Enzyme Kinetics (activity)

Effect of Substrate 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:

      1. In 4 cuvettes, ADD 4 ml of the diluted starch solution
        • label them 1x, 1/2x, 1/4x, 1/8x
      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 amylase solution to each tube simultaneously and mix.
      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

1/2X

% Trans

1/4X

% Trans

1/8X

% Trans

0

2

4

6

8

10

12

14

16

18

20

22

Plot the results

  1. Using a plot.ly, plot the data on the same chart.
  2. Calculate the line of best fit of each dataset.
  3. The slope represents the activity of the enzyme in each condition. What is the unit of this activity?
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