biology
BIOLOGY 211 LA-4 Enzymes Page 2 of 2
Effect of temperature on the activity of turnip peroxidase
(If you are struggling with any of the questions, I recommend thoroughly reading through the first two pages of Enzyme Lab introduction.)
Question: How does temperature affect the absorption of an enzyme?
Null hypothesis (avoid using the word “will” in your hypothesis): Enzyme activity is not affected by temperature.
(If you need help with hypothesis or independent and dependent variables, I suggest reviewing background information from Diffusion Lab.)
In this experiment, what are the independent and dependent variables?
independent: temperature of the solution
dependent: reaction rate (light absorption)
What is the name of the enzyme in this enzyme-mediated reaction? Peroxidase
What is the substrate molecule in this enzyme-mediated reaction? ________ Hydrogen peroxide ________
What is/are the product(s) produced in this enzyme-mediated reaction? H2O & O2
Which tube served as a negative control? ______6____________________
How do you know it is a negative control? 6 is missing substrate in both 6a and 6b.
Paste in your graph below:
Do the data support the null hypothesis? Explain, using the data (numbers) you obtained to support your conclusion.
The data does not support the null hypothesis because the temperature was a determining factor of light absorption. When the temperature was 32 °C, the light absorption peaked at 200 nm (0.4*500 nm), and decreased at a steady incline after that, dropping to 50 nm at 65 °C, and denaturing at 100 °C. This shows that there was a peak temperature that aided in enzyme absorbance. In addition, the light absorption before this peak temperature was low when it was a colder temperature, reading at about 50 nm at 4 °C. This shows that when it is too cold or too hot, the light absorption is affected, proving that temperature is a determining factor of enzyme reaction rate, which is measured through light absorption.
Of those tested, what is the optimum temperature for turnip peroxidase? ______32 °C _________
Why is activity lower at other temperatures? The activity at very high temperatures is affected because the enzyme is denatured due to the breaking of bonds which cannot be repaired, which alters the shape of the protein. Enzymes have a desired temperature range and when it is too cold, the shape tightens, and this makes it harder to perform proper functions. When it is too hot, the shape of the enzyme loosens. This affects how easily the enzymes perform their function and will slow down the process. At lower temperatures, the enzyme reaction rate is also decreased, and less light is shown to be absorbed. This is because there is less kinetic energy when the temperature is colder, and when the particles collide there will be less activation energy, or reaction rate.
Effect of pH on the activity of turnip peroxidase
Question: When the pH is very acidic or alkaline will this negatively affect the productivity of the enzymes?
Null hypothesis (avoid using the word “will” in your hypothesis): pH is not a determining factor of enzyme functionality.
In this experiment, what are the independent and dependent variables?
independent: pH
dependent: reaction rate (color of the solution)
Which tube served as a negative control? ______tube 5____________________
Which tube served as a positive control? (which would you to predict to have optimal pH for turnip peroxidase)
_________ 2_________________
Paste in your graph below:
*Note: The top of the graph shows pH 9 at 0, it is not left blank.
Do the data support the null hypothesis? Explain, using the data (numbers) you obtained to substantiate your conclusion.
The data does not support the null hypothesis since there was no reaction at very low and at higher pH levels. Since the color determined whether a reaction occurred or not, we can see that at pH 3, there was some reaction, but much lower than the others. This shows that the reaction was a lot weaker since it was 0.1 and therefore it was affected by pH, but still possible. We can see that the peak pH level is next for this reaction, measured at pH 5 as 0.4 which proves that there is an optimum pH level, hence pH is a determining factor. The next best recorded pH level was 7, where the color of the reaction was 0.25, which is better than the reaction at pH 3, showing that the reaction is best in an acidic environment more towards the neutral spectrum (7 pH). Finally, we see an alkaline solution, pH 9, which has no color, showing that there was no reaction. This proves that a basic solution is not viable for a reaction with turnip peroxidase. Thus, pH is a determining factor for enzyme reactions and whether or not they function properly.
Of those tested, what is the optimum pH for turnip peroxidase? _____ pH 5__________
Why is activity lower at other pH values?
Activity is lower at other pH values because enzymes have an ideal pH level to function properly. A protein may denature by pH sticking to polar amino acids. In addition, the enzymes will slow down and eventually stop in non-ideal pH values.
EXPERIMENT PROPOSAL
Question: How does salt concentration affect the absorbance of an enzyme?
independent: Salt
dependent: reaction rate (color of the solution)
Null hypothesis: Reactivity of enzymes does not decrease as salt concentration increases
Materials:
· Test tubes
· Micropipette
· Spectrophotometer
· pH buffer
· Turnip peroxidase (enzyme)
· Guaiacol (indicator)
· Hydrogen Peroxide (substrate)
· NaCl solution (0%, 5%, 10%, 15%)
Methods: Make a control group with everything but the NaCl. Then, add 0.5 mL of NaCl (different percentages) in each tube, plus the same solution that is in the control tube. Mix and put the tubes by the spectrophotometer at 500 nm and record absorbance after 2 minutes.
|
Sodium chloride percentage |
0% |
5% |
10% |
15% |
20% |
|
Color at 2 minutes |
0.4 |
0.25 |
0.20 |
0.15 |
0.1 |
Graph:
Results: The NaCl caused a decrease in enzymatic absorption. The absorbance peak was at 0% and was lowest at 20% NaCl, and as the sodium chloride decreases as more sodium is added this shows that NaCl hinders the reaction rate of the peroxidase. This is shown from a decrease in the color, which measures the enzymatic reactivity.
How Temperature Affects the Absorption of Light Which Measures Reaction Rate
Absorbance 4 23 32 65 100 0.1 0.25 0.4 0.1 0
Temperature (°C)
Absorbance (500 nm)
How pH Affects the Color of the Solution to Determine Whether a Reaction Occured
Series 1 3 5 7 9 0.1 0.4 0.25 0
pH level
Color at 2 Minutes
How Sodium Chloride Concentration Affects the Color of the Solution Indicating Reaction Rate
Series 1 0 0.05 0.1 0.15 0.2 0.4 0.25 0.2 0.15 0.1
Sodium Chloride Concentration
Color at 2 Minutes