Enzyme Kinetics (activity)

Effect of Enzyme Concentration on Enzyme Activity

      1. Add 5 ml of water to an empty tube (this is the BLANK)
      2. Add 5 ml of pH 7 buffer to 3 separate tubes
      3. Follow the dilution scheme below:
amylase-dilution
Dilution scheme for amylase (enzyme)
      1. In 4 separate tubes, ADD 4 ml of starch solution
        • label them 1x, 1/5x, 1/25x, 1/125x
      2. Add 2 drops of iodine to each starch tube and the Blank
      3. Read the Blank in the spectrophotometer and calibrate it to 100% transmittance at 560nm
      4. Read each tube in the spectrophotometer. This is time 0 min
      5. Add 35 drops of diluted amylase solutions to the appropriately labeld tubes simultaneously and mix.
        • Each tube receives a different Amylase dilution
      6. At 2 minute intervals, quickly read ALL tubes in the spectrophotometer.
      7. Continue reading the samples every 2 minutes until you reach 22 minutes on the table below.

Time

(min)

1X

% Trans

1/5X

% Trans

1/25X

% Trans

1/125X

% Trans

0

2

4

6

8

10

12

14

16

18

20

22

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