Group D -Amylase Lab


Part D. Determination of the effect of pH and temperature on Amylase

1. Select 5 test tubes labeled D1 through D5 place them in a test tube rack.

2. Using the pipette pump dispenser, add 1 ml of amylopectin to each tube. Check off each chemical in the table below as you add it.

3. Using an adjustable pipetter, add the amount of the appropriate buffer as indicated in the table to each tube. Be sure to switch tips when you change buffers. Check off each one as you add it.

4. Obtain a Styrofoam cup of ice and place tube D5 in the ice.

5. Using the pipette pump dispenser quickly add 0.2 ml of amylase solution to each tube as quickly as possible. Immediately place tube D4 in the hot water bath and leave D5 in the ice bath. Note the time when the amylase is added to tube D5.

6. After exactly 10 minutes, use the pipette pump dispensers to add 1 ml of DNS reagent and 1 ml tartrate solution to each tube. Caution: DNS reagent should be washed off your skin immediately.

Test Tube #
Amylopectin
1000 µl Buffer
Amylase
DNS Solution
Conversion to µl of Maltose
D1
1 ml

.

pH 4

.

0.2 ml

.

1 ml

.

1 ml

.

D2
1 ml

.

pH 7

.

0.2 ml

.

1 ml

.

1 ml

.

D3
1 ml

.

pH10

.

0.2 ml

.

1 ml

.

1 ml

.

D4
1 ml

.

pH 4

.

0.2 ml

.

1 ml

.

1 ml

.

D5
1 ml

.

pH 4

.

0.2 ml

.

1 ml

.

1 ml

.

7. Mix the contents of each tube using the laboratory mixer and place each tube into a hot (use caution) water bath for five minutes. Check off each step in the table below.

Test tube #
Vortex Mixer ()
Temperature
pH
Hot Water Bath ()
Absorbance Measured
Conversion to µl of maltose
D1

.

Room Temp.

4

.

.

.

D2

.

Room Temp.

7

.

.

.

D3

.

Room Temp.

10

.

.

.

D4

.

95oC Water Bath

4

.

.

.

D5

.

0oC Ice Bath

4

.

.

.

8. Remove the tubes from the water bath and allow them to cool to room temperature. Observe and make note of the color change - use color pencils if available. Store the tubes in the refrigerator until the next lab period.

9. Take your samples to the spectrophotometer. Measure the absorbance at a wavelength of 540 nm of the contents of each tube. Record the absorbance in the above table. Your teacher will provide instructions on the use of the spectrophotometer. The absorbance is proportional to the amount of maltose produced by the enzyme in ten minutes.

10. Prepare a graph of absorbance versus pH for samples D1, D2, and D3 as indicated in the student worksheet.

11. Prepare a second graph of absorbance verses temperature for samples D5, D1, and D4 (in this order) Why?

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Go to directions for Part [A] [B] [C]