NAME: Shernece Kalicharan

ID NUMBER: 815008866

DEMONSTRATOR: Steve
LAB STREAM: Tuesday 1-6

LAB PARTNER: STAYAVI MOHIP

DATE: 25th October, 2016.

Title: Measurement of Arginase Activity

Aim: To prepare a crude preparation of the enzyme arginase from rat liver using protein
purification, and to spectrophotometrically estimate enzyme activity using fixed a time incubation
method and to better appreciate the role of urea cycle in living systems.
Introduction
The urea cycle takes place in five reactions.

The primary reaction transpires in the

mitochondrial matrix. The successive reactions take place in the cytosol. The reaction in the
mitochondrial matrix undergoes catalysis by ornithine transcarbamoylase. A carbamoyl group
is transferred from a carbamoyl phosphate to ornithine to form citrulline. In another reaction
of the urea cycle, argininosuccinate synthetase serves as the catalyst. It utilizes ATP which
activates citrulline by producing a citrullyl-AMP intermediate. An amino group of an
aspartate residue attacks the intermediate to generate argininosuccinate. The third reaction
involves catalysis by argininosuccinate lyase. This splits argininosuccinate to fumarate and
arginine. Arginase catalyzes the last step of the reaction. The enzyme catalyzes the reaction
of the guanidine group to form urea and ornithine. Ornithine is transported to the matrix and
the cycle is complete.

Figure 1: Urea Cycle

Arginase is known to be highly specific for its substrate due to its high number of hydrogen
bonds. It consists of a carboxy terminal tyrosine residue which is needed for maximum
catalytic activity. It has an active site and 2 Mn 2+ bridged by O2. The proposed mechanism of
this enzyme is a nucleophilic attack by the metal connecting the hydroxide of the
guanidinium carbon of the substrate.

Figure 2: Reaction of arginine to ornithine and urea

Enzyme activity is referred to as the measure the quantity of active enzyme present in a
reaction. It is defined in terms of units which is the mol product formed min -1. There are two
ways enzyme activity can be measured. They are observing the loss of the substrate or the
development of a product.
In this experiment, arginase is present in the liver extract. The liver extract containing
Arginase iss added to water and arginine. The quantity of urea produced is measured using a
spectrophototmetre. The enzyme and substrate are combined and incubated for 10 minutes.
At the end of this 10 minutes, the catalysis of the reaction must be stopped by denaturing the
enzyme. Perchloric acid is added to achieve this step. A fraction of the reaction solution is
extracted and reacts with -INPP to produce a pink coloured solution and its absorbance is
measured. The enzyme assay measures the total amount of urea in the extract. Urea
contained in the extract can also be measured. In the zero minute control tube the perchloric
acid denatures the enzyme so no urea is made and further enzyme activity is prevented.
In humans, excess ammonia is channeled to the mitochondria of liver cells and converted into
carbamoyl phosphate. Carbamoyl phosphate enters the nitrogen into the urea cycle. The urea
produced in the liver is released into the blood stream. The kidneys filter out the urea. The
urea is ultimately excreted in the urine. The liver is known as a complex organ and performs
many vital functions. Urea is generated in the liver and is a metabolite of amino acids.

Ammonium ions are formed in the breakdown of amino acids and some are utilized in the
biosynthesis of nitrogen compounds. An excess of ammonium ions are converted to urea. The
liver also serves as a filter for blood and processes it as it is circulated throughout the body. It
manufactures blood clotting proteins and performs other vital functions. The cells of the liver
contsist of proteins that catalyze these chemical reactions.
Procedure:
Extraction of liver for enzyme assay
200 300 mg of liver was weighed on the top pan balance. It was cut into small pieces with
scissors and homogenized in 10mL of ice-cold cacodylate buffer. The homogenizing tube was
kept on ice. The liver extract was filtered through cheesecloth. The filtered liver extract was
diluted in 1 in 20 with ice-cold distilled water and this diluted extract was used for the
enzyme. It was kept on ice until ready to use.
The following mixtures were prepared in duplicate and equilibrated at 37C for 10 minutes.
MIXTURE 1 reaction tube
Arginine (0.5mol/L pH 9.7)

1.0mL

MnSO4 (4mmol/L prepared fresh) 0.5mL

MIXTURE 2 zero-time control

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

3mL of your diluted liver extract was placed in a clean and labeled test tube and equilibrated
at 37C for 5 minutes. The reaction was started when 0.5mL of your equilibrated, diluted
liver extract was to the mixtures from above. (You should have 4 tubes). The tubes were
capped with parafilm and mixed thoroughly by gentle inversion and incubated at 37C for 10
minutes. The reaction was stopped by adding 5mL of the perchloric acid to mixture 1 and mix
thoroughly. The tubes were centrifuged on the bench top centrifuge, at 2900rpm for 10

minutes, to remove the precipitated protein and 1mL of the supernatant was transferred to a
clean, labeled test tube for the urea assay
. C.

Determination of urea

Preparation of test samples

1mL of the sample from above was mixed with 6mL of -isonitrosopropiophenone (-INPP)
reagent and heated in a boiling water bath for 1 hour. The test tubes were wrapped in
aluminum foil and tops were covered. After 1 hour, the tubes were removed , transferred to a
beaker of cold water and placed in a dark cupboard for 15 minutes. The tubes were allowed to
cool for 15 minutes, the absorbance at 520nm were read.
Preparation of the calibration curve
0, 0.2, 0.2, 0.4, 0.6, 0.8, 0.8 and 1.0mL of the standard urea solution (2.5mmol/L) were
pipetted into clean labeled test tubes and the volume was brought to 1.0mL with distilled
water. (Remember to wrap your tubes in foil) Pipette 6 mL -INPP reagent was pipetted
into each tube, mixed thoroughly and heated in a boiling water bath for 1 hour.
Results:
Table 1: Calibration curve for urea
Tube no.

Urea stock
Distilled
solution (mL)
water (mL)
1
0
1.0
2
0.2
0.8
3
0.2
0.8
4
0.4
0.6
5
0.6
0.4
6
0.8
0.2
7
0.8
0.2
8
1.0
0
Table 2: Absorbance values for samples
Sample
A1
A2
B1
B2

A520 nm
0.028
0.012
0.030
0.017

-INPP
reagent (mL)
6
6
6
6
6
6
6
6

Urea content
( mol)
0
0.5
0.5
1.0
1.5
2.0
2.0
2.5

A520 nm
0.00
0.44
0.101
0.230
0.298
0.510
0.510
0.632

Calculations
Sample calculation for urea content in mol for tube no. 2
1000 mL contains 2.5 mols
1 mL will contain 2.5/1000 mmols
= 2.5 x 10-3 mmols
No. of mmols in 0.2 mL in urea stock solution
0.2 mL = 0.2 x (2.5 x 10-3) mmols
= 0.0005 mmols
mols = 0.0005 x 1000
= 0.5 mols

Average of A = 0.028 + 0.012/2 = 0.02

Average of B = (0.030 + 0.017)/2=0.0235
Determination of urea content of samples
Average absorbance of A = 0.02
Y= B-A
= -3.5x 10-3
Using equation of calibration graph: y = mx
y = 0.2538x
x = y/m
= 3.5 x 10-3/0.2538
= 0.0148 mol urea
Units of enzyme in mol m/min/mL
Total volume of supernatant = 7 mL
0.0148 mols of urea are found in 1 mL of supernatant
7 mL = 0.0148 mol x 7
= 0.1036 mols
10 minutes = 0.1036 mols
1 minute

= 0.1036/10
= 0.01036 mols

0.5 mL = 0.01036 mols

1 mL

= 0.01036 x 2
= 0.02072 mols min-1mL-1

Activity per gram

Volume of homogenate = 180 mL x 0.02072
= 3.7296 units
3.7296 units is found in 0.3 g of liver
3.7296/0.3 = 12.432 g
Total activity
Total mass of liver = 14.66 g
Total activity = 12.432 x 14.66
= 182.25 U
Discussion
According to table 1, the absorbance values for each standard solution increased. The solutions
that were in test tubes 6 and 7 had the same absorbance value of 0.510 nm. This may have
occurred because in the test tubes there were equal volumes of each reagent.
The liver extract was kept on ice because it maintains the integrity of the enzymes because they
are highly sensitive. The cooling of the extract reduced the activity of the enzyme which resulted
in a decrease of molecular degradation. Cooling also lowered metabolic rate which prevented
their structures from breaking down. Bacterial growth was also avoided because of the cool
temperature.
The test tubes were wrapped in foil because the red colour produced is sensitive to light.
In the presence of -INPP, the urea solution turns pink. With increasing urea content the pink
colour intensified and the absorbance values increased. The darker pink colour detected occurred
because the micromoles of urea increased causing more molecules to be present in each solution.
These molecules blocked the light which resulted in a darker coloured solution because light

cannot pass through. Light is absorbed at a specific wavelength because electrons in chemical
bonds are able to absorb certain wavelengths of light. When the urea content increased, more
molecules exist for the light to hit when it passed through the solution.
In reaction tubes A and B, arginine was mixed with manganese sulphate and was allowed to
incubate at 37o C to avoid aggregation at higher temperatures. The Mn2+ in the manganese
sulphate recovered the arginase activity lost when the liver extract was dissolved and diluted.
After the incubation, perchloric acid was added to reaction mixture B. This was known as the
zero time control. It stopped the reaction of arginine to ornithine and urea. The perchloric acid
reacted with the amine groups on arginine to produce ammonium perchlorate which destroyed
the arginine substrate. The enzyme was denatured and further activity was prevented.
-INPP was combined with the supernatant of tubes A and B and a pink colour produced. This is
colour is noted when urea is present in solution. The urea measured in tube B was present in the
extract.
The values in tube B are supposed to be 0 because of the perchloric acid which denatured the
enzyme and averted enzyme activity. No urea was supposed to be present in the solution. The
cuvette could have been contaminated by solution A. The cuvette structure tends to trap solution
even after it is rinsed with distilled water.
References
9603, Lakewood, and NJ 08701 1. Arginase - Worthington Enzyme Manual. 2016.
Accessed October 30, 2016. http://worthington-biochem.com/ar/default.html.
Neurospora, In and Richard Stockton. Enzyme and Protein Assays Kelly Keenan. n.p., n.d.
http://www.fgsc.net/teaching/keenan.pdf.
Chem 125 - Experiment II. Accessed November 30, 2016.
http://www.umich.edu/~chem125/softchalk/Exp2_Final_2/Exp2_Final_2_print.html.
Lab #3: Spectrophotometry. n.p., 2005.
http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab3.pdf.
Accessed October 30, 2016.
http://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kinetics/Catalysis_and
_Enzymes/Enzymes/Enzyme_Assays.
, 2005. http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab4.pdf.

Accessed October 30, 2016.

file:///C:/Users/SherneceK/Downloads/article_wjpps_1432989797.pdf.
Citations, Quotes & Annotations
19603, Lakewood, and NJ 08701 1. Arginase - Worthington Enzyme Manual. 2016.
Accessed October 30, 2016. http://worthington-biochem.com/ar/default.html.
Arginase - Worthington Enzyme Manual, 2016, accessed October 30, 2016,
http://worthington-biochem.com/ar/default.html.
Neurospora, In and Richard Stockton. Enzyme and Protein Assays Kelly Keenan. n.p., n.d.
http://www.fgsc.net/teaching/keenan.pdf.
In Neurospora and Richard Stockton, Enzyme and Protein Assays Kelly Keenan, (n.p., n.d.),
http://www.fgsc.net/teaching/keenan.pdf.
Chem 125 - Experiment II. Accessed November 30, 2016.
http://www.umich.edu/~chem125/softchalk/Exp2_Final_2/Exp2_Final_2_print.html.
Chem 125 - Experiment II, accessed November 30, 2016,
http://www.umich.edu/~chem125/softchalk/Exp2_Final_2/Exp2_Final_2_print.html.
Lab #3: Spectrophotometry. n.p., 2005.
http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab3.pdf.
Lab #3: Spectrophotometry, (n.p., 2005),
http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab3.pdf.
Accessed October 30, 2016.
http://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kinetics/Catalysis_and
_Enzymes/Enzymes/Enzyme_Assays.
accessed October 30, 2016,
http://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kinetics/Catalysis_and
_Enzymes/Enzymes/Enzyme_Assays.
, 2005. http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab4.pdf.
(n.p., 2005), http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab4.pdf.
Accessed October 30, 2016.
file:///C:/Users/SherneceK/Downloads/article_wjpps_1432989797.pdf.
accessed October 30, 2016,
file:///C:/Users/SherneceK/Downloads/article_wjpps_1432989797.pdf.
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