PRACTICAL 1

POLYPHENOL OXIDASE ACTIVITY OF BANANAS

Polyphenol oxidase (-diphenol:O2 oxidoreductase) is a copper containing enzyme commonly found
in plant tissues. It catalyses the oxidation of -diphenols (e.g. catechol) to -quinones (e.g. -
benzophenone). Quinones so formed are highly reactive and polymerise spontaneously to give
yellow or brown products. These products can be observed when plant tissue, such as potato or
apple is cut and the enzyme is exposed to atmospheric oxygen. Polyphenol oxidase is of interest
with regard to the role of phenols in the resistance of plants to infection, as a rise in the level of the
enzyme is often noted in infected tissue and the polymerized products from the reaction are
fungiostatic.

Materials.
1. Bananas
2. 0.1M acetate buffer, pH 5.6
3. Pestle and mortar
4. Picollo centrifuge
5. Glasswool
6. Ice bucket
7. Spectrophotometer
8. 0.06M catechol buffer
9. Stopwatches
10. Funnels

Method.

Establishing a linear progress curve.

Take a banana and carefully peel it. Take the fruit and mull in 0.1M acetate buffer pH 5.6 in a chilled
mortar. The amount used will depend on the material available, usually 2ml of buffer is required for
1g of tissues. Centrifuge on a picollo for 1 minute. Filter the supernatant through glasswool and store
in a test tube on ice. This serves as the enzyme preparation. Zero the spectrophotometer using the
catechol-buffer. Mix 0.5ml of enzyme preparation to 4.5ml of catechol-buffer in a test tube, and
immediately read at 450nm every 30 seconds for at least 5 minutes

pH optimum.
Mix 0.5ml of enzyme preparation from the fruit to 4.5ml of catechol-buffer (0.1M acetate pH 3.7;
4.8; 5.6 and 0.1M phosphate pH 6.4; 7.0); in a test tube and incubate for 5 minutes at room
temperature. Immediately put the tubes in boling water for 1 minute. Read the absorbance at
450nm, after calibrating the spectrophotometer using catechol-buffer.

Temperature optimum.
Mix 0.5ml of enzyme preparation (fruit fraction) to 4.5ml of catechol-buffer (0.1M acetate pH 5.6)
and incubate for 5 minutes in the fridge, at room temperature, 37oC, 50oC and 70oC. Immediately
put the tubes in boiling water for 1 minute. Read the absorbance at 450nm, after calibrating the
spectrophotometer using catechol-buffer.

Determination of Km.
Km is measured at room temperature from the initial velocity of attack on catechol over the substrate
concentration range: 0.1mM, 0.5mM, 1mM, 3mM, 7mM, and 10mM. Enzyme solution is diluted until
the reaction progress curve for the lowest substrate concentration is linear for at least 3.5 minutes.
Average velocity over 3.5 minutes is then taken as a measure of initial velocity at this and higher
substrate concentrations.

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PRACTICAL 2

THE PRODUCTION OF PYRUVATE AND ACETALDEHYDE DURING THE FERMENTATION OF

GLUCOSE BY YEAST.

Fermentation.
The first stage of glucose metabolism in yeast involves the production of pyruvate. This process,
which takes place in a series of reactions with a large number of intermediates, is known as
glycolysis. The overall reaction can be represented as the transfer of two pairs of hydrogen atoms
from glucose to NAD+ to form NADH. Since NAD+ is present in only catalytic amounts, NADH must
be reoxidized to replenish NAD+if the process is to continue. NADH is converted to NAD+ by reaction
with molecular oxygen via the respiratory chain. Under anaerobic conditions, this is not possible and
reoxidation takes place when pyruvate is converted to acetaldehyde, then ethanol.

Demonstration of metabolic intermediates.

The metabolites pyruvate and acetaldehyde are normally present at only low concentrations, so, in
order to demonstrate their existence as intermediates on the pathway, some means has to be found
to prevent the reaction proceeding any further. This is a general approach used widely in
investigating metabolic pathways and usually involves blocking the enzyme that catalyses the
conversion of the compound under investigation, by adding an inhibitor. Alternatively, the
physiological conditions can be changed so that the enzyme operates at well below its maximum
activity, or a 'trapping' agent can be added which reacts with the intermediate to form a compound
that is not metabolized further. One of these latter two approaches are illustrated in the following
experiment.

Pyruvate decarboxylase is inactive in slightly alkaline solution, so pyruvate accumulates and its
presence is demonstrated by the reaction with sodium nitroprusside.

Materials
1. Disodium hydrogen phosphate (0.5 mol/L).
2. Potassium dihydrogen phosphate (0.5 mol/L).
3. Yeast suspension (100 g/L in Na2HP04).
4. Yeast suspension (100 g/L in KH2PO4).
5. Yeast suspension (100 g/L in water).
6. Glucose (100 g/L).
7. Sodium nitroprusside (50 g/L, prepare just before use).
8. Ammonia.
9. Ammonium sulphate.
10. Sodium sulphite.
11. Sodium hydroxide (100 g/L).
12. Piperidine (30 g/L aqueous solution).
13. Trichloracetic acid (100 g/L).
14. Water bath at 37C.

Method

Formation of pyruvate from glucose.

Pipette 5 ml of glucose solution into two boiling tubes (A and B), add 5 ml of the yeast suspension
in the slightly alkaline solution of Na2HPO4 to tube A and 5 ml of the yeast suspension in the acid
solution of KH2PO4 to tube B. Place the tubes in a water bath at 37 C for 1 h. Observe the tubes
during the incubation period and note their appearance. Are there any differences? What do you
think is the reason for that? At the end of the incubation period, add 2 ml of trichloracetic acid solution
to each tube, mix thoroughly, and centrifuge for 20 min on a benchtop centrifuge. Remove the
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supernatant and test for the presence of pyruvate.

Sodium nitroprusside test for pyruvate.

Add 2 ml of boiled supernatant to about 1 cm of solid ammonium sulphate in a test tube. Add two
drops of freshly prepared sodium nitroprusside solution (50 g/L), mix thoroughly, and run
concentrated ammonia carefully down the side of the tube so as to form two layers. If pyruvate is
present, a green or blue ring forms at the junction of the two liquids. A transient pink ring may appear
before the characteristic blue or green colour given by pyruvate.

Formation of acetaldehyde from glucose.

Pipette 5 ml of the glucose solution into two tubes C and D, add 5 ml of the yeast suspension in
water to both tubes, and 0.5 g of sodium sulphite to tube D. Mix thoroughly and incubate the two
tubes at 37 C for 1 h. Observe the tubes during the incubation period and note their appearance.
Are there any differences? What do you think is the reason for that? At the end of the incubation
period, centrifuge the tubes, remove the supernatant, and add 0.5 ml of freshly prepared sodium
nitroprusside to 2 ml of the supernatant followed by 2 ml of aqueous piperidine and mix. If
acetaldehyde is present then a blue colour is observed.

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PRACTICAL 3

EXAMINATION OF SUCCINIC ACID DEHYDROGENATION.

One of the reactions in the citric acid cycle is the dehydrogenation of succinic acid to produce fumaric
acid. One of the first discoveries about this cycle was that certain small organic acids increased the
rate of aerobic respiration. Other small acids did not speed the reaction and instead acted as
inhibitors. We will check this with our yeast suspensions today.

The added methylene blue will act as an artificial indicator and electron acceptor. Oxidized
methylene blue is blue, but when reduced it becomes colorless. Here we will use it in place of the
normal electron acceptor FAD+. So, the speed at which the blue color disappears is an indication of
the metabolic rate of the yeast (or at least the activity of the citric acid cycle).

Materials
1. 0.5 M glucose
2. 0.5 M fructose
3. 0.5 M succinic acid
4. 0.5 M malonic acid
5. 0.1% (w/v) Methylene blue
6. Mineral oil
7. Clean test tubes
8. Test tube racks
9. An incubator at 37oC
10. Ice buckets

Method
1. Label 8 small test tubes.
2. Prepare tubes as outlined in the next table. (you will decide with your team what to add to tubes
E through I)
3. Add 100l of 0.1% methylene blue to each tube and mix well. The solution should be a light blue
in color.
4. Hold each tube at a slight angle and add 500l mineral oil to form a thin film over the top of the
solution. This will slow the diffusion of oxygen into the system.
5. Place tubes in 37C incubator and observe at 5 minute intervals. Note any color changes.

Tube # Yeast Glucose Fructose Succinic Malonic Water Incubation

suspension acid acid temperature
A 1ml - - - - 2ml 37oC
B 1ml 1ml - 1ml - - 37oC
C 1ml 1ml - - 1ml - 37oC
D 1ml 1ml - 0.5ml 0.5ml - 37oC
E 1ml - 1ml 1ml - - 37oC
F 1ml - 1ml - 1ml - 37oC
G 1ml - 1ml 0.5ml 0.5ml - 37oC
H 1ml 1ml - - - 1ml 37oC
I 1ml - 1ml - - 1ml 37oC

Record the length of time it took each tube to turn from blue to colourless.

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Tube number Time to colourless
(minutes)
A
B
C
D
E
F
G
H
I

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2016 BCH223 practical guide NT Mazomba
PRACTICAL 4

THE HILL REACTION IN ISOLATED CHLOROPLASTS

Illuminated chloroplasts generate NADPH and evolve oxygen. During this process, water undergoes
photolysis (the Hill reaction) to produce protons and oxygen. In this practical, illuminated spinach
chloroplasts will be shown to reduce 2,6-dichlorophenolindophenol (DCPIP) an artificial H-acceptor.

Materials
1. Fresh spinach leaves
2. Isolation medium: 0.4M sucrose in 0.06M potassium phosphate buffer, pH 6.5
3. Reaction medium: 0.01M KCl in 0.03M potassium phosphate buffer, pH 6.5
4. Blender
5. Muslin
6. 0.1mM DCPIP in reaction medium
7. Sodium dithionite
8. Light box

Method

Preparation of chloroplasts.
Wash spinach leaves in distilled water, allow to drain and place them in a plastic bag and keep at
5oC for a few hours until they are turgid. (All subsequent manipulations are performed with ice-cold
materials.) Remove mid-ribs, and homogenize leaves (50g) in isolation medium (50ml) in a blender
for 2 min. Filter the resulting suspension through 5 layers of muslin, and centrifuge (200g, 5oC, 2
min) to sediment unbroken cells, nuclei and leaf debris. Centrifuge (1000g, 5oC, 10min) the
supernatant fraction to sediment chloroplasts.

The Hill reaction.

Dilute the chlorophyll suspension with reaction mixture to give a chlorophyll concentration of ca.
5mg ml-1 and mix the diluted suspension (1.0ml) gently with DCPIP solution (9ml). Prepare three
tubes in this way. Place one tube is in the dark. Read the absorbance at 520nm of the second tube
immediately against that of the third tube to which a few crystals of sodium dithionite have been
added to reduce, and therefore to decolourize, the dye. Prepare a fourth tube using triple quantities
and expose this tube to light from a lightbox. Withdraw samples (4ml) from the illuminated tube at 7
min intervals and read their absorbance at 520nm against the third tube. Take the last sample 28min
after the start of illumination. Determine the absorbance of the tube kept in the dark at the end of
the experiment.

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PRACTICAL 5

SPECTROPHOTOMETRIC QUANTITATION OF ANTIOXIDANT CAPACITY OF A PLANT LEAF

EXTRACT

Production of free radicals takes place as a result of normal metabolic processes in the body
especially that which happens during cellular respiration in the mitochondria. Reactive oxygen
species are detrimental to biological molecules in the cells, hence destroying cell membranes,
nucleic acids and proteins which in turn lead to aging and other diseases such as cardiovascular
diseases and cancer.

The search for free radical scavengers is thus an important component in drug discovery. Most of
the antioxidants are part of our diet, but some are in ointments, etc. In this experiment, antioxidants
will be extracted from plant leaves. Leaves will be air dried, homogenized, and soaked in 95%
ethanol for 48 hours. The suspension will be, filtered and concentrated in vacuum to yield the crude
ethanol extract.

You will determine the antioxidant capacity of the leaf extract using -tocopherol (Vitamin E), one of
the well-known antioxidants, as a standard.

Materials

1. Tocopherol solution (10mM)

2. Phosphomolybdenum reagent
3. Tubes
4. Incubator at 80OC
5. Spectrophotometer

Method

1. Extract your leaf as follows.

2. Look for a simple leaf extract method, such as hexane followed by ethanol.
3. Label seven tubes 1-7. Thoroughly mix various solutions as follows;

Tube number 1 2 3 4 5 6 7
Phosphomolybdenum reagent (ml) 5.0 5.0 5.0 5.0 5.0 5.0 5.0
Ethanol (ml) 0.5 0.4 0.3 0.2 0.1 0.0 0.0
-Tocopherol solution (ml) 0.0 0.1 0.2 0.3 0.4 0.5 0.0
Leaf extract (ml) 0.0 0.0 0.0 0.0 0.0 0.0 0.5

4. Do this in duplicate.
5. Incubate the tubes at 80OC for 15 minutes.
6. Cool the tubes, dilute to 25ml with distilled water and read absorbance at 695nm.

Practical writeup.
1. Determine the concentration of the antioxidant in the unknown sample using your results.
2. What is an antioxidant, and what is its importance?
3. Write briefly about another method you may use to prepare an antioxidant extract from plant
leaves.

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2016 BCH223 practical guide NT Mazomba