Enzyme Lab Write Up

Procedure 2: Determining the Effect of pH on Enzymatic Activity

Research Question: What do you predict will occur if the pH in the reaction changes?

Hypothesis: If the pH in the reaction changes it will have an effect on the enzyme’s rate of reaction. The color change and speed of the reaction will be affected by the different pH buffers. I predict that pH 7 will be the most efficient pH solution (the neutral pH we used in procedure 1 as the baseline), while the pH buffers that are both lower (more acidic) and higher (more alkalinity) will begin to denature the enzyme and the enzyme will no longer work sufficiently.

Procedure:

• Create test tubes of the identical solutions from procedure 1 of the lab replacing the distilled water with different pH buffers in the enzyme mixtures, while keeping the water in the substrate mixture the same. We will be testing pH levels 3, 5, 7, 8, and 10. The control group will be the neutral buffer pH 7 (baseline from procedure 1). The constants are the amounts of guaiacol, hydrogen peroxide, water in the substrate tubes, and turnip peroxidase.  
• Mix the new enzyme solutions with the substrate solutions to create a 15 mL mixture. Use results from procedure 1 to determine the time set and record for procedure 2. We will record results of the color change every 20 seconds for two minutes.  
• Form conclusions based on the color change over time for the different pH solutions.

Claim: The enzyme proved to be most efficient in the ph 7 solution while the pH buffers that were both lower (3 and 5) and higher (8 and 10) began to denature the enzyme and the enzyme did not work as well under these conditions. The pH of the solution has an effect on the enzyme’s rate of reaction.

Evidence:

Relative Color Change of Solutions with Different pH Buffers

pH	Time	20s	40s	60s	80s	100s	120s
3		1	1	1	2	2	2
5		1	2	3	4	5	6
7		2	3	4.5	5.5	6	8
8		2	3	4	5	5.5	6
10		1	1	1	1	1	1

Justification: For the solution with the pH 3 buffer (most acidic) the relative color change remained very low, only reaching the second color on the chart. For the solution with the pH 10 buffer (most alkaline) had the same reaction but this time only reaching the first color change on the chart. For the most acidic and basic buffers there was very little change which means the enzyme was not very efficient. This was because the pH values that we were testing were not the optimum conditions and the enzyme was denaturing. The pH buffer that showed the most activity in the experiment was the neutral pH 7. This reaction showed the most color change in the fastest amount of time. It reached the eighth color on the chart in two minutes while pH 3 reached 2, pH 5 reached 6, pH 8 reached 6, and pH 10 reached 1. The solution with pH 7 seemed to show the most enzymatic activity.

Procedure 3: Effect of Different Enzyme Concentration on Enzymatic Activity

Research Question: What do you predict will occur if the enzyme concentration changes?

Hypothesis: If the enzyme concentration changes it will have an effect on the enzyme’s rate of reaction. The color change and speed of the reaction will be affected by the amount of enzyme we put into the solution. I predict that if we add more of the enzyme to the solution, then the reaction will work faster and the solution will turn darker much quicker just as if we add less enzyme to the mixture, then the reaction will work much slower and not turn as dark.

Procedure:

• Make identical substrate solutions just as we did in procedures 1 and 2. The constants are the amounts of guaiacol, hydrogen peroxide, and water in the substrate tubes. Make 3 different enzyme solutions as follows:
• Enzyme 1 (baseline/control group): 6 mL of pH 7 buffer, 1.5 mL of turnip peroxidase
• Enzyme 2 (2x): 4.5 mL of pH 7 buffer, 3 mL of turnip peroxidase
• Enzyme 3 (0.5x): 6.75 mL of pH 7 buffer, .75 mL of turnip peroxidase
• We will record results of the color change every 20 seconds for two minutes.  
• Form conclusions based on the color change over time for the different enzyme concentrations.

Claim: Adding twice as much enzyme to the solution proved to make the reactions go faster. Adding half the amount of enzyme to the reaction proved to slow the reaction time. The amount of enzyme in the solution has an effect on the enzyme’s rate of reaction.

Evidence:

Relative Color Change of Solutions with Different Enzyme Concentrations

Enzyme Concentration	Time	20s	40s	60s	80s	100s	120s
0.75 mL (0.5x)		2	3	4	6	6	7
1.5 mL (1x)		1	3	4	5	6	7
3 mL (2x)		3	4	5	6	8	9

Amount of Time Required for Relative Color Change 9 for Different Enzyme Concentrations

Enzyme Concentration	0.75 mL	1.5 mL	3 mL
Time at which color change 9 was reached	2 minutes, 35 seconds	2 minutes, 3 seconds	57 seconds

Justification: The solution we made with twice as much of the enzyme as the original solution proved to show the most color changes in the shortest amount of time which means it showed the most enzymatic activity. In two minutes the solution with 3 mL of turnip peroxidase made it to the ninth color change on the chart while the other solutions with 0.75 mL and 1.5 mL of turnip peroxidase only made it to the seventh color change in the same amount of time. In another trial we tested how fast it would take for the three solutions to make it to the ninth color change. The 0.5x solution took 2:35, the x solution took 2:03, and the 2x solution only took 57 seconds. This shows that the enzyme concentration has a direct effect on the amount of enzymatic activity.