Enzyme Kinetics (activity)

Effect of pH on Enzyme Activity

      1. Add 5 ml of water to an empty tube (this is the BLANK)
      2. To 3 separate tubes, add 2.5 ml of buffer pH 3, pH 5 or pH 7
        1. Add 2.5 ml of starch (substrate) to each of these tubes (excluding BLANK)
      3. Add 2 drops of iodine to each tube and mix: Blank, pH 3, pH 5, pH 7
      4. Read the Blank in the spectrophotometer and calibrate it to 100% transmittance at 560nm
      5. Read each tube in the spectrophotometer. This is time 0 min.
      6. Add 35 drops of amylase solution to each tube simultaneously, mix to homogeneity.
      7. At 2 minute intervals, quickly read ALL tubes in the spectrophotometer.
      8. Continue reading the samples every 2 minutes until you reach 22 minutes on the table below.

Time (min)

pH 3

% Trans

pH 5

% Trans

pH 7

% Trans

0

2

4

6

8

10

12

14

16

18

20

22

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