The Effect of Amylase Concentration and pH Levels on Time of Starch Disappearance With an Additional focus on Carboxypeptidase By: Pauline Hickey, Amanda.

The Effect of Amylase Concentration and pH Levels on Time of Starch Disappearance

With an Additional focus on

Carboxypeptidase

By: Pauline Hickey, Amanda Clark, Peter Crane, Anya Novikova

Carboxypeptidase (1)

Amylase

Results Section

• In both scatter plots, the time required to digest starch decreased as the concentration of amylase increased.

• Both data bases (our/class) show that there was an inverse relationship between the two variables.

Our Results Table

Percent Concentration of Amylase

Time of Starch Disappearance (in seconds)

0.5 10

0.25 20

0.125 50

0.075 60

Class Average Results Table

Percent Concentration of Amylase

Time of Starch Disappearance (in seconds)

0.5 16.6

0.25 53.6

0.125 81.6

0.075 223.6

• Both graphs show that more time was needed to digest starch at the highest and lowest pHs.

• The graphs form a V shape that visualize how the rate digestion was more rapid towards the middle pH of 6.8 and then slowed down again.

Our Results Table

The pH Time of Starch Disappearance (in minutes)

2 9

4 5

6.8 0.5

8 2

10 9

Class Average Results Table

The pH Time of Starch Disappearance (in minutes)

2 9

4 5

6.8 0.47 (28 seconds)

8 1.83 (1 minute 50 seconds)

10 8

Discussion Section

Hypothesis #1

• As the concentration of the enzyme Amylase increases, the rate of starch disappearance increases.

• A.K.A. The rate of starch disappearance is directly proportional to the concentration of Amylase acting on it.

Supported?

• Hypothesis WAS SUPPORTED, as seen in graph 1 the time of starch disappearance increases relatively steadily as the dilution/concentration of amylase decreased from .50 to .075

• A.K.A The idea that amylase helps to digest starch is supported. Also, when there is more (higher concentration) of amylase, starch will be broken down more quickly.

Supporting Background Info

• Supporting background info: “It [amylase] catalyzes the breakdown of starch. When amylase reacts with starch, it cuts off the disaccharide maltose (two glucose molecules linked together). As the reaction progresses, less starch will be present and more sugar (maltose) will be present.” (2)

• This helps explain why starch breakdown begins in the mouth; amylase is found in human saliva, so as soon as a starchy food like a potato enters the oral cavity and interacts with saliva, amylase begins to break it down into sugar.

• Amylase is also secreted by the pancreas to continue starch digestion in the stomach. (3)

(4)

Hypothesis #2

• Amylase will be most active at a small, middle range of pH (6-8), and will not be active at too high or too low a pH.

• A.K.A: Starch will disappear most quickly at a middle pH range (6-8) and will take much longer to disappear at too high or too low a pH.

Supported?

• Hypothesis WAS SUPPORTED. As seen in graph 2, starch disappeared most quickly (only 30 seconds) at a middle-range pH of 6.8.

• The starch disappeared most slowly (around 9 minutes or more) at the extreme lows (2-strongly acidic) and extreme highs (10.0-strongly basic) pH levels.

Background Info

• The effective range of pH was hypothesized to be within 6 and 8 because the body consists largely of water with a pH of 7, and the different bodily fluids largely range between 6 and 7.4. (5)

Sources of Error

• Graduated cylinders, like most forms of measurement, have some degree of error, so what looked like 5mL measured out may have varied slightly and may have caused the time of disappearance to go from 10 to 20 to 50 instead of 10 to 20 to 40- which would directly correspond to the decreasing concentration of amylase from .5 to .25 to .125.

• When the dilution was .075, the blue color continued to be slightly apparent even after 60 seconds but the lack of spaces in the tray did not allow for the experiment to continue.

• When measuring the amylase in cleaned-out graduated cylinders, some water droplets may have been leftover and could have slightly diluted the amylase more than it was supposed to be diluted, which could have lengthened the time of starch disappearance, possibly accounting for the jump from 16.6 to 53.6 seconds instead of the predicted proportional increase of approximately 16 to around 32 or 33 seconds.

What would happen at…

80 °C?

• Amylase becomes “denatured…a major change from the native state to another state without changing the primary structure. ” (6)

• The speed of the atoms and the strength of their collisions increase, which can damage the molecular structure- sometimes irreversibly. (6)

37°C.?

• This is the average body temperature and therefore the most optimal environment for amylase to react with starch.

22 °C.?

• This is about room temperature, which relatively cold in terms of amylase. This colder temperature, however has not been observed to greatly affect the reaction of amylase with starch. (6,7)

• Some theories suggest that amylase evolved to be able to survive in colder temperatures. It is believed that those enzymes which could not stand the cold died off, allowing for the more temperature-resistant molecules to survive. This did not occur with extremely hot temperatures because such temperatures are rarely found in any climate on earth.(6)

4°C.?

• Even at this somewhat extreme cold, amylase continues to react effectively with starch.(6)

• It is believed that amylase evolved to be able to function at such low temperatures due to naturally-occurring cold climates. (6)

Digestive Application: Carboxypeptidase

Carboxypeptidase: Source, Location of Enzyme Activity and Substrates

• The source of this digestive enzyme is the pancreas 4

• The location of the enzyme’s activity: small intestine 4

• Substrates for carboxypeptidase are protein fragments 5

What is Produced From This Hydrolysis Reaction?

• In order to break down a protein (polymer) into amino acids (monomers), cells use a hydrolysis reaction a protein reacts with a water molecule to produce an amino acid and a protein 7

• Carboxypeptidase is secreted by the pancreas, and speeds up the hydrolysis reaction between the protein and the water molecule 8

• The main products of this hydrolysis reaction are amino acids which are then small enough to be absorbed by the villi in the small intestine7

What Factors Effect Enzyme Activity?

• As the pH level changes in the small intestine and gradually becomes basic, more enzymes are activated further that chemically break down various nutrients into smaller molecules to allow absorption 10

• The ideal pH of the duodenum is 6 to 6.5 11

• The ideal pH of the jejunum and illium is 7.8

pH Levels in the Digestive System

Digestive enzymes that are located in the duodenum are most active at a pH of 6 8

Digestive enzymes that are located in the jejunum and illium are most active at a pH of 7.8 8

pH’s Effect on Carboxypeptidase’s Activity

7.8

100%

Temperature’s Effect on Carboxypeptidase’s Activity

• The temperature of the small intestine is approximately 37 degrees Celcius7

• Small fluctuations in temperature change will not effect carboxypeptidase’s activity9

• However, a big increase or decrease in temperature will decrease the amount of activity by carboxypeptidase11

Effect of Temperature on Carboxypeptidase’s Activity

References

1. http://www.chemistry.wustl.edu/~edudev/LabTutorials/Carboxypeptidase/images/cpep.jpg2. “Laboratory #5: Enzymes,” http://www.und.nodak.edu, October 19 2010,

http://www.und.nodak.edu/dept/jcarmich/101lab/lab6/lab6.html3. “Amylase,” wikipedia.ord, October 19 2010, http://en.wikipedia.org/wiki/Amylase4. http://www.istockphoto.com/file_thumbview_approve/4643127/2/istockphoto_4643127-starchy-food.jpg5. “What is the Normal pH of Blood, Urine and Saliva?”, www.enotes.com, October 19 2010, http

://www.enotes.com/science-fact-finder/human-body/what-normal-ph-blood-urine-saliva 6. “Amylase Enzyme: The Effects Of Temperature,” www.allsands.com, October 18 2010,http

://www.allsands.com/science/amylaseenzymeh_wpp_gn.htm7. “The Effect That Temperature Has on Enzyme Activity,” www.docshare.com, October 17 2010,http://www

.docshare.com/doc/163073/The-effect-that-temperature-has-on-enzyme-act8. Starr, Cecie and Ralph Taggart. Biology: The Unity and Diversity of Life. Pacific Grove, CA: Brooks/Cole, 2001.9. Casiday, Rachel and Regina Frey. “Enzyme-Substrate Binding: Carboxypeptidase,”

http://www.chemistry.wustl.edu October 18 2010, http://www.chemistry.wustl.edu/~edudev/LabTutorials/Carboxypeptidase/carboxypeptidase.html

10. Winsemius, David. “What are the pH Levels in the Stomach During a Titration of Saliva and Food?” http://madsci.org October 18, 2010, "http://madsci.org/posts/archives/may98/892927858.Ch.r.html" http://madsci.org/posts/archives/may98/892927858.Ch.r.html

11. Boudinot, Sally. “Anatomy of the Gastrointestinal Tract and Drug Absorption.” www.chemcases.com, October 19 2010, http://www.chemcases.com/pheno/pheno14.htm

12. “Temperature of a Healthy Human Body,” www.hypertextbook.com, October 19 2010, http://hypertextbook.com/facts/LenaWong.shtml