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Lab Partner: Date: Thursday 28th October 2010

Course Code & Title of Lab: BIOL 236 Measurement of Arginase Activity Aim: To determine the amount of urea produced via spectrophotometric analysis with isonitrosopropiophenone when varying amounts of arginine is converted to orthinine using arginase contained in rats liver extract. Theory: The urea cycle is the process by which ammonia is converted to urea. It takes place within the mitochondria and cytosol of hepatocytes (Nelson and Cox 2008). Fig. 1 shows the urea cycle.

(Hames and Hooper 2005) Fig. 1: The urea cycle. Enzymes involved include: 1) carbamoyl phosphate synthesase; 2) ornithine transcarbamoylase; 3) argininosuccinate synthetase; 4) arginosuccinase and 5) arginase. During the urea cycle, the amino acid arginine is converted to ornithine another amino acid upon addition of water. This reaction occurs in the cytosol of hepatocytes. During this reaction urea is produced, see Fig. 2.

Fig.2: Urea Cycle reaction, Arginine to Ornithine (Hames and Hooper 2005) The ornithine produced in the reaction seen in Fig. 2 is then transported into the mitochondria to initiate another round of the urea cycle (Nelson and Cox 2008). In this experiment, the enzyme arginase is present in the liver extract. Therefore the reaction can be investigated by adding the liver extract containing arginase to solutions of arginine and water. The amount of urea produced can then be determined using spectrophotometric analysis with isonitrosopropiophenone. Manganese sulphate was added to the tubes as the exogenous manganese ions in reaction recovered the activity of arginase, which was lost in dissolving and dilution of the liver extract (Xie, et al. 2004). Perchloric acid was added to the tubes to stop the reaction of arginine to ornithine and urea. This occurs since the perchloric acid reacts with the amine groups on arginine to produce ammonium perchlorate hence destroying the arginine substrate.

Procedure:

Reagents and Materials 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Arginine (0.5mol/L, pH 9.7) Manganese sulphate (4mmol/L prepare fresh) Perchloric acid (5% (v/v) prepare by diluting the conc. Perchloric acid with water) 2.0g of -isonitrosopropiophenone containing 170mL of conc. sulphuric acid and 40ml of syrup phosphoric acid (orthophosphoric acid) made up to 1L with water. Standard urea solution 2.5mmol/L Water bath at 37C Boiling water bath hot plate Aluminum foil Liver tissue from rat, pig or cow Glass test tubes to fit the Beckman bench top centrifuge

Extraction of liver for enzyme assay: 200-300mg of liver was weighed out on a top pan balance, cut into small pieces and homogenized in 10mL of ice-cold cacodylate buffer. The liver extract was filtered through cheesecloth and diluted by a 1:20 ratio with ice-cold distilled water. Incubation: 4 test tubes were obtained and labelled 1a, 1b, 2a and 2b. To test tubes 1a and 1b the following reagents were added: 1.0mL of arginine and 0.5mL of MnSO4. To test tubes 2a and 2b the following reagents were added: 1.0mL of arginine, 0.5mL of MnSO4 and 5mL of perchloric acid. 3mL of diluted liver extract was added to a test tube and left to equilibrate at 37C for 5 minutes. The reaction was started by adding 0.5mL of equilibrated diluted liver extract to tubes 1a, 1b, 2a and 2b. The tubes were mixed and incubated at 37C for 10minutes. After 10minutes, the reaction in tubes 1a and 1b were stopped by adding 5mL of perchloric acid. The tubes were centrifuged at 2900rpm for 10minutes and 1mL of supernatant from each tube was transferred to separately labelled tubes. Determination of urea: 6mL of -INPP was added to the tubes containing 1mL of supernatant. The tubes were thoroughly mixed and placed in a boiling water bath for 1 hour. After 1 hour, the tubes were allowed to cool and the absorbance of its contents read at 520nm. Preparation of the calibration curve: Eight test tubes were obtained and labelled 1-8 and the following volumes of standard urea solution were added to each tube respectively: 0mL, 0.2mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 0.8mL and 1.0mL. The volume of each tube was made up to 1mL by distilled water. 6mL of -INPP was pipetted into each tube and the tubes were mixed and placed in a boiling water bath for 1 hour. The tubes were allowed to cool and the absorbance of its contents read at 530nm.

References

Hames, David, and Nigel Hooper. 2005. Instant Notes Biochemistry 3/e. New York: Taylor & Francis Group. Nelson, David L, and Michael M Cox. 2008. Lehninger Principles of Biochemistry. New York: W.H. Freeman and Company. Xie, Xiu-Yin, Xia Li, Zhi-Yong Wang, and Cun-Xin Wang. "Thermokinetic studies on the activation of bovine liver arginase by manganese ions." Thermochimica Acta, 2004: 19-23.