BCH 3110 Practical Manual

FACULTY OF BIOTECHNOLOGY AND

BIOMOLECULAR SCIENCES

BCH3110

PROTEIN AND NUCLEIC ACID METABOLISM

PRACTICAL MANUAL
 BCH 3110 Practical Manual

LIST OF PRACTICAL
2

1) ANALYZE DIGESTIVE ENZYME ACTIVITY OF PROTEIN HYDROLYSIS

2) ANALYZE THE ACTIVITY OF ARGINASE IN UREA CYCLE AND ITS
PRODUCT
3) DETERMINATION OF URIC ACID CONCENTRATION IN BLOOD SERUM
4) HYDROLYSIS OF DEOXYRIBONUCLEOPROTEINS AND DEFINITION OF
DNP COMPONENTS IN HYDROLYSATE
5) MEASURING UREASE ACTIVITY
6) ANALYZE GLUTAMATE DEHYDROGENASE OXIDOREDUCTION
ACTIVITY
7) ESTIMATION OF GLUTATHIONE (GSH) BY USING ELLMAN’S
REACTION
8) DETERMINATION THE REACTION OF GLUTAMINASE
 BCH 3110 Practical Manual

PRACTICAL 1 (NAY)
3
ANALYZE DIGESTIVE ENZYME ACTIVITY OF PROTEIN HYDROLYSIS

INTRODUCTION

The function of the digestive system is to break down large food molecules (macromolecules)
of carbohydrates, proteins and lipids into micromolecules; small enough to be absorbed into
the blood stream where the nutrients can be utilized by body cells. Food is broken down by
mechanical action such as chewing and by the chemical action of digestive enzymes.
Enzymes are proteins that catalyze reactions. They increase the rate of the reactions without
being consumed by the reaction.

Enzymes in the human digestive tract catalyze the conversion of proteins into short chains of
amino acids called peptides, as well as individual amino acids. Pepsin is an enzyme that
breaks down proteins into smaller peptides (that is, a protease). It is produced in
the stomach and is one of the main digestive enzymes in the digestive systems of humans and
many other animals, where it helps digest the proteins in food. It functions at a very low pH,
which makes it ideal to use for studying the effect of pH on enzyme activity. Pepsin is most
efficient in cleaving peptide bonds between hydrophobic and preferably aromatic amino acids
such as phenylalanine, tryptophan and tyrosine. Pepsinogen is secreted by gastric glands of
the stomach into the stomach. There, in the acid environment of the stomach, pepsinogen is
converted into pepsin.

Enzyme activity is also affected by temperature and pH. Human enzymes work best at human
body temperature (37°C). Since enzymes are proteins, their molecular structure is denatured
by heating to 60°C or higher and they no longer function. Conversely, as enzymes are cooled,
their activity slows as the temperature decreases and activity ceases at 0°C.

MATERIALS

Pepsin solution; hard-boiled egg white; water; HCL; NaOH; water bath

METHODS

This experiment will examine the effects of the presence of enzymes, variable pH, and low
temperatures on the digestion of protein using a gastric protease (pepsin)

1. Obtain a set of five labeled test tubes

2. Add a small sliver of hard-boiled egg white to each of the five tubes

3. Add the following solutions to the tubes (see Fig)

TUBE 1: Add 5 ml pepsin solution and 10 drops of water

TUBE 2: Add 5 ml pepsin solution and 10 drops of HCl
TUBE 3: Add 5 ml pepsin solution and 10 drops HCl, then place on ice.
TUBE 4: Add 5 ml water and 10 drops HCl
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TUBE 5: Add 5 ml pepsin solution and 10 drops of NaOH

4
4. Place Tubes 1, 2, 4 and 5 in a 37 °C water bath to incubate for 90 min. (The speed
of this reaction can be increased by using very thin strips of egg white and/or
incubating at 30°C0.

5. Examine the egg white following incubation.

DISCUSSION

Note any digestion that occurs to the egg white and any color changes that may have
happened to the solution.

QUESTION

1. If proteases such as pepsin and trypsin digest protein, why don't they digest the stomach
and small intestine, which are made from protein?

2. What kinds of chemicals might you add to try to speed the action of an enzyme or to
inhibit its action?

3. Would pepsin be active in the mouth? Explain your answer.

4. Describe the significance of the optimum pH for pepsin observed in the experiment and the
secretion of pepsin by the gastric glands.
 BCH 3110 Practical Manual

PRACTICAL 2 (NAY)
5
ANALYZE THE ACTIVITY OF ARGINASE IN UREA CYCLE AND ITS PRODUCT

INTRODUCTION
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). Multicellular organisms
have evolved a variety of systems to eliminate excess nitrogen from the body and animals
may be classified according to their major nitrogenous excretion product. The benefit of
excreting urea to terrestrial animals is that it lessens the osmotic excretory load and, thus,
prevents excessive water loss. The primary function, however, of the urea cycle is to remove
the potentially toxic metabolite ammonia from the body.
Fig 1 shows the urea cycle. During the urea cycle, the amino acid arginine is converted to
ornithine another amino acid upon addition of water. This reaction occurs in cytosol of
hepatocytes. During this reaction, urea is produced (Figure 2).

Figure 1: The urea cycle. Enzyme involved include: carbamoyl phosphate synthase I (CPS I);
ornithine transcarbamoylase (OTC); argininosuccinate synthase (ASS); arginosuccinase and
 BCH 3110 Practical Manual

arginase. The two nitrogen and one carbon that are synthesize into urea are shown in red to
illustrate the functional parts of the pathway. 6
The ornithine produced in the reaction seen in Figure 2 is then transported into mitochondrial
to initiate another round of the urea cycle (Nelson and Cox, 2008).

Figure 2: One of urea cycle reaction particularly involve arginase.

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 alpha-isonitrosopropiophenone.
Perchloric acid is 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.

MATERIALS
Arginine (0.5 mol/L, pH 9.7); Manganese sulphate (4 mmol/L prepare fresh); NaCl (0.2%
w/v); Perchloric acid (5% (v/v) prepare by diluting the conc. perchloric acid with water); 2.0
g of alpha-isonitrosopropiophenone containing 170 ml of conc. sulphuric acid and 40 ml of
phosphoric acid (orthophosphoric acid) made up to 1 L with water; Standard urea solution 2.5
mmol/L; Water bath at 37°C; Boiling water bath – hot plate; Aluminium foil; Liver tissue
from rat or cow; Falcon tubes to fit the beckman bench top centrifuge; cuvettes; UV
spectroscopy.
 BCH 3110 Practical Manual

METHODS
7
A) Extraction of liver for enzyme assay:

Weigh out 200-300 mg of liver on the top pan balance, cut into small pieces in 10 ml of
ice-cold cacodylate buffer. Filter the liver extract through cheesecloth and dilute it by a
1:20 ratio with ice-cold distilled water.

B) Incubation:

Obtain 4 test tubes and label 1a, 1b, 2a, 2b. Add following reagent to following test
tubes respectively;

To test tubes 1a and 1b: 1.0 ml of arginine and 0.5 ml of NaCl.

To test tubes 2a and 2b: 1.0 ml of arginine and 0.5 ml of MnSO4.
To test tubes 3a and 3b: 1.0 ml or arginine, 0.5 ml of MnSO4 and 5 ml of perchloric
acid.

In another test tube, add 4 ml of diluted liver extract and equilibrate at 37°C for 5
minutes.

Start the reaction by adding 0.5 ml of equilibrated diluted liver extract to tubes 1(a-b),
2(a-b), and 3(a-b). Mix the tubes and incubate at 37°C for 10 minutes. After 10
minutes, stop the reaction in tubes 1(a-b) and 2(a-b) by adding 5 ml of perchloric acid.

Replace each reaction in test tubes to each of falcon tubes. Centrifuge the falcon tube at
3000 rpm for 10 minutes and transfer 1 ml of supernatant from each tube to separately
labeled tubes.

C) Determination of urea:

Add 6 ml of α-INPP to the tubes containing 1 ml of supernatant. Thoroughly mix the

tubes and place in a boiling water bath for 1 hour.

After 1 hour, allow the tubes to cool and read the absorbance of its content at 520 nm.

D) Preparation of calibration curve:

Obtain and label 8 test tubes with 1-8 and add the following volumes of standard urea
solution to each tube respectively; 0 ml, 0.2 ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 0.8 ml
and 1.0 ml. Bring the volume of each tube to 1 ml by distilled water. Pipet 6 ml of α-
INPP into each tube and mix the tubes and place in a boiling water bath for 1 hour.
Allow the tubes to cool and read the absorbance of its contents at 530 nm.
 BCH 3110 Practical Manual

DISCUSSION
8
Observe and explain any changes take place in every step of reactions. Discuss the changes
and explain the relationship of those reactions to the urea cycle. Read the absorbance in each
reaction tubes and determine the urea content based on the standard curve prepared. Explain
in details the different results from test tube 1(a-b), 2(a-b) and 3(a-b). Attach the standard
curve and calculation on urea concentration in the lab report.

QUESTIONS

1. What is the molecular formula arginine and name the properties of this amino acid?
2. Explain the metabolism of arginine and how it is produced in urea cycle?
3. Describe the importance of H2O (hydrolysis) in the urea cycle, arginine to ornithine.
4. If there are excess of other amino acids eg; alanine, glutamate, histidine, lysine, and
aspartate in the diet, are there possibilities urea cycle enzymes will be affected? Explain.
5. Explain the effect of arginine-free diets on hepatic levels of urea cycle enzymes.
6. Liver plays an important role in whole body nitrogen (and carbon) homeostasis. Explain.
7. Arginase is a homotrimer. Each subunit contains an active site, and the two essential
manganese ions are bridged by oxygens. What is the function of manganese ions in arginase
activity?
8. What is ornithine and what is the function of ornithine in urea cycle?
9. Describe how urea cycle disorder can affect detoxification of ammonia among children and
adults.
10. How urea cycle is linked to the citric acid cycle?1-241- Explain.
 BCH 3110 Practical Manual

PRACTICAL 3 (NAY)
9
DETERMINATION OF URIC ACID CONCENTRATION IN BLOOD SERUM

INTRODUCTION

Nucleic acids are made up of nucleotides. A component of each nucleotide is the purine base
(adenine or guanine) or pyrimidine base (cytosine, uracil or thymine), pentacarbon sugar:
ribose or deoxyribose and orthophosphoric acid residue. The base binds the sugar by the β-N-
glycosidic bond, orthophosphoric acid residue binds the sugar component by an ester bond
through the -OH group at carbon 3' or 5' of ribose or deoxyribose. Individual nucleotides are
bound with phosphodiester bonds between carbons 3' and 5’.

The acidic character of the nucleic acids is caused by orthophosphate residues, each of which
contains H+ capable of dissociation. Due to this, nucleic acids are polyanions - carriers of
many negative charges, and this makes them capable of interacting with polycations,
particularly with alkaline proteins - which are carriers of positive charges. They also bind
with micromolecular compounds of an alkaline nature, such as methylene blue. In the natural
environment, DNA mainly binds with alkaline proteins - histones, whereas RNA mainly
binds with neutral proteins, which are part of the ribosome. Complexes of nucleic acids with
proteins are called nucleoproteins. In the student laboratory conditions, artificial
nucleoproteins can be obtained by mixing a solution of nucleic acid with blood serum.
Nucleoproteins dissociate into its constituents in concentrated salt solutions. In an alkaline
environment, nucleic acids form salts - nucleates, which are soluble in water. They can be
precipitated from solution with ethanol.

Sugar constituents of nucleic acids: ribose and deoxyribose can be detected directly in the
solutions of these acids or their salts without prior hydrolysis. Ribose contained in the RNA,
purine nucleosides and nucleotides, heated with concentrated HCl dehydrates to furfural,
which with orcin in the presence of Fe3+ ions forms a complex of a stable green colour.
Deoxyribose, contained in the DNA, when heated with concentrated sulphuric acid is
converted into hydroxylevulinyl aldehyde. This compound forms a blue colour complex in
reaction with diphenylamine. Purine bases can only be detected in the hydrolysis products of
nucleic acids. The sample of nucleic acid, intended for the detection of purines, should be
hydrolysed in sulphuric acid at 100°C. Nucleic acids are hydrolysed, initially to
mononucleotides. Purine mononucleotides are further hydrolysed to bases, pentoses and
orthophosphoric acid. The action of this acid leads to the hydrolytic breakdown of the β-N-
glycosidic bonds between the purine and the ribose or deoxyribose. The following purine
bases are released: adenine and guanine. Purine bases precipitate easily as insoluble
complexes with the ions of copper or silver. In the same conditions, pyrimidine nucleotides
are stable and do not decompose.

The final product of purine bases' transformations in the human organism is poorly soluble
uric acid. Sodium salts of this acid (sodium urate) are quite soluble, while ammonium, silver
and copper urates are sparingly soluble in water. The reducing uric acid properties make it an
important biological antioxidant. It inactivates reactive forms of oxygen and prevents them
from forming.
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10

Uric acid is oxidized by the action of nitric acid (V) to alloxan and urea. Condensation of two
molecules of alloxan with ammonia induces the formation of murexidic acid. Its salts are
called murexidates. In reaction with the NH4+ ion a purple-coloured murexide is formed, and
in reaction with the Na+ ion, a blue-coloured sodium murexidate is formed.

The method is based on the fact that uric acid reduces the phospho-tungstic reagent (Folin's
reagent) yielding coloured products whose coloration intensity is presumed to be proportional
to the uric acid concentration.

MATERIALS

Distilled water; Blood serum; Solution of H2SO4 (0.35 M) 0.4 M NaOH; Solution of tungstic
sodium; Standard solution of uric acid; Solution of sodium carbonate; Phospho-tungstic
reagent; quartz cuvettes; UV spectroscopy.

METHODS

Reagents Control Standard Solution Sample

Distilled water - - 4ml
Blood serum - - 0.5ml
Mix
Solution of H2SO4 - - 0.25ml
NaOH (0.35 M 0.4
M)
Solution of tungstic - - 0.25ml
sodium
 BCH 3110 Practical Manual

Mix, centrifuge the mixture at 3000 rpm for 5 min

Centrifugate - - 2ml 11
(Supernatant)
Standard solution of - 2ml -
uric acid
Distilled water 2ml - -
Solution of sodium 1ml 1ml 1ml
carbonate
Phospho-tungstic 0.5ml 0.5ml 0.5ml
reagent

Mix, wait 30 minutes and measure the absorbance for sample and standard solutions against
distilled water on a photocolorimeter at 510-560 nm 710 nm (green light filter) using 1cm
thick cells.
Calculation is carried out by the equation:

C=A sample/A standard ×30×10

C= A sample / A standard x conc. of standard uric acid in mg/100 ml

where C is the concentration of uric acid in blood serum, µmol/litre; A sample is the
absorbance measured for sample solution; A standard is the absorbance measured for
standard solution; 30 µmol - is the mass of uric acid contained in standard solution, used for
reaction; 10 - is the scaling factor for conversion to per litre blood serum.

Norm for male – 240-500 µmol/l; for female – 160 –400 µmol/l.

DISCUSSION

Calculate the concentration of uric acid in blood serum by making use of the measured
absorbance values and record the result in a report sheet. Compare the found uric acid
concentration to the norm. Suggest an explanation for the difference (if any) between these
two indices from the standpoint of disturbed purine metabolism.

QUESTIONS

1. Explain the degradation of purine nucleotide.

2. Explain in detail the concentration of uric acid in the blood serum and the amount in the
daily urine.
3. What is hyperuricemia. What is xanthinuria.
4. Describe gout and its causes.
 BCH 3110 Practical Manual

PRACTICAL 4 (NAY)
12
HYDROLYSIS OF DEOXYRIBONUCLEOPROTEINS AND DEFINITION OF DNP
COMPONENTS IN HYDROLYSATE

INTRODUCTION

Yeast cells contain a lot of nucleoproteins. Peptides, nitrogenous bases, pentoses and
phosphatic acid are formed by acid catalyzed hydrolysis of yeast. The end products of
hydrolysis may be detected by qualitative reactions.

MATERIALS

Dry yeast; 10 % sulfuric acid; 10% solution sodium hydroxide; 1% solution of copper
sulphate; solution ammonium; 2% solution silver in ammonia; molybdic reagent; Benedict’s
reagent, quartz cuvettes; UV spectrophotometer; Falcon tube; Water bath; Ice; Adenine;
Guadinine; Cytosine; Thymine; Uracil; E. coli DNA and RNA; sodium citrate; NaCl; DNAse

METHODS

Expose the dry yeast (0.5 g) to a wide tube with reverse condenser. Add 10 ml of 10% sulfate
acid. Place the tube into boiling water for 1 hour then cool the tube and determine compounds
of DNP.

1. Peptides Biuret test. Place test sample (0.5 ml) into the tube, add 500 ul of 10% solution
sodium hydroxide and 200 ul of 1% solution of copper sulfate. Write down the results and
conclusion.

2.”Silver” test on purine nitrogenous bases. Place test sample (1ml) into the tube, add 500
ul of solution ammonium (for alkaline media) and 1 ml of 2% solution silver in ammonia.
Incubate them for 5 min at room temperature to let the colour develop. Write down the results
and conclusion.

3. Benedict’s test. Place test sample (1mL) in clean test tube. Add 1mL of Benedict’s
reagent. Heat the mixture in boiling water for 3-5 minutes and observe the colour change.
Write your results and conclusion.

4. UV Spectrum for Nucleotide Bases The aromatic bases: Adenine, guanine, cytosine, and
thymine of DNA as well as uracil of RNA all absorb ultraviolet light but with different
spectra. In this experiment you will take a wavelength scan to obtain the spectrum of
mononucleotides from 300 nm down to 220 nm. The concentration of nucleotides will be
0.05 mM. Plot the graph from the absorbance obtained.

5. UV Assay of DNA Repeat the procedure in (4) using DNA from E. coli. Plot the graph.
Identify the differences between graph in (4) and (5).
 BCH 3110 Practical Manual

7. Effect of Heat on DNA Measure and record the absorbance of the DNA solution at 260
nm. Heat 100 ul of the DNA solution in a microcentrifuge tube at boiling temperature for 2 13
min. After cooling for ~5 minutes, read the absorbance at 260 nm.

8. Effect of NaOH on DNA On ice, add 20 ul of 2 M NaOH to 200 ul of DNA. As a control

add 20 ul of 2 M NaCl to 200 ul of DNA. Measure the absorbance of each solution at 260
nm. Warm each tube to 70°C and read the absorbance again.

9. Effect of DNase on DNA To 100 ul of DNA, add DNase (from a 10 mg/mL stock) to a
final concentration of 7.5 μg/mL and MgCl2 (from a 25 mM stock) to a final concentration of
25 μM. Incubate at 37oC for 10 minutes and measure the absorbance at 260 nm.

Exercise

1. Describe the principles of the Biuret test.

2. Will the ‘silver’ test be applicable to pyrimidines?
3. Draw the oxidation mechanism reaction that occur in the Tollen’s Test.
4. Explain the reaction that happens during a Benedict’s test.
5. How is spectrophotometry applied to determine nucleic acid’s purity?
6. Describe the wavelength of absorption of a DNA and RNA.
7. Describe the effect of heat, NaOH and DNAse on the structure of DNA.
8. What is the function of DNase (deoxyribonuclease)?
 BCH 3110 Practical Manual

PRACTICAL 5 (NDMN)
14
MEASURING UREASE ACTIVITY

INTRODUCTION

Urea is a small molecule formed as proteins are broken down. It’s excreted in urine, but isn’t
particularly toxic at low levels so it’s found in cells throughout the body. The molecular
structure of urea is below (Figure 1), and as it contains nitrogen (N) several pathogens have
adapted to use it as a nitrogen source using an enzyme called urease to break it down.

Figure 1

Nitrogen is a crucial element for plant growth, but most plants can only use it in the form of
ammonium or nitrate. Only legumes (thanks to the bacteria they live in symbiosis with) and
cyanobacteria can use elemental nitrogen from the air.Many animals excrete urea in their
urine. Soil micro-organisms feed on animal urine, producing urease to transform the urea to
ammonia, which is then readily accessible to plants. This is part of the nitrogen cycle, the
process by which the nitrogen from proteins and other compounds is constantly recycled.

Urease (urea amidohydrolase: EC 3.5.1.5) catalyzes the hydrolysis of urea to yield ammonia
and carbamate. Found in large quantities in jack beans, soybeans, and other plant seeds, it
also occurs in some animal tissues and intestinal microorganisms. Urease is significant in the
history of enzymology as the first enzyme to be purified and crystallized (by James B.
Sumner in 1926). The latter compound spontaneously decomposes to yield another molecule
of ammonia and carbonic acid:

In aqueous solutions, the released carbonic acid and the two molecules of ammonia are in
equilibrium with their deprotonated and protonated forms, respectively. The net effect of
these reactions is an increase in pH.
 BCH 3110 Practical Manual

A. CRUDE UREASE EXTRACTION

15
MATERIALS

~60 g soya beans

Water 150 ml
Mortar and pestle

METHODS

1. Soak the soya beans for at least 4 hours or overnight

2. Drain the soya beans and grind them into small pieces/powder with a mortar and pestle.
3. Add water to free a milky liquid.
4. Centrifuge the crude extract at 15,000 ×g for 10 minutes at 4ºC to remove debris.
5. Discard the pellet and collect the supernatant for next procedures. All procedures are
performed at 4°C.

B. UREASE ASSAY

MATERIALS

750 mM Sodium Phosphate Buffer, pH 7.0 at 25°C (Buffer)

500 mM Urea with 0.05% (w/v) Bovine Serum Albumin Solution (Urea)
0.40% (w/v) p-Nitrophenol (Indicator)
100 mM Standardized Hydrochloric Acid (HCl)
20 mM Sodium Phosphate Buffer, pH 7.0 at 25°C (Enzyme Diluent)
Urease (Enzyme)
Crude Urease

METHODS

1. Pipet (in milliliters) the following reagents into suitable containers:

Reagent Blank Test 1 Test 2 Test 3

Urea

2. Equilibrate to 25°C using a thermostatically suitable waterbath. Staggering each aliquot a

minimum of two minutes add

Reagent Blank Test 1 Test 2 Test 3

Enzyme - 0.10 0.10 0.10

3. Mix by swirling and incubate at 25°C for exactly 5 minutes. Then add:

Reagent 0.20 0.20 0.20 0.20

(Indicator)
 BCH 3110 Practical Manual

Reagent 0.10 - - -
(Enzyme) 16

4. Using a suitable magnetic stirrer, titrate immediately with HCl by adding small amounts
until the color of the indicator turns from yellow to colorless. This is the endpoint. Record the
volume of HCl used for both the Test and Blank solutions.

CALCULATIONS

ΔVHCl = VTest - VBlank

Units/ml enzyme = (HCl Molarity) (∆VHCl) (1000) (dF)

(5) (0.01)
Where:

ΔVHCl = Total Volume (in milliliters) of HCl used in titration

1000 = Conversion factor from millimoles to micromoles as per unit definition
df = Dilution Factor, if applicable
5 = Time of assay (in minutes) as per unit definition
0.10 = Volume (in milliliters) of enzyme used

Units/mg solid = Units/ml enzyme

mg solid/ ml enzyme

Units/g solid = Units/mg solid x 1000

5. Final assay concentration

In a 1.10 ml reaction mix, the final concentrations are 684 mM Sodium Phosphate, 455 mM
Urea, 0.05% (w/v) Albumin, Bovine and 20-40 units Urease.

Question

1. What is the purpose of urease test?

2. Explain the biochemistry of the urease action.
3. What can happen if ammonia builds up in the body?
4. What is the benefit of an organism capable of producing urease?
5. Urea formation begins with breakdown of protein to amino acids. What is this reaction
called?
 BCH 3110 Practical Manual

PRACTICAL 6 (NDMN)
17
ANALYZE GLUTAMATE DEHYDROGENASE OXIDOREDUCTION ACTIVITY

A. CRUDE ENZYME PREPARATION

INTRODUCTION

Glutamate dehydrogenase is known to be located in the liver of catfish. The first step in
enzyme purification is to make a cell-free extract from the source material so that the target
enzyme is in solution. The crude extract will contain a complex mixture of proteins from the
cell cytoplasm, and some additional macromolecules, cofactors and nutrients.

Crude enzyme extracts are prepared by cell disruption or cell lysis, which is achieved by:

1. Non-mechanical methods, including freezing-thawing, osmotic shock, lysozyme treatment

2. Mechanical methods, including ultrasonication, homogenizer, liquid extrusion (French
Press)

Differential centrifugation can be used to separate homogenates components by size.

Normally to purify soluble cytoplasmic proteins we do not need to remove small organelles
like ribosomes or all the membranes from the crude extract. What we only A 7 | Page BCH
3102 Enzymology 2015 need to remove is the big particles (bigger than 10 µm, which is the
mesh size of the net used in a chromatographic column adaptor). The unbroken cells and
other large organelles can be spin down at 10 000 ×g for 5-10 minutes. The debris will be
pelleted down, and the supernatant is recovered

MATERIALS

Buffers and Solutions : 0.1 M sodium phosphate, pH 8.0

1 mM phenylmethylsulfonyl fluoride (PMSF)

Special equipment: Homogeniser

Refrigerated centrifuge
Ultracentrifuge

METHODS

1. Clarias batrachus (catfish) is used as the fresh water test organisms. The fish weighing
>250 g and length ∼15 cm were obtained from the commercial dealers. Glutamate
dehydrogenase (GDH) is extracted from catfish liver.

2. Acclimatize the catfish to laboratory conditions for two days in aquaria filled with 20 liter
of chlorinated-free tap water at 25ºC and aeration of oxygen. Change water daily to maintain
the cleanliness and to eliminate the interfering products of respiration and excretion.
 BCH 3110 Practical Manual

3. Kill the fish by freezing in ice for 20 minutes.

18
4. After 20 minutes, immediately decapitate by cutting with a sharp knife to remove liver
samples.

5. Homogenize approximately 1.0 g of the liver in 20% (w/v) of 40 ml 0.1 M sodium

phosphate buffer pH 8.0 containing 1 mM phenylmethylsulfonyl fluoride (PMSF) using a
homogenizer fitted with a pestle. Homogenizer generates a lot of heat, and it will be
necessary to use an ice bath to preserve the activity of the target enzyme. PMSF is used to
inactivate and remove unwanted serine proteases. (Caution: PMSF is toxic. Do not inhale)

6. Homogenize the liver suspension and centrifuge the crude extract at 15,000 ×g for 10
minutes at 4ºC to remove debris. (Note: When carrying out centrifugation, balancing the
tubes is very important).

7. Discard the pellet and collect the supernatant for purification procedures. All procedures
are performed at 4°C.

B. ENZYME ASSAY

I. NAD-SPECIFIC GDH (OXIDATIVE DEAMINATION) ACTIVITY

INTRODUCTION

GDH catalyzes the reversible NAD-linked oxidative deamination of glutamate into α-

ketoglutarate and ammonia. To explore GDH kinetics in vitro, in the first experiments we
apply different concentrations of glutamate as a substrate in combination with the coenzyme
NAD+ on the so-called NAD-specific GDH activity. The time-dependent appearance of
NADH is measured at 340 nm by spectrophotometry, where the increase in the absorbance
measured is proportional to the GDH activity.

L-Glutamate + NAD⁺ + H₂O -----GDH-----------► α-Ketoglutarate + NH₃ + NADH + H⁺

The appearance of NADH is measured at 340 nm by spectrophotometry.

MATERIALS

A. Buffer solution: 50 mM K-phosphate buffer, pH:7.4 containing 2,5 mM EGTA

B. NAD⁺solution: 100 mM (dissolved in distilled water)
C. L-Glutamate solution: 1 M and 0.05 M (dissolved in distilled water)
D. GDH (GDH1, liver-type): (dissolved in 50 mM K-phosphate buffer, pH:8.3)

• Recording spectrophotometer, able to measure at 340 nm,

• semi-microcuvettes with a light path of 1 cm,
• L-shaped stirring rod made of glass, or plastic (it should fit into the cuvettes, and be able to
carry at least 30 µl of reagent solution),
 BCH 3110 Practical Manual

METHODS
19
1. Prepare the following reaction mixtures in semi-microcuvettes (d=1.0cm)

Reaction mixtures 1 2 3 4 5
Buffer solution (A) 945 µl 935 µl 915 µl 945 µl 935 µl
100 mM NAD (B) 25 µl 25 µl 25 µl 25 µl 25 µl
0.05 M L- 10 µl 20 µl 40 µl
Glutamate (C)
1 M L-Glutamate 10 µl 20 µl
(C)
Calculate the
concentration of L-
glutamate in the
reaction mixtures

The enzymatic reactions are initiated by adding the enzyme at last. Before that, the
absorbance of the cuvettes is set to 0.00 value and this way they serve as blanks. A reagent
blank serves for the correction for the small amount of error in the assay results that would
show up and derive from the reagents (NAD and L-glutamate) themselves displaying some
absorbance at 340 nm.

To initiate the reaction mix in 20 µl of the GDH enzyme solution (E) with a stirring rod and
gently mix the samples to achieve homogeneity. Start measuring reaction time.

2. Record the increase in absorbance at 340 nm for 5 minutes in a spectrophotometer, and

calculate the ΔA per minute from the initial linear portion of the curve (ΔA test).

Time 1 2 3 4 5
(minute)
0

3. Calculations

Reaction rate (v) can be calculated by using the following formula:

 BCH 3110 Practical Manual

Reaction rate (v) (U/ml GDH) = ∆A/min x Vt

20
6.22 x 1.0 x Vs
Were:
Vt: total volume (1ml)
Vs: volume of the added enzyme also in ml (0.02ml)
6.22: millimolar extinction coefficient of NADH at 340nm (㎠/micromole)
1.0: light path length (cm)

GLUT ∆A per 5 minutes ∆A per minutes V (µmol/min/ml)

concentration (mM)
1

DISCUSSION

Graphical display of the results: Plot the values for substrate (L-glutamate) on the x-axis
(abscissa) and the corresponding calculated values for V on the y-axis (ordinate). Estimate
the values of apparent Km and Vmax.

II. NADP-SPECIFIC GDH (OXIDATIVE DEAMINATION) ACTIVITY

INTRODUCTION

In the second experimental set up we assay the NADP-specific GDH activity, where the
enzymatic reaction is carried out applying glutamate as a substrate in combination with
NADP as a coenzyme.

L-Glutamate + NADP⁺ + H₂O -----GDH----------► α-Ketoglutarate + NH₃ + NADPH + H⁺

The formation of NADPH is measured at 340 nm by spectrophotometry.

MATERIALS

A. Buffer solution: 50 mM K-phosphate buffer, pH:7.4 containing 2,5 mM EGTA

B. NADP⁺solution: 100 mM (dissolved in distilled water)
C. L-Glutamate solution: 1 M (dissolved in distilled water)
D. GDH (GDH1, liver-type) diluent: (dissolved in 50 mM K-phosphate buffer, pH:8.3)
 BCH 3110 Practical Manual

METHODS
21
1. Prepare the following reaction mixture in semi-microcuvette (d=1.0cm):

Reaction mixtures 1
Buffer solution (A) 935 µl

100 mM NADP+ (B) 25 µl

1 m L-glutamate (C) 20 µl

Calculate the concentration of

L-glutamate in the reaction
mixtures

The above-prepared reaction mixture serves as absorbance reference (reagent blank).

Therefore you must set the zero-absorbance value of the photometer with these.

The enzymatic reactions are initiated by adding the enzyme at last.

To initiate the reaction mix in 20 µl of the GDH enzyme solution (E) with a stirring rod and
gently mix the sample to achieve homogeneity. Start measuring reaction time.

2. Record the increase in absorbance at 340 nm for 5 minutes in a spectrophotometer, and

calculate the ΔA per minute from the initial linear portion of the curve (ΔA test).

Time 1
(minute)
0
1
2
3
4
5

3. Calculations

Reaction rate (v) can be calculated by using the following formula:

Reaction rate (v) (U/ml GDH) = ∆A/min x Vt

6.22 x 1.0 x Vs
Were:
Vt: total volume (1ml)
Vs: volume of the added enzyme also in ml (0.02ml)
 BCH 3110 Practical Manual

6.22: millimolar extinction coefficient of NADH at 340nm (㎠/micromole)

1.0: light path length (cm) 22

III. NADH-SPECIFIC GDH (REDUCTIVE ANIMATION) ACTIVITY

INTRODUCTION

Finally, assaying NADH-specific GDH (reductive amination) activity is based on the fact that
the disappearance of NADH, which is measured at 340 nm by spectrophotometry, when the
enzyme assay is carried out using α-ketoglutarate, NH₃+, and NADH.
α-Ketoglutarate + NH₃ + NADH + H⁺ --------GDH------------► L-Glutamate + NAD⁺ + H₂O

The oxidation of NADH is measured at 340 nm by spectrophotometry.

MATERIALS

A. Buffer solution: 100 mM K-phosphate buffer, pH:7.4 containing 2,5 mM EGTA

B. NH₄Cl solution: 0.5 M (dissolved in distilled water)
C. α-Ketoglutarate solution: 1 M (dissolved in distilled water, pH to 7.0 with Tris)
D. NADH solution: 10 mM (dissolved in distilled water)
E. GDH (GDH1, liver-type): (dissolved in 50 mM K-phosphate buffer, pH:8.3)

METHODS

1. Prepare the following reaction mixture in semi-microcuvette (d=1.0cm):

Reaction mixture 1
Buffer solution (A) 920 µl

NH4Cl solution (B) 20 µl

α-ketoglutarate solution (C) 20 µl

This reaction mixture (A+B+C) is used to zero the instrument as a reagent blank. (When
zeroing the instrument on a reagent blank, only the color of NADH that decreases from
reaction with the GDH is measured).

Add 30 µl of the NADH solution (D) with a stirring rod and gently mix the sample to achieve
homogeneity. After that record this baseline for about 0.5 min.

The enzymatic reactions are initiated by adding the enzyme last.

To initiate the reaction, mix in 10 µl of the GDH enzyme solution (E) with a stirring rod and
gently mix the sample to achieve homogeneity. Start measuring reaction time.
 BCH 3110 Practical Manual

Record the decrease in absorbance at 340 nm for 5 minutes in a spectrophotometer, and 23

calculate the ΔA/minute from the initial linear portion of the curve (ΔA test).

Time 1
(minute)
0
1
2
3
4
5

2. Calculations

Reaction rate (v) can be calculated by using the following formula:

Reaction rate (v) (U/ml GDH) = ∆A/min x Vt

6.22 x 1.0 x Vs
Were:
Vt: total volume (1ml)
Vs: volume of the added enzyme also in ml (0.02ml)
6.22: millimolar extinction coefficient of NADH at 340nm (㎠/micromole)
1.0: light path length (cm)

CONCLUSION AND PROBLEMS

1. Give the apparent Km for glutamate and vmax of GDH activity using NAD+ as coenzyme
2. Compare the reaction rates of both of NADP-dependent GDH (oxidative deamination) and
NADH-dependent specific GDH (reductive amination) reaction
 BCH 3110 Practical Manual

PRACTICAL 7 (NDMN)
24
ESTIMATION OF GLUTATHIONE (GSH) BY USING ELLMAN’S REACTION

Introduction
In human body, the cells contain tripeptide glutathione (GSH), which is a substance made
from three amino acids. The principle behind the assay is that the sulfhydryl group of
glutathione reacts with DTNB, thereby producing a yellow-coloured 5-thio-2-nitrobenzoic
acid (TNB). The mixed disulphide GSTNB (GSH and TNB), that is concomitantly produced
is reduced by glutathione reductase to recycle the GSH and produce more TNB. The rate of
TNB production was directly proportional to the concentration of rGSH in the sample. Thus,
measurement of the absorbance of TNB at 412 nm provides an accurate estimation of the
amount of glutathione in the sample.
Materials/Chemicals/ Reagents;
1. 25% Trichloroacetic acid (TCA)
2. 5% TCA
3. 0.1 M potassium phosphate buffer of pH 7.6 (should be prechilled)
4. 1.0 mM DTNB (5’5’-dithio bis 2-nitrobenzoic acid; 396.36 M.W.) in phosphate
buffer of pH 7.6
5. Standard GSH solution (L-Reduced glutathione; 307.32 M.W.); 0.2 – 1.0 mM in 5 %
TCA
6. EDTA
7. Catfish tissue liver

Methods
A. Preparation of crude extract

1. Weight about 5 g of catfish liver. Trim fat, connective tissue, and blood vessels from
the liver tissue and dice into pieces of a few grams prior to homogenize using
homogenizer in 10 ml 0.1 M potassium phosphate buffer pH 7.6 containing 1 mM
EDTA.
2. Add 2.5 ml of 25% TCA to liver homogenate to precipitate the protein. Make sure to
work all the way in a prechilled condition.
3. Cool the tubes in ice for 10 minutes and dilute the mixture with 7.5 ml of 5% TCA
and centrifuge at 5000 X g for 15 minutes.
4. Take out 0.2 ml of the supernatant for the assay.
5. Initiate the reaction by adding 0.2 ml of supernatant to 2.3 ml of phosphate buffer (pH
7.6) followed by the addition of 0.5 ml of 1 mM DTNB solution.
6. Shake the reaction and incubated at room temperature for 5 minutes.
7. Read the absorbance at 412 nm by using a spectrophotometer. Prepare three reactions
and read the absorbance for each.
 BCH 3110 Practical Manual

B. Standard curve of GSH

25
1. Prepare GSH at the concentration ranging from 0.2, 0.4, 0.6, 0.8 and 1.0 mM of GSH
(reduced glutathione) in 5% TCA. Make a serial dilution from 1 mM stock solution of
GSH.
2. Follow same procedures in ‘A’ above by replacing 0.2 ml of supernatant with 0.2 ml
standard GSH from each serial dilution in triplicates.
Notes:
1. The fatty tissue surrounding the organ/tissue must be scrupulously removed prior to
homogenization, as it can often interfere with subsequent protein isolation from the
homogenate.
2. Homogenize at full speed for 1-3 min depending on the toughness of the tissue. For
long periods of homogenization, it is best to blend in 40-s to 1 min burst with a few
minutes in between to avoid excessive heating. This will also reduce foaming.

Discussion
Complete information of serial dilution, the real concentrations and absorbance of the
standard GSH and samples as in the table below:
Standard Serial Actual ABS 1st ABS 2nd ABS 3rd Average Actual
GSH or dilution conc of ABS ABS
sample of GSH GSH in ((1+2+3)/3)
(mM) the
mixture
(uM)
1 0.2
2 0.4
3 0.6
4 0.8
5 1.0
Sample

Construct your own standard curve by using those values and determine the concentration of
GSH in the samples by using the curve. Determine how much concentration of GSH in µM
for the sample liver and also express the concentration in nmole GSH/g, wet weight of the
liver tissue, based on the standard curve of GSH.
 BCH 3110 Practical Manual

Questions
26
1. In human body, the cells contain tripeptide glutathione (GSH), which is a substance
made from three amino acids. Name the amino acids and explain how those amino
acids working each other to produce glutathione?
2. In its reduced form, glutathione plays key roles in the cellular control of reactive
oxygen species. What is the importance of glutathione to human body?
3. Glutathione also plays a role in a variety of other metabolic phenomena and is a
coenzyme for certain enzymic reaction. Name the enzymic reaction and how
glutathione acts in the reaction.
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PRACTICAL 8 (NDMN)
27
DETERMINATION THE REACTION OF GLUTAMINASE

INTRODUCTION

The conversion of glutamate to glutamine, catalysed by glutamine synthase, is of great

significance in the utilization of ammonia. This reaction converts glutamate and ammonia to
glutamine with the hydrolysis of ATP.

L-glutamate + NH3 + ATP  L-glutamine + ADP + Pi

This reaction takes place in a number of mammalian tissues, eg; liver, brain, kidney, muscle
and intestine. Glutamine which is widely distributed in mammalian tissues, is not only an
essential building block of proteins, but is a central compound in nitrogen metabolism.
Glutamine functions in the uptake, storage, and formation of ammonia, the homeostatic
control of amino acid balance and also synthesis of the purine and pyrimidines moieties of
nucleic acids, ATP and other nucleotides. Glutamine also vital for the formation of amino
sugars and the biosynthesis of a number of amino acids and other compounds of biological
importance.

Glutaminase is present in the mitochondria of many animal organs. It is activated by

inorganic phosphate. In the absence of phosphate, enzyme activity is very low. There are two
isoforms of glutaminase: the first one only occurs in liver, and the other one in the kidney and
other organs. The accumulated evidence indicates that mammalian tissues have a
homeostatic metabolic mechanism for preserving amino acid balance in which the dietary
nonessential amino acids, especially glutamine, function to maintain the tissue levels of the
other amino acids by preventing the loss of essential carbon chains.

With the presence of glutamine synthase and glutaminase, production of glutamate and
glutamine play a central role in the metabolism of amino acids and ammonia.
The reactions catalysed by glutamine synthase and glutaminase are presented in the following
figure.
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28
In this experiment, a glutamine consumption indicator is the amount of ammonia released as
a result of its hydrolysis. Other than kidney, liver is the organ that consumes glutamine. It
contains glutaminase, therefore the incubation of liver with glutamine, ammonia is released.

There are many ways to detect presence of ammonia. One of them is colorimetry method
based on its reaction with phenol in the presence of chlorate (I). A coloured reaction product
forms, and the color intensity of the solution is proportional to the concentration of ammonia.
Presence of ammonia in the reaction relatively related to the presence of glutaminase in the
homogenized liver.

In detection of glutamine synthase activity, the assay relies on the y-glutamyl transferase
reaction, eg; formation of glutamyl-γ-hydroxamate from glutamine and hydroxylamine.

As shown in formula, the relation is 1:1, which allow to calculate the enzyme activity directly
from the concentrations in the standard curve of glutamic acid-γ-hydroxamate.

MATERIALS

Homogenized tissue liver, L-glutamine, 0.15 M phosphate buffer pH 8.0, Eppendorf tubes,
test tubes, stopper for test tube, trichoroacetic acid, table top centrifuge, incubator, phenol
reagent, 95% ethyl alcohol, 0.5% (w/v) sodium nitroprusside, 0.02 M sodium chlorate (I), 0.9
M Potassium hydroxide (KOH), 0.5% (w/v) Sodium hydroxide (NaOH), MilliQ water,
spectrophotometer.

METHODS

I. To measure glutaminase activity

A) Tissues preparation
Use prepared homogenized liver of catfish.

B) Glutamine solution, phenol reagent, sodium nitroprusside & sodium chlorate

reagent preparation

Glutamine solution: Dissolve 142 mg of glutamine in 50 ml of 0.15 M phosphate buffer

pH 7.5.
 BCH 3110 Practical Manual

Phenol reagent: Dissolve 6 g of phenol in 60 ml of 95% ethyl alcohol.

29
Sodium nitroprusside (SNP): Dissolve 0.3 g SNP in 60 ml of dH2O. Must be prepared
in dark bottle / bottle covered with aluminium foil as SNP is light sensitive. (BE
CAREFUL, Sodium nitroprusside is TOXIC, wear proper glove and mask during
preparation)
0.02 M Sodium chlorate (I) in 120 ml of 0.5% (w/v) NaOH: Prepare 120 ml of 0.5%
(w/v) NaOH. Next, weigh sodium chlorate (I) to prepare 120 ml of 0.02 M sodium
chlorate in 0.5% (w/v) NaOH. (M.W NaClO3; 106.44 g/mol)
C) Assay preparation and incubation
1) Prepare 12 Eppendorf tubes for marked with the symbols I-5, I-10, I-15, I-20, I-25,
I-30, II-5, ... II-30. The first digit indicates the number of the reactive system (see
table), the second- the incubation time in minutes. To each of them, add 0.1 ml of 1
M trichloroacetic acid.
2) In two test tubes labelled as I and II (to differentiate reaction system), put 5 ml of
homogenized liver. To the test tubes containing homogenized liver, add 5 ml of the
appropriate incubation liquid, with glutamine or without glutamine (according to
the Table I), and then incubate the content at 37°C for 30 minutes.

Table I: Volume of two different reaction system (I and II) for the experiment.
Reaction Organ tissue 0.15 M phosphate Glutamine solution
buffer pH 7.5 in 0.15 M phosphate
system
buffer pH 7.5
[ml]
[ml]
I Liver 5 -
II Liver - 5

3) After interval of 5, 10, 15, ..and 30 minutes of incubation, collect from the test tube
(I and II), 1 ml of incubation fluid and transfer to the previously prepared, labelled
respective Eppendorf tubes containing trichloroacetic acid (to stop the reaction by
precipitation of protein). After five minutes end of incubation time, add to each tube
0.1 ml of 0.9 M KOH solution in order to neutralized the trichloroacetic acid.

4) Mix the contents in each Eppendorf tubes and centrifuge at a speed of 2000 rpm for
10 minutes.

D) Colorimetric determination of ammonia

Prepare 14 test tubes. To two test tubes (for control samples), transfer 0.5 ml of phenol
reagent, 0.5 ml of sodium nitroprusside and 0.1 ml of ammonia-free water (MilliQ
water).
 BCH 3110 Practical Manual

To the properly labelled remaining 12 test tubes (labelled according reaction system
and time of incubation), transfer 0.5 ml of phenol reagent, 0.5 ml sodium nitroprusside 30
and 0.1 ml of supernatants.

Next, to each test tube of samples and two controls, add 1 ml chloride reagent (0.02 M
sodium chlorate (I) in 0.5% NaOH), close with the stoppers, mix, and place for 20
minutes in a water bath at 50°C. After that time, cool (do not open) and measure the
absorbance to the control samples at the wavelength of 625 nm.

DISCUSSION
Why phosphate buffer is used as the reaction buffer?
After reaction was stopped by TCA and KOH, the reaction was centrifuged results in
separation of pellet and supernatant. What is the pellet in the tube represents? Between the
two reaction systems, which one produces more pellet? Why glutamine is added to one of the
reaction systems? What can you conclude from the results that has been observed?
Based on the results obtained, present in one coordinate system diagrams of the absorbency
dependence on the incubation time for all two incubation systems. Discuss your results.

QUESTIONS

1. In muscle, glutamine synthase is very active, catalysing the formation of glutamine

from glutamate and ammonia at the expense of a molecule of ATP. In the liver or
kidney, the rate of formation of glutamine is very low, but a high level of glutaminase
activity, which generates ammonia and glutamate, is observed. How would you
explain the difference in the levels of enzyme activity in these two organs?
2. Most of the proteins synthesized in mammals contain all 20 amino acids. More
protein is degraded than is synthesized when even one essential amino acid is missing
from the diet. A) under such conditions, how could an increase in the rate of protein
degradation provide the missing amino acid? B) how does an increase in the rate of
protein degradation contribute to increased levels of nitrogen excretion?
3. Normal human blood plasma contains all the amino acids require for the synthesis of
body proteins, but not in equal concentrations. Alanine and glutamine are present in
much higher concentration than any other amino acids. Suggest why.
4. Ammonia is highly toxic to animal tissue. Describe in details in what condition is
ammonia degraded by urea and what is the other condition cause ammonia to be
degraded by glutamine synthesis?
5. What is the normal blood ammonia concentration in human? What is the effect of
excessive cerebral ammonia uptake to human body?
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