How does Ph level and NaCl impact the effectivness of the en

How Does Salinity Impact the Effectiveness of the enzyme, Peroxadase.

Materials & Methods

Methods: Procedure 2- pH

Materials pH:

1) Collect all materials

2) Prepare all of the enzyme tubes by putting all these chemicals into a test tube

a) 6 mL of distilled water

b) 1.5 mL of peroxidase

3) Prepare the substrate test tubes

a) add 0.2 mL guaiacol

b) add 0.3 mL Hydrogen Peroxidec (H202)

c) 7 ml of the pH 2 chemical

4) repeat step 3 with the various levels of PH

5) Cut out the 6 parafilm squares and take the wrapper off

6) combine all the solutions into the enzyme test tubes as quickly as possible and put the parafilm on top

7) Invert the test tubes twice

8)Start your stop watch

9) At 0,1,2,3,4, and 5 minutes compare each of the test tubes to the chart to match the color and write down the results on a table

10) clean all the testubes with dish soap and return the materials to the correct spot

• Turnip Peroxide
• 0.1% hydrogen peroxide
• Guaiacol
• Distilled (deionized) water
• 12 test tubes (approximately 16 x 150 mm)
• Appropriate test tube rack
• Timer
• 1, 5, and 10 mL graduated pipettes, pipette pumps or syringes
• Buffers with range of pH

Method: Extension- Sea Salt (NaCl)

Materials: NaCl

1) Collect all materials

2) Make all 4 of the sea salt and water solutions **

a) 1%- 9.9 grams of water and 0.10 grams of sea salt

b) 2%- 9.8 grams of water and 0.20 grams of sea salt

c) 5%- 9.5 grams of water and 0.50 grams of sea salt

d) 10%- 9 grams of water and 1 grams of sea salt

3) Prepare all 4 of the enzyme test tubes

a) 6 mL of distilled water

b) 1.5 mL of peroxidase

4) Prepare all 4 of the substrate test tubes

a) 0.2 mL of guaiacol

b) 0.3 mL of hydrogen peroxide (H2O2)

c) 7 mL of sea salt solution

5) Cut 4 squares of Parafilm

6) Combine the substrate with the enzyme tubes for all 4 pairs

7) Cover with Parafilm.

8) Invert twice, and place back onto the test tube rack.

9) Immediately start timing the reactions.

10) Record and observe the color of each tube from 0 to 5 minutes. Refer to the color chart and write down the results on a table following each minute (0,1,2,3,4,5).

Optional- continue to to watch if color changes.

• Turnip Peroxide
• 0.1% hydrogen peroxide
• Guaiacol
• Distilled (deionized) water
• 8 test tubes (approximately 16 x 150 mm)
• Appropriate test tube rack
• Timer
• 1, 5, and 10 mL graduated pipettes, pipette pumps or syringes
• Sea Salt (2 grams) **

**= specific altercations to the lab which we designed

Salt Concentration Lab

pH Lab

-Independent Variable-

Time (Minutes)/

pH

Time (Minutes)/ Salt

Color Change (Amount of Oxygen

Produced)/

effectiveness of the enzyme

Color Change (Amount of Oxygen Produced)/ effectiveness of the enzyme

-Dependent Variable-

Discussion

Results

The graphs show that both pH and Salt are inhibitors to the enzyme Peroxadase. As shown through graph 1, the enzyme works best at a pH closer to 7. This is due to the fact that the enzyme works best in water and the farther you get from pH 7 the less effective the enzyme is and the closer the enzyme gets to becoming denatured as shown through the ph of 10. Then for the salt lab, one observes through graph 2 that the higher the salt conenctration the less oxygen produced/ the less effective the enzyme is which coincides with our background information that salt is an inhibitor to the enzyme Peroxadase.

Conclusion

PH

For the second hypothesis,The more amount of potential hydrogen the more Oxygen will be produced in the same amount of time; while extreme levels of pH will not yield any results, this was dissproved. The data shows that the enzyme Peroxadase works best at a pH level closer to 7, At the data point of 2 minutes with a pH of 10 the color change is only 1 versus with a pH of 2 the color change is 6 but for the pH of 6 the color change is 8. The data shows that the farther the enzyme gets from a pH of 7 the less effective the enzyme is and the closer it becomes to being denatured as it is at a pH of 10. Due to the fact that the baseline with no denaturing was approximately 10 after 5 minutes it what is expected with otu any denaturing. Another factor that could also inhibit the enzyme is salt, which is what the extension is on.

NaCl

(graph 2)

(graph 1)

The extension was successful because the results supported previous research and the hypothesis. According to the hypothesis, the farther we get away from 1% of salt concentration, around blood level, the more denatured the enzyme will become, was supported by the data. The color change at 3 minutes with a salt concentration of 5% was 6 versus a salt concentration of 1%, which was at 9. The data shows that the more salt concentration in a solution the more the enzyme Peroxidase is inhibited. This is due to the fact that with no salt to inhibit you expect a 10 for color change but, the salt inhibits the enzyme to work. This experiment was a success because the group was able to identify another inhibitor to the enzyme Peroxidase and the group was able to support the standing theory regarding Peroxidase.

- (table 1)

(table 2)-

The Baseline (ph 7 & salt conetration 0) is different from the other data points because there was no denaturing and it was in the optimum environment for the enzyme to work.

Introduction

Questions

Hypothesis

1) How does Hydrogen Peroxide interact with the enzyme Peroxidase?

2) How does the pH level of a liquid impact the amount of oxygen produced and how quickly?

3) How does NaCl (salt) impact the effectivness of the enzyme Peroxidase?

1) The color of the water will change to about a 7 and need substantial time.

2) The more amount of potential hydrogen the more Oxygen will be produced in the same amount of time; while extreme levels of pH will not yield any results.

3) The farther we get away from 1% of salt concentration, around blood level, the more denatured the enzyme will become.

Background

A catalyst is a substance that increases the rate of a chemical reaction without itself undergoing a chemical change. An enzyme is typically a protein in a bulbous shape with a specific three dimensional shape. On the enzyme there is something called an active site, this area allows certain substances to attach and create a specific reaction as they catalyze (2). The active site of an enzyme is very selective, which means only certain substances can bind to the site. If the enzyme’s shape is changed in anyway, or the protein is denatured, then the acitve site has also chaged its shape. The previously fitting substance will then no longer bind with the enzyme at the active site, hence disrupting enzymatic functions (3).

Since enzymes are catalysts of biological systems, they speed up chemical reactions used by cells without permanently changing their shape and they can be used over and over again. Enzymes are needed or cellular reactions for life would not work fast enough to keep the organism alive and functioning (1).

Peroxidase is an enzyme that can be found in a wide variety of organisms, from plants to humans to bacteria. It specifically breaks apart hydrogen peroxide (H2O2), which is one of the toxins produced as a byproduct of using oxygen for respiration.

2 H2O2 + peroxidase ---> 2 H2O + O2 + peroxidase

As peroxidase breaks down hydrogen peroxide water and oxygen gas is formed (1).

The hydrogen peroxide latches onto an active site of the peroxidase enzyme. There it will catalyze and break down into water and oxygen (gas). As it breaks down into water and oxygen, it will leave the active site and a new hydrogen peroxide will latch onto the active site (Martin?). Because the scale of this reaction is very small and not visible to the human eye, an indicator like guaiacol is used. Guaiacol has a high affinity for oxygen, which is easily available after hydrogen peroxide and peroxidase interact, and when guaiacol is instantly binded with oxygen it forms tetraguaiacol, which is a brown color. The more oxygen there is the more darker brown of a color the solution will become. (3)

pH is used to measure the acidity and bascity of a solution, and it is also a measure of Hydrogen Ion (H+) concentration. Thus it is a good indicator for Hydroxide Ion (OH-) concentration. pH ranges from a pH of 1 to a pH of 14. A pH of 7 is considered “neutral” and deionised water has a pH value of 7. Acids have pH values below 7, and basic solutions have a higher pH value than 7. The lower the pH value the higher the H+ concentrations and lower the OH- concentrations, vice versa for the higher the pH value. (4)

Because H+ and OH- ions have a charge, they interfere wiht hydrogen and ionic bonds that hold enzymes together. The bonds and the ions will be attracted or repelled due to the charges that were created by the bonds. This causes a change in the shape of the enzyme, the protein is now denatured, and the active site is no longer able to do its original function. Enzymes have different optimum pH values. The optimum pH value is the value at which the bonds within the enzyme shape their active site to have the best fit to the shape of the substrate. At optimum pH the rate of reaction is at its optimum as well. On average, enzymes function best in pH levels of 5 to 8. Any change in pH affects the reaction rate of the enzyme and its substrate by changing the shape of the enzyme. (4)

Salt concentrations also affects an enzymes function. Just like there is an optimum pH value for an enzyme to function the best, enzymes also have an optimum amount of salt it needs to have the fastes reaction rate. Changes in salinty adds or removes cations (positive charge) and anions (negative charge) and affects the R-group bonding of amino acids. Also like pH, these charges will cause bonds and ions to be either attracted or repelled. The shape will change, which ultimately changes the active site and the enzyme can no longer function. Lower salt concentrations lead to the charged amino acid side chains of the enzyme molecule to attract to each other. This enzyme will then denature and no longer be able to bind with substances. On the other hand, if the salt concentrations are too high, the normal interaction will not occur and a different interaction will occur before the enzyme percipiates. (5)

Optimum level of salt for a enzyme to function is around 0.9%. The salt concentration in human blood is also around 0.9%. (5) We chose to use salt because we know conditions aren’t always optimum, and we wanted to see how different concentrations affect the effectiveness of an enzyme. (Especially considering salt is such a common commodity)

Refrences/ Bibliography

1) Whitson, Mary Kathryn, Dr. "Enzymes." Enzymes. North Kentucky University, n.d. Web. 21 Oct. 2016. <http://www.nku.edu/~whitsonma/Bio150LSite/Lab%2011%20Enzymes/Bio150LEnzymes.html>.

2) Kareska, Susan (2009) "Factors A ecting Hydrogen Peroxidase Activity," ESSAI: Vol. 7, Article 27. Available at: <http://dc.cod.edu/cgi/viewcontent.cgi?article=1122&context=essai>.

3) "Big Idea." Dictionary of Marketing Communications (n.d.): S153-160. Media.collegeboard. College Board. Web. 21 Oct. 2016. <http://media.collegeboard.com/digitalServices/pdf/ap/bio-manual/Bio_Lab13-EnzymeActivity.pdf>.

4) Adam-Day, Sam. "Factors Affecting Enzyme Activity." A Level Notes. Sam Adam-Day, n.d. Web. 21 Oct. 2016. <https://alevelnotes.com/Factors-affecting-Enzyme-Activity/146>.

5) "Enzyme Catalysis." Enzyme Catalysis. AP Lab Page, n.d. Web. 21 Oct. 2016. <http://www.biologyjunction.com/enzyme_catalysis.htm>.

By,

Sky Choi &

LJ Kolodge