5, 1, or 2mL of potato juice, 1mL of water and either 6, 5.5, 5, or 4mL of pH 7 phosphate buffer (note: mL of each tube totaled 7mL). Four "experimental- test tubes were also prepared in the same protocol as the blanks but instead of 1mL of water, 1mL of catechol (the substrate) was added. As soon as the catechol was added, readings of absorbance were taken on the spectrophotometer. Readings were taken at 0 minutes and then every 2 minutes up until 10 minutes.
To determine the effect of substrate concentration on the rate of reaction (Experiment 2), one blank test tube was prepared with no catechol but 1.5mL of enzymatic potato juice and 6.9mL of pH 7 phosphate buffer. Four experimental tubes with 0.1, 0.4, 1.2, or 1.6mL of catechol were prepared. The appropriate amount of pH 7 phosphate buffer was added in order to fill the tubes to a volume of 6.9mL. To each experimental tube 1.5ml of potato juice was added and the readings on the spectrophotometer were taken in the same method as Exercise 1.
In Experiment 3, the effect of temperature on enzyme activity was found by preparing 5 blank test tubes with 5mL of pH 7 phosphate buffer and 1mL of water. Five experimental tubes were prepared in the same method except the 1mL of water was replaced with 1mL of catechol. The experimental tubes and the blanks were placed in their appropriate temperature treatment of 6 ∘C, 20 ∘C (room temperature), 30 ∘C, 40 ∘C, or 50 ∘C. After 5 minutes, 1mL of enzymatic potato juice was added to all tubes. After returning them to their temperatures for 5 minutes (mixing every minute) the absorbance was measured in the spectrophotometer against their blanks.
The effect of pH on enzyme action (Experiment 4) was tested by preparing 5 blank test tubes containing 1ml of water, 1mL of potato juice and 6mL of different pH phosphate buffer: pH 4, 6, 7, 8, or 10. The 5 experimental tubes were prepared in the same method as the blanks except 1mL of catechol was added instead of the1mL of water.