Hill Reaction

The physiological reactions of mitochondria and chloroplast can be reduced to a series of electron transfers,
catalyzed by specific enzymes found within the organelles. Thus, we can study the component processes of
photosynthesis and respiration by isolating the organelles and measuring specific enzyme activity associated with
that organelle. Photosynthesis requires two processes which can be functionally dissociated, although they work as
a unit within the chloroplast. The first process is known as the light reactions, while the second is known by
analogy as the dark reactions. The light reactions are rapid changes in the subatomic arrangement of molecules
that ultimately split water in the presence of light (photodissociation). Hydrogen atoms from the water are use to
reduce NADP to NADPH2, and are then used to reduce CO2 to CH2O.
In the light reaction, O2 is also photochemically released from water together with ATP and NADPH. This
is known as the PHOTOCHEMICAL or HILL REACTION. Robert Hill showed that when isolated chloroplasts
were supplemented with an artificial hydrogen-acceptor molecule and then illuminated, evolution of oxygen and
simultaneous reduction of the hydrogen acceptor took place, according to the reaction:
2H2O + 2A → 2AH2 + O2
(Light)
A = artificial hydrogen acceptor
AH2 = the reduced form
The reaction proved that the oxygen evolved does not come from CO2, and the reaction became known as the Hill
Reaction. The Hill Reaction can be followed by either measuring the oxygen evolved, or by measuring the rate of
reduction of the hydrogen acceptor “A”. If “A’ is a dye that changes color as if accepts electrons (reduction) then
the change in color intensity can be conveniently determined spectrophotochemically. DPIP or 2,6-
dichlorophenolindophenol is suitable for the measurement of the rate of Hill Reaction. DPIP is blue in the
oxidized form and becomes colorless when reduced

Oxidized Reduced

Figure 7.1. The structure of DPIP in its oxidized and reduced forms

Objectives
At the end of the exercise, the students should be able to:
1. Discuss the Hill Reaction
2. Measure the occurrence of the Hill reaction spectrophotometrically using 2,6-dichlorophenolindophenol as
hydrogen acceptor.
3. Determine the role of chloroplast are light in the Hill Reaction
Materials
1. Cone Type coffee filter paper
2. Acetone (a common nail polish remover) You may also use rubbing alcohol if needed.
3. Glass container or shallow pan
4. 2 empty pickle jars with lids that can be sealed (this can be substituted with a glass and aluminum foil)
5. pencil
6. tape
7. paint brush
8. fresh spinach, flower petals, vegetable shavings, etc.
 9. Fresh spinach leaves
10. Metal paper hole punch
11. 10 mL or larger plastic syringe (without needle) - get one from your local pharmacy
12. Baking soda solution (dissolve some baking soda powder in water)
13. Liquid dish soap solution (dissolve 5 mL in 250 mL of water)
14. 3 clear plastic cups or beakers (250 mL to 500 mL)
a. Cup 1: Detergent solution
b. Cup 2: Baking soda solution (treatment)
c. Cup 3: Water (control)
15. Light source (fluorescent is good because it produces light without much heat)

Procedure
A. Isolation of Chloroplasts
1. Weight 10 grams of fresh papaya leaves, which have been previously washed with distilled water and
removed of big viens.
2. Add the leaves into 50 ml cold 0.35M NaCl solution in an ice-cold blender.
3. Blend at high speed for 30 seconds, then cool the jar for 30 seconds. Do this three times.
4. Strain the extract through double-layered cheesecloth into an ice-cold beaker.
5. Transfer the suspension into four cold centrifuge tubes making sure that they are of equal weight.
6. Centrifuge at 200 X g for 5 minutes
7. Decant the supernatant into four clean centrifuge tubes and discard the pellet.
8. Centrifuge at 1400 x g for 15 minutes. Discard the supernatant and resuspend the pellet by adding 5 ml
0.35M NaCl to each tube. Collect all suspensions in a small beaker. (Note: Compute for rpm value of he
angular speeds given in g using r = 8 cm).
B. Determination of chlorophyll concentration
1. Dilute 0.1 ml of the prepared chloroplast suspension to 20 ml of 80% acetone to extract the chlorophyll
(1:200 dilution)
2. Set the wavelength selector of the spectrophotometer at 652 nm. Zero the instrument with a blank of 80%
acetone and measure and record the absorbance of the chlorophyll solution. After measuring the
absorbance discard this 1:200 dilution.
3. Calculate the amount of total chlorophyll per ml of the chloroplast suspension:

c = A/ab (DF)
mg chlorophyll/ml = absorbance/34.5 (200)
mg chlorophyll/ml = 5.7 x absorbance

4. Dilute the original suspension with the proper amount of cold 0.35M NaCl solution to obtain a
concentration of 0.05 mg chlorophyll/ml. Use the relationship:

C1V1 = C2V2
Example: After the addition of acetone, a 1:200 dilution of chlorophyll extract registered an
absorbance of 0.06. (a) Calculate the amount of total chlorophyll/ml of suspension. (b) Calculate
the volume of 0.35M NaCl needed to obtain 25 ml of 0.05 mg/ml chlorophyll concentration.
(a) mg chlorophyll/ml = 5.7 (0.06) = 0.342
(b) C1V1 = C2V2
 (0.342 mg/ml)(V1) = (0.05 mg/ml) (25 ml)
V1 = 3.65 ml of the original chloroplast suspension
Vol. NaCl needed = 25 ml – 3.65 ml
= 21.35 ml 0.35M NaCl to be added.

C. Light Reaction vs. Light-Independent Reaction (Home base experiment)

1. Use the metal hole punch to cut out 20 circular disks from the fresh spinach leaves, 10 for a control and 10
for a treatment.
2. Separate the two parts of the syringe, drop 10 of the spinach disks inside, reassemble the syringe.
3. Push the plunger almost to the bottom but don't crush the disks.
4. Control or treatment
• For the treatment, draw up a small amount ~1 mL of detergent solution, and then draw the baking
soda solution up to ~3-5 mL
• For the control, draw up a small amount ~1 mL of detergent solution, and then draw the water up
to ~3-5 mL
5. Point the syringe upward, tapping the sides, so that any air bubbles rise, and gently squeeze the syringe
until liquid begins to come out.
6. Put a finger on the end of the syringe, and draw the plunger back slightly, creating a partial vaccum.
7. Repeat until the leaf disks are suspended in the solution. This action forces the liquid into the interior of the
leaf.
8. Pour the contents of control and treatment syringes into two labelled clear plastic cups.
9. Swirl the liquid to try to keep the disks from sticking to each other or the sides of the cups and then let them
sit.
10. Turn on a bright light and monitor the disks every minute. Count how many disks are floating during each
of the next 15 minutes.
11. After all (or most) of the disks are floating, put the cups in the dark (a shoebox or a closet) and monitor for
the next 15 minutes.
12. Record how many disks remain floating after each minute until all (or most) of them have sunk.

Questions and Answers

1. In this experiment, the progress of the Hill Reaction was measured by spectrophotometrically following the
reduction of the dye. Discuss other methods that can be employed. Compare the advantages and
disadvantages of these different techniques.
2. Would the Hill Reaction proceed at a significantly faster rate if the reaction proceeded at a temperature 10
degrees centrifuge higher? Explain.
3. Would the Hill Reaction proceed at a significantly faster rate if the reaction is conducted at higher light
intensities? Explain.

1. How does the suction help the leaf disks to sink?

When you apply a gentle vacuum to the leaf disks in solution, this air is forced out and replaced
with solution, causing the leaves to sink.
2. How does the detergent help the leaf disks to sink?

The soap breaks down the molecules on the leaf's surface (because the leaf repels the solution)
to allow the bicarbonate solution to penetrate the cells. That way, the leaf disk can absorb the
solution.

3. Why don't the leaf disks soaking in the water (control) float?

The spongy mesophyll layer is normally infused with gases, oxygen and carbon dioxide. Leaves
(or disks cut from leaves) will normally float in water because of these gases.

4. What is the purpose of the baking soda solution?

Bicarbonate ion in the baking soda serves as the carbon source for photosynthesis. As
photosynthesis proceeds oxygen is released into the interior of the leaf, which changes the
buoyancy, causing the disks to rise.

5. What is the purpose of the light reaction?

The overall purpose of the light-dependent reactions is to convert light energy into chemical
energy. This chemical energy will be used by the Calvin cycle to fuel the assembly of sugar
molecules.

6. Why do the leaf disks in the baking soda solution (treatment) begin to float?

This is because leaves have air in the spaces between cells, which helps them collect CO2 gas
from the baking soda solution to use in photosynthesis.

7. Why do the leaves begin to sink again in the dark?

When you put floating leaf disks in the dark, they will eventually sink. Without light energy, no
photosynthesis will occur, so no more O2 gas will be produced.

8. Why don't the leaves in the baking soda solution continue to produce oxygen in the dark?

There is no light source to provide energy for the reaction.

9. Why do we use the half-way mark as a point of comparison rather than the point at which all
the disks are floating?

To get the time half of the disks are floating as a point of reference for graphing.

10. If the light-independent reaction can run without light, why does oxygen production (and
presumably glucose production) stop?
 Although the light-independent reaction is not directly dependent on light, it is indirectly
dependent on light since the necessary energy carriers (ATP and NADPH) are products of light-
dependent reactions.

Results

In the light, the disks in the control solution (water) stay on the bottom of the cup, but the disks in the treatment
solution (baking soda) begin to rise as they use the CO2 to undergo photosynthesis and produce oxygen bubbles
(after 9 mins, half of the disks were floating and after 15 mins, all of them were floating). The bubbles should
cause the disks to float. After you remove the light and place the cups in the dark, the treatment disks stop
undergoing photosynthesis and the disks begin to sink (after 7 mins, 5 of the disks sunk and after 15 minutes, all of
them sunk).

Figure 1 After being in the dark Figure 2 Under the light

Integration of Faith and Learning

Seven times in the gospel of John, Jesus makes a powerful statement beginning with the phrase "I am." These
comments echo the words of God to Moses in Exodus 3:14. There, when Moses asks who he should say has sent
him to Israel, God tells Moses to tell the people "I AM WHO I AM…Say this to the people of Israel: 'I AM has
sent me to you.' In John chapter 8:12 "Then spake Jesus again unto them, saying, I am the light of the world: he
that followeth me shall not walk in darkness, but shall have the light of life." Jesus is our source of salvation. When
we focus on him and soak in his light, like the leaves, we will float in his love but if we cut ourselves off from that
light, we will sink in the darkness of sin and death.