase - Lugol's Test for Starch

Observations (Color and/or precipitate)

Results (Positive or negative)

Tube 7

Blue

Positive

Tube 8

Blue/Black

Positive

Tube 9

Blue/Black

Positive

Tube 10

Clear, Yellow

Negative

Tube 7 turns blue after the addition of Lugol's which indicates a positive result, and the presence of starch. Although the pH is not proper in this tube, starch is still indicated. Tube 8 and 9 turns blue/black which also indicates a positive result. Tube 8's test however, was terminated at 30 minutes. This should have been a negative result between 20 to 22 minutes and it should have been clear or yellow. Tube 9's pH was too high but, did show starch. Tube 10 shows a negative result with a clear/yellow color because the wrong substrate was used.

Starch Digestion with Pancreatic Amylase - Benedict's Test for Reducing Sugars

Observations (Color and/or precipitate)

Results (Positive or negative)

Tube 7'

Brown

Positive

Tube 8'

Red-Orange

Positive

Tube 9'

Red-Orange

Positive

Tube 10'

Blue

Negative

Tube 7' shows a brown color, indicating reducing sugars however, it was not working properly and should have remained blue and been negative. The pH in tube 7' is too low, making the enzyme in active. Tube 8' and 9' show a red-orange color, indicating positive results for reducing sugars. Tube 8' has the proper pH and substrate, and tube 9' has the proper substrate but the pH is too high, resulting in the enzyme working partially. Tube 10' shows blue, indicating that it is negative for reducing sugars, which was expected because the proper substrate was not used.

Part D

The enzyme tested in part D was pepsin. The proper substrates are proteins. The products formed are small polypeptides. All of this takes place in the stomach and the proper pH range is 1.5 to 3.5.

A coagulation test for proteins was preformed, as well as Biuret, which were both explained above.

Protein Digestion with Pepsin - Test for Coagulation

Observations (Type of precipitate)

Results (Digestion or not)

Tube 11

Small Particles

Negative (Control)

Tube 12

Flocculation

Digestion

Tube 13

None

No digestion

Tube 11 is our negative control. The small particles are a false positive. Tube 12 had the proper pH, enzyme and substrate, resulting in flocculation, indicating digestion. Tube 13's pH was too high resulting in no precipitate and no digestion because of denaturing.

Protein Digestion with Pepsin - Biuret Test

Observations (Color)

Results (Positive or negative)

Tube 11'

Blue

Negative

Tube 12'

Light Violet

Positive for polypeptides

Tube 13'

Light Violet

Positive for polypeptides

Tube 11', being our negative control shows a negative result for the Biuret test. Tube 12' and 13' both show light violet color, being positive for polypeptides. The pH in 13 is too high but the enzyme works partially.

Part E

Salivary amylase is the enzyme tested in this part. This takes place in the oral cavity. The substrates are starch and glycogen. The products formed are smaller polysaccharides and maltose and the proper pH is 6.35 to 6.85.

Benedict's is used in this part, which was explained above.

Sucrose and Salivary Amylase

Observations (Color and/or precipitate)

Results (Positive or negative)

Analysis of Enzyme Action

Tube 15

Clear Blue

Negative

Did not work

Tube 15 did not work because the wrong substrate was used.

Starch Digestion with Salivary Amylase - Tube 16

Start Time: 1:45 End Time: 2:01 Actual Time: 16 minutes

Analysis: The salivary amylase works to completion

Lugol's Observation: Yellow Results: Negative

Benedict's Observation: Red/Brown Results: Positive

The starch digestion with salivary amylase in tube 16 works to completion, which is shown in our lab by the negative Lugol's and positive Benedict's tests. The pH and substrates were proper in this tube.

Starch Digestion with Salivary Amylase - Tube 17

Start time: 1:45 End Time: 2:45 Actual Time: terminated at 1 hour

Analysis: The salivary amylase works at a slower pace because

Lugol's Observation: Black Results: Positive

it was put in an ice bath. Given more time, it would work to

Benedict's Observation: Blue Results: Negative

completion. At this point it appears to not work.

Starch digestion with salivary amylase in tube 17 works partially because it was cooled in an ice bath, resulting in slower rate of reaction.

The six major constituents of bile are water, bilirubin, bile acids, cholesterol, phospholipids, and electrolytes. (Bowen 2001) Bile is produced in the liver and stored in the gallbladder. (Campbell et al. 2008) Bile salts function as a detergent or emulsifier for fats, virtually trapping them and making it easier for the body to rid itself of the fat. (Bowen 2006)

When an enzyme is boiled, the heat acts as a catalyst and speeds the reaction, therefore an ice bath (40 to 50 C) would deter the enzyme from working properly and would inhibit its reaction or make it inactive. This is evident from our lab procedure in which we boiled tube 3 and got a fast reaction, and put tube 17 in an ice bath, which delayed and inhibited results.

The zymogen for pepsin is pepsinogen and is activated when it comes in contact with acid, such as the hydrochloric acid of the stomach. (Bowen 1996) The zymogen for trypsin is trypsinogen and is activated by enterokinase which is inside the lining of the small intestine. (Bowen 2006) The zymogen for chymotrypsin is chymotrypsinogen which is activated by trypsin and trypsinogen. (Bowen 2006) The zymogen for carboxypeptidase is pro-carboxypeptidase A and is activated by trypsin and chymotrypsin. (Szmola et al. 2011)

Bolus - a ball of food that has been masticated and wadded up by the tongue for passage through the esophagus (Campbell et al. 2008)

Chyme - food once it has been partially digested in the stomach and enters into the small intestine (Campbell et al. 2008; Taber et al. 2005)

Colipase - "A small protein in pancreatic juice that is essential for the action of pancreatic lipase" (Medical-Dictionary 2007)

Ghrelin - a polypeptide produced in the stomach that aides in the release of the growth hormone from the pituitary gland, regulating appetite and weight (Taber et al. 2005)

Micelle - a ball of fat surrounded by bile salts within the small intestine (Taber et al.2005)

Negative control - a standard set up that lacks a reaction (Taber et al. 2005)

Parietal cells - "A large cell on the margin of the gastric glands of the stomach that secretes hydrochloric acid and intrinsic factor."(Taber et al. 2005)

Peristalsis - the involuntary movement of smooth muscle within the body to help move food along the digestive tract (Campbell et al. 2008)

Secretin - a hormone released by the duodenum which stimulates the pancreas and gallbladder to release bile and sodium bicarbonate (Taber et al. 2005)

Upon being ingested into the mouth, a piece of food is masticated by the teeth, increasing surface area for better digestion. As it is being masticated, it is also being moistened by saliva, and digestive enzymes are possibly starting to work on the food. During mastication, the tongue, which is a large muscle, is wadding the food into a bolus and pushing it to the back of the oral cavity, past the hard and soft palate and into the pharynx. Once swallowed, the bolus passes through the pharynx and over the epiglottis and trachea, moving down the esophagus which is a tube lined with smooth muscle, by means of peristalsis. At the cardiac sphincter, the opening of the stomach from the esophagus, the food enters the stomach where it is broken down by pepsin and hydrochloric acid, and it is also being broken into smaller pieces by the rugae. The food is now called chyme, as it is passing through the pyloric sphincter and into the duodenal portion of the small intestine. There it passes the pancreas, and gallbladder which release pancreatic proteases and bile to further break down the chyme. Inside the small intestine are villi which increase surface area for better absorption of nutrients. After the duodenal portion of the small intestine, the chyme enters the jejunum and then the ileum, the second and third portions of the small intestine. Here absorption of nutrients takes place. From the distal portion of the ileum, the chyme now enters the large intestine through the cecum, and passes by the appendix, an organ of unknown function. The chyme enters the ascending colon, travels through the transverse colon, and down the descending colon, then through the sigmoid colon. While in the colon, the water from the chyme is being reabsorbed. After the sigmoid colon, the waste product proceeds through the rectum and out the anal opening. Bacteria are also contained within the small and large intestine which aid in the digestion process. (Campbell et al. 2008; Your Digestive 2008)

Literature Cited

Bowen, R "Pepsinogens and Pepsins." Arbl.cvmbs.colostate.edu. 23 Nov. 1996. Web. 05 Mar. 2011. <http://www.vivo.colostate.edu/hbooks/pathphys/digestion/stomach/pepsin.html>.

Bowen, R "Secretion of Bile and the Role of Bile Acids In Digestion." Arbl.cvmbs.colostate.edu. 23 Nov. 2001. Web. 05 Mar. 2011. <http://www.vivo.colostate.edu/hbooks/pathphys/digestion/liver/bile.html>.

Campbell NA, Reece JB, Urry LA, Cain ML, Wasserman SA, Minorsky PV, Jackson RB. 2008. Biology, Eighth Edition. San Francisco: Pearson Education, Inc. Pp 884-890

Szmola, R., M. Bence, A. Szabo, CE Costello, J. Samuelson, and M. Sahin-Toth. "Chymotrypsin C Is a Co-activator of Human Pancreatic Procarboxypeptidases A1 and A2." Chymotrypsin C Is a Co-activator of Human Pancreatic Procarboxypeptidases A1 and A2. 21 Jan. 2011. Web. 05 Mar. 2011. <http://www.ncbi.nlm.nih.gov/pubmed/21098023>.

Taber, CW, and Thoms, CL Taber's: Cyclopedic Medical Dictionary. 21st ed. Philadelphia: F.A. Davis, 2005. Print.

"Your Digestive System and How It Works." National Digestive Diseases Information Clearinghouse. NIH Publication No. 08-2681, Apr. 2008. Web. 05 Mar. 2011. <http://digestive.niddk.nih.gov/ddiseases/pubs/yrdd/index.htm>.

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