DEPARTMENT OF LIFE SCIENCES

BIOC 2069
PRACTICAL #4
MEASUREMENT OF ARGINASE ACTIVITY

Student Learning Outcomes

You should be able to:
 Use your knowledge of protein purification to prepare a crude preparation of the enzyme
arginase from rat liver
 Spectrophotometrically estimate enzyme activity using fixed a time incubation method
 Better appreciate the role of the urea cycle in living systems

PRINCIPLE
Arginase catalyzes the hydrolysis of arginine to ornithine and urea.

Arginine +H2O → Ornithine + Urea

The activity is determined by measuring the amount of urea released in the reaction.

Reagents and Materials

1. Arginine (0.5mol/L, pH 9.7)

2. Manganese sulphate (4mmol/L prepare fresh)

3. Perchloric acid (5% (v/v) prepare by diluting the conc. Perchloric acid with water)

4. 2.0g of α-isonitrosopropiophenone containing 170mL of conc. sulphuric acid and 40ml of

syrup phosphoric acid (orthophosphoric acid) made up to 1L with water. EXERCISE
CAUTION! Add the solid to a little water in a beaker with a magnetic stirrer. Add the
phosphoric acid slowly followed by the sulphuric acid, also added slowly. Filter through
glass wool and make up to 1L with distilled water –final solution should be pale yellow.

5. Standard urea solution 2.5mmol/L (if the urea is not anhydrous then dry in an oven at 102°C
overnight, after grinding the pellets in a mortar and pestle. Cool in a desiccator)

6. Water bath at 37°C

7. Boiling water bath – hot plate

8. Aluminum foil

9. Liver tissue from rat, pig or cow

10. Glass test tubes to fit the Beckman bench top centrifuge

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BIOC2069
Lab 4 – Arginase Activity
Semester I 2014/2015
PROCEDURE

A. Extraction of liver for enzyme assay

1. Weigh 200 – 300 mg of liver on the top pan balance.

2. Cut into small pieces with scissors and homogenize in 10mL of ice-cold cacodylate
buffer (very poisonous; contains arsenic which is absorbed through the skin)
0.1mol/L, pH 6.5 at 0°C. i.e. keeping the homogenizing tube on ice.

3. Filter the liver extract through cheesecloth.

4. Dilute the filtered liver extract 1 in 20 with ice-cold distilled water and use this diluted
extract for the enzyme. Keep on ice until ready to use.

B. Incubation

Prepare the following mixtures, IN DUPLICATE and equilibrate at 37°C for 10 minutes.

MIXTURE 1 – reaction tube MIXTURE 2 – zero-time control

Arginine (0.5mol/L pH 9.7) 1.0mL Arginine (0.5mol/L pH 9.7) 1.0mL
MnSO4 (4mmol/L prepared fresh) 0.5mL MnSO4 (4mmol/L prepared fresh) 0.5mL
Perchloric acid 5mL

1. Place 3mL of your diluted liver extract in a clean and labeled test tube and equilibrate at
37°C for 5 minutes.

2. Start the reaction by adding 0.5mL of your equilibrated, diluted liver extract from step 1 to
the mixtures from above. (You should have 4 tubes).

3. Cap your tubes with parafilm and mix thoroughly by gentle inversion and incubate at 37°C
for 10 minutes.

4. Stop the reaction by adding 5mL of the perchloric acid to mixture 1 and mix thoroughly.

5. Centrifuge the tubes on the bench top centrifuge, at 2900rpm for 10 minutes, to remove the
precipitated protein and transfer 1mL of the supernatant to a clean, labeled test tube for the
urea assay.

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BIOC2069
Lab 4 – Arginase Activity
Semester I 2014/2015
C. Determination of urea

You have a calibration curve and test samples to prepare. This should be done
simultaneously.

Preparation of test samples

1. Mix 1mL of the sample from step 5 above with 6mL of α-isonitrosopropiophenone (α-
INPP) reagent and heat in a boiling water bath for 1 hour. The red colour produced is
sensitive to light so wrap all of the test tubes in aluminum foil, which must also cover
the top of the tubes.

2. After 1 hour, remove the tubes, transfer them to a beaker of cold water and place in a dark
cupboard for 15 minutes.

3. After allowing the tubes to cool for 15 minutes, read the absorbance at 520nm.

Preparation of the calibration curve

4. Pipette 0, 0.2, 0.2, 0.4, 0.6, 0.8, 0.8 and 1.0mL of the standard urea solution (2.5mmol/L)
into clean labeled test tubes and bring the volume to 1.0mL with distilled water.
(Remember to wrap your tubes in foil)

5. Pipette 6 mL α-INPP reagent into each tube, mix thoroughly and heat in a boiling water
bath for 1 hour.

6. Allow the tubes to cool for 15 minutes and read the absorbance at 520nm.

PREPARE YOUR CALCULATIONS AND BE AWARE OF THE VOLUMES OF

EACH REAGENT THAT YOU REQUIRE FOR EACH STEP.

D. TREATMENT OF RESULTS AND WRITE UP

1. Plot a calibration curve of absorbance vs. urea content in μmoles.

2. Calculate the amount of urea in the sample used.

3. Calculate the units of enzyme in μmol/min/mL enzyme extract.

4. Determine the activity of the enzyme as units of enzyme per gram of liver i.e.
μmoles/minute/g tissue.

5. What is the total activity of the enzyme present in the liver.

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BIOC2069
Lab 4 – Arginase Activity
Semester I 2014/2015
 Final Results

1 2

A. Units of enzyme
(μmol/min/mL enzyme extract) =

B. Units of enzyme per g. tissue

(μmol/min/g tissue) =

C. Total Activity of Enzyme in Liver

(Results of B x mass of whole liver) =

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BIOC2069
Lab 4 – Arginase Activity
Semester I 2014/2015
 DATA SHEET

PRACTICAL #4: THE MEASUREMENT OF ARGINASE ACTIVITY

NAME:………………………………… LAB PARTNER:…………………………

DATE:………………………………….

Calibration Curve for Urea

Tube # 1 2 3 4 5 6 7 8
Urea stock solution (mL) 0 0.2 0.2 0.4 0.6 0.8 0.8 1.0
Distilled water (mL)
α- INPP reagent (mL)
Urea content (μmol)
A520nm

Plot your calibration curve in the lab (Absorbance at 520 nm vs. urea content)

Urea Determination on samples

1 2-(zero-time control)

Absorbance at 520nm (i)

(ii)

Use your calibration curve to determine the urea content in your samples

Urea content (μmol) (i)

(ii)

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BIOC2069
Lab 4 – Arginase Activity
Semester I 2014/2015