Unit 13

Unit 13: Biochemistry and Biochemical Techniques
Assignment title: Enzymes
Name: Hani Oladeinde
Student I.D: OLA17005525
Task 1
Experiment 1: The effects of Temperature on Enzyme Activity
Materials:
Test tubes
Test tube holder
Graduated pipette
Egg white suspension
pH 2 buffer solution
Water baths
Pepsin
Indicator paper
Dropping pipette
Method:
Label 5 test tubes 1-5.

Using a graduated pipette place 3cm3 of egg white suspension into each of the test tubes.

Using a clean graduated pipette place 3cm3 of the pH 2 buffer solution to each test tube.

Leave test tube 1 in the test tube holder and place test tube 2-5 in water baths with 30-60oC respectively for five minutes.

After 5 minutes remove the test tubes 2-5 from the water baths.

Using the 1ml graduated pipette place 1ml of pepsin into each of the test tubes 1-5 and place the test tubes 2-5 into their respective water baths immediately and leave for five minutes
Remove the test tubes from the water baths after the 5 minutes have elapsed and record their appearance in the table below.

Use indicator paper to test the pH of each sample using a dropping pipette and record the pH.Errors:
Other students took my test tube samples so this effect my results because everyone had different amounts in their test tube. To avoid this error, label the test tubes with your name on it or put your samples in separate water baths from other samples.

Reading the measurement from the graduated pipette was hard to see so this affect my results. This error can be avoided by having two or more people reading the graduated pipette.

I didn’t use a clean graduated pipette for the different substances that I used so this affected my results because it cross-contaminated the sample. And to avoid this error, you must use clean graduated pipettes for each substance that you are measuring.

Results:
Tube number Temperature (oC) pH The appearance of tube contents after 5 minutes
1 20 4 Cloudy
2 30 5 Cloudy
3 40 5 Cloudy
4 50 10 Cloudy
5 60 4 Cloudy
Test tube 2 and 3 appearances should have been clear because enzymes rate of reaction is faster between 30oC and 40oC. Also test tubes 4 and 5 appearances should have been clear because enzymes denature after 40oC. Nevertheless, all five test tubes turned cloudy but there were no changes because of the above errors.

Conclusion:
The temperature affects the enzyme pepsin rate of reaction. It affects the enzyme activity however human input affected the quality of the results. Redoing the experiment may be needed to find what the optimum temperature is on a more precise range.

Experiment 2: The effect of pH on the reaction between pepsin and egg white
Materials:
Test tubes
Test tube holder
Graduated pipette
Egg white suspension
Acid or alkali
Water baths
Pepsin
Indicator paper
Dropping pipette
Stopwatch
Universal indicator
Method:
Label 5 test tubes 1-5.

Using a graduated pipette place 3cm3 of egg white suspension into each of the test tubes.

Using a graduated pipette, add acid or alkali to the test tubes as indicated in the results table below.

Using a graduated pipette, add 1cm3 of 1% pepsin solution to each test tube.

Place all five of the test tubes into the water bath at 40oC and start the timer on the stopwatch.

After five minutes, remove the test tubes from the water bath and record the appearance of the samples.

Using a dropping pipette, take a sample from test tube 1. Place a small drop onto the indicator paper and compare the colour produced to a universal indicator. Note down the pH of the solution in test tube 1 in the table below. Repeat the procedure for test tubes 2-5 making sure you use a clean dropping pipette between solutions.

Errors:
Other students took my test tube samples so this effect my results because everyone had different amounts in their test tube. To avoid this error, label the test tubes with your name on it or put your samples in separate water baths from other samples.

Reading the measurement from the graduated pipette was hard to see so this affect my results. This error can be avoided by having two or more people reading the graduated pipette.

I didn’t use a clean graduated pipette for the different substances that I used so this affected my results because it cross-contaminated the sample. And to avoid this error, you must use clean graduated pipettes for each substance that you are measuring.

Results:
Tube number Egg white suspension and pepsin plus: pH The appearance of contents after five minutes
1 1.5cm3 of sodium carbonate solution 4 Cloudy
2 0.5cm3 of sodium carbonate solution 5 Cloudy
3 Nothing 6 Cloudy
4 0.5cm3 of hydrochloric acid 9 Clear
5 1.5cm3 of hydrochloric acid 10 Clear
Test tubes 1 and 2 appearances should have been clear as enzyme pepsin works best at low pH values which are the optimum condition. In test tube 4 and 5 the appearance should have been cloudy because enzyme pepsin denatures at high pH values. However, for all test tubes 1, 2, 4 and 5 turned out to be the opposite because of errors.

Conclusion:
The pH affects the enzyme pepsin rate of reaction. It affects the enzyme activity as the pH increases however human input affected the quality of the results. Redoing the experiment may be needed to find what the optimum pH is on a more precise range.

Every enzyme has a temperature at which it works fastest. For most human enzymes this is about 37.5oC (optimum temperature). If the temperature is increased up to this optimum value then the rate of the reaction it controls also increases. The temperature increase means that the substrate and enzyme molecules have more energy so are more likely to collide with each other. More molecules also have the minimum amount of energy that is needed for a reaction to occur upon collision is called the activation energy. Below the optimum temperature enzyme and substrate molecules have less energy so fewer collisions occur and the rate of reaction is slower. Above the optimum temperature, the rate of reaction also decreases. The heat energy breaks the hydrogen bonds which form the active site and loses its shape. The substrate can’t fit into the active site so the reaction can’t be catalysed. At very high temperatures this change is permanent. When this change in shape happens the enzyme is said to be denatured.

Enzymes also have an optimum pH at which they work fastest which increases the rate of reaction. This is about pH 7-8 (optimum pH). Some enzymes can work at more extreme pH values such as protease enzymes in the stomach. These need to work in acidic conditions so they have an optimum pH of 1. Enzymes’ tertiary structure is fixed in position by H bonds and ionic bonds. Hydrogen ions have a + charge and are drawn to negatively charged sets on amino acids affecting with the H bonds and ionic bonds that clutch the tertiary structure and the active site in position. The pH affects the charge on the amino acids that make the active site on the enzyme. If the pH value is above or below the optimum this will quickly decrease the rate of reaction. If the pH changes then the charges on the active site change and the substrate may no longer be attracted to the active site. For example, an amino acid containing a carboxyl group will be uncharged at low pH (COOH) but charged at high pH (COO-). So a substrate with a positive charge can’t be made to react quickly at a low pH. At very acidic and very alkaline conditions an enzyme can be denatured. Also at very minimal pH quantities, this restriction triggers the enzyme to unroll the form of the active site can’t match to the substrate anymore and the reaction can’t be catalysed by the enzyme. This also denatures the enzyme.