TO CALCULATE THE

ENZYME ACTIVITY OF
CATALASE PRESENT IN
RABBIT BLOOD SAMPLE
DR.ADNAN IQBAL
3 11 B I O C H E M I S T RY L A B
REQUIREMENT

• Test tube
• Water bath
• Hemolysate
• Phosphate buffer
• Sulphuric acid
• Potassium permanganate solution
THEORY
• Enzymes are the catalysts of biological systems.
• They speed up chemical reactions in biological systems by lowering the activation
energy, the energy needed for molecules to begin reacting with each other.
• We will focus on the enzyme catalase. It is present in blood
• Catalase is one of several enzymes that break down peroxide, a toxic metabolic waste
product of aerobic respiration.

2H2O2 → 2H2O + O2 (gas)

PROCEDURE
1. Take 2 test tubes.
2. Mark one test tube as “T” (test) and other one as “C”
(Control).
3. Add 4.5ml of phosphate buffer and 0.2 ml of H2O2 in
both test tubes and put in the water bath for 2 mins
at 40ºC.
4. Then add 0.5 ml of hemolysate in “T” test tube and
water bath both test tubes for 5 mins at 40ºC
5. Add H2SO4 and then place the both test tubes at
70ºC in water bath for 5 mins to stop the enzyme
activity.
6. Cool down the test tubes and titrate the both test
tubes with KMnO4 present in the burette
TITRATION
• To determine how much hydrogen peroxide (substrate) has been broken down by catalase at varying
times, measure the amount of peroxide remaining in each sample.

• To do so, slowly add KMnO4, which is purple, to the sample.

• The peroxide in the sample causes the KMnO4 to lose color when the solution is mixed thoroughly. When
all the peroxide has reacted with KMnO4, any additional KMnO4 will remain light brown or pinkish even
after you swirl the mixture. This is the endpoint.

• Record the amount of KMnO4 you have used. (The more KMnO4 you use, the more peroxide is left in the
sample.
JUSTIFICATIONS
• Reason for heating the test tubes at 40ºC is to start the enzyme activity.

• Phosphate buffer is used to keep the PH constant at 7 because catalase enzyme activity is
maximum at this PH.

• Sulphuric acid and heating at 70ºC is done to stop the enzyme activity.

• Hemolysate is prepared by adding 0.1 ml of blood in 24.9ml distilled water. So the

dilution factor for hemolysate is 250.
FORMULA FOR ENZYME ACTIVITY

26.5x (C-T)x 250÷10x0.5

• 26.5 is the micromolar concentration of H2O2

• C is the control
• T is the test
• 250 is the dilution of hemolysate
• 10 is the incubation time in minutes
• 0.5 is volume in ml of hemolysate
• Unit of enzyme activity = micromole/ml/min