Effect of pH on amylase activity

Prof. Wilson: You will first test the amylase activity in different pH buffer solutions. We will be using iodine solution to colour the starch dark blue. Do you remember what color change denotes the presence of maltose?

James: Isn’t it a light-yellow, professor? I remember I watched a video about this just the other day after your lecture. 

Prof. Wilson: That’s right! What pH range should we test?

You: Considering that this is an enzyme found in the human body, it seems reasonable that the optimal pH would be in the range of 3.0-8.0.

Prof. Wilson: Very good, then, let’s set up.

You are provided with the following to begin preparing your samples

       1. Stopwatch

       2. Ice

       3. Glass tubes

       4. Marker

       5. Pasteur pipettes

       6. I2/KI (iodine) solution

       7. Amylase solution

       8. 1% starch solution

       9. 1M phosphate buffer solutions at pH 3.0

      10. 1M phosphate buffer solutions at pH 7.0

      11. 1M phosphate buffer solutions at pH 8.0

      12. 1M phosphate buffer solutions at pH 10.0

You follow the steps below:

1. Take one glass test tube and label ‘Control’ using a marker. Using a pasteur pipette, add one drop of I2/KI solution and using another pasteur pipette add 1 ml of 1% starch solution. This solution should turn dark blue. Remember to always use different pasteur pipettes for each solution. 

2. Prepare six glass test tubes with one drop of I2/KI solution and 4 ml of 1% starch solution. Label them as 0 min, 2 min, 4 min, 6 min, 8 min, 10 min respectively, using a marker. Add 0.5ml 1M phosphate buffer solutions at pH 3.0. Mix well.

3. Add 0.5ml of 1% amylase (enzyme) solution and mix. Start the stopwatch.

4. Immediately collect 0.5 ml of the mixture solution and add it in the glass test tube labelled 0 min. Observe the colour change. 

5. Repeat step 4 for the remaining glass test tubes until 10 minutes have been completed. Observe any changes in the colour of the mixture and record the time it took for the dark blue colour to turn light-yellow for each reaction.

6. Repeat steps 2 – 5 for the 1M phosphate buffer solutions at pH 7.0

7. Repeat steps 2 – 5 for the 1M phosphate buffer solutions at pH 8.0

8. Repeat steps 2 – 5 for the 1M phosphate buffer solutions at pH 10.0

9. Calculate the rate of each reaction using the formula 1/t