Laboratory Report

Microbial Biochemistry

(BIC2BMB)

Experiment 5:
Photoinduced Proton Transport Through Chloroplast
Membranes

Name: Tshediso

Surname: Malakoane

Student number: 216078572

Date: 18 October 2019.

 Abstract
The aim of the experiment was to prepare chloroplast, determine chlorophyll content and
measure proton uptake. The events of the electron transport chain involve NADH and FADH,
which act as electron transporters as they flow through the inner membrane space. In
complex I, electrons are passed from NADH to the electron transport chain, where they flow
through the remaining complexes. NADH is oxidized to NAD in this process. Complex II
oxidizes FADH, garnering still more electrons for the chain. At complex III, no additional
electrons enter the chain, but electrons from complexes I and II flow through it. When
electrons arrive at complex IV, they are transferred to a molecule of oxygen. Since the
oxygen gains electrons, it is reduced to water. The chloroplast was prepared, chlorophyll
content determined by checking the absorbance at 652nm and the proton uptake measured
using a pH meter with the help of a lamp as a light source, three conditions were set up,
chloroplast with no cofactor, chloroplast with cofactor and chloroplast with two co factors.
The observation was that the addition of co factors leads to a drop in the pH. The results
obtained were partially accurate and partially inaccurate. The inaccuracy was due to factors
such as human error. To improve the accuracy of the results, number of students in a group
must be reduced.

Introduction
The electron transport chain is a series of proteins and organic molecules found in the inner
membrane of the mitochondria, electrons are passed from one member of the transport
chain to another in a series of redox reactions (Bansal, 2006). Energy released in these
reactions is captured as a proton gradient, which is then used to make ATP in a process
called chemiosmosis, together, the electron transport chain and chemiosmosis make up
oxidative phosphorylation (Rao, 2008). The key steps of this process, Delivery of electrons
by NADH and FADH2. Reduced electron carriers (NADH and FADH2) from other steps of
cellular respiration transfer their electrons to molecules near the beginning of the transport
chain. In the process, they turn back into NAD+ and FAD, which can be reused in other
steps of cellular respiration (Bansal, 2006).

(Bansal, 2006) Further elaborated on the different steps of electron transfer and proton
pumping. Stating that as electrons are passed down the chain, they move from a higher to a
lower energy level, releasing energy. Some of the energy is used to pump H + ions, moving
them out of the matrix and into the intermembrane space. This pumping establishes an
electrochemical gradient. Splitting of oxygen to form water. At the end of the electron
transport chain, electrons are transferred to molecular oxygen, which splits in half and takes
up H+ to form water. Gradient-driven synthesis of ATP. As H + ions flow down their gradient
and back into the matrix, they pass through an enzyme called ATP synthase, which
harnesses the flow of protons to synthesize ATP.

The proton uptake of thylakoid membranes during photosynthesis was analysed through
measuring pH, as light reaches the plant, the chlorophyll begins to absorb and release
excited electrons (Berry and Rumberg, 1999). Further stating that as the electrons pass
through the Quinone, they increase the Proton concentration on one side of the thylakoid
membrane. This then drives the ATP synthase to form ATP from ADP by means of
channelling the protons to turn the synthase.

As the light enters the chlorophyll, emission of electrons begins a cycle of reactions, the
evolution of oxygen and protons is completed by the splitting of water molecules by the light
activated chlorophyll electron (Fuks and Homble, 1996). The electron is then processed by
Quinone structures, which also integrate a proton gradient on the inside of the thylakoid
membrane. The electron chain ultimately finishes with the reduction of NADP to NADPH.
This increase of protons on one side of the membrane is driven to diffuse to a lower
concentration, and it is here where ATP synthase channels are used (Berry and Rumberg,
1999). The Protons are used to turn the synthase in order to form ATP from ADP and
phosphates. The electron is restored to the chlorophyll by the above-mentioned process,
which is known as photolysis. The net reaction goes as follows: 2 H2O + 2 NADP+ + 3 ADP
+ 3 Pi + light → 2 NADPH + 2 H+ + 3 ATP + O2 (Ho and Po, 1996).

Materials and Methods

The materials that were used in this experiment were: Homogenizing buffer, 0.02 M Tris, pH
8, containing 0.01 M NaCl and 0.4 M sucrose, sharp knife, homogenizer, cheesecloth,
refrigerated centrifuge tubes, chloroplast suspension solution, 0.4 M sucrose containing 0.01
M NaCl, 80% acetone solution in water, conical centrifuge tube, glass cuvette,
spectrophotometer, tabletop centrifuge, pH meter, 500-watt tungsten lamp, CUSO4 to
remove infrared radiation magnetic stirrer and small stir bar, phenazine methosulfate, timer,
large test tube (2 x 20 cm), phosphate solution (0.01 M) and adenosine diphosphate (0.02
M)

For the preparation of chloroplasts, the midribs were cut and discarded from 50 g of spinach
leaves and the teared leaves into small pieces. The leaves were immediately put into a
precooled homogenizer containing 100 mL of ice-cold homogenizing buffer. The leaves were
then grinded. The homogenate was strained through four layers of cheesecloth. All the liquid
from the cheesecloth was squeezed. The filtrate in precooled tubes was centrifuged at 1000
x g for 1 minute to remove whole cells. The supernatant transferred to precooled centrifuge
tubes and spun at 6000 x g for about 15 seconds. The supernatant decanted and
suspended the chloroplasts in 50 mL of Tris homogenizing buffer. The suspension
centrifuged at 6000 x g for about 10 seconds. The supernatant was then poured off and
discarded. Resuspend the isolated chloroplasts by stirring into 50 mL of suspension solution
(sucrose containing NaCl). The chloroplasts were suspended in ice for later use.

For the determination of chlorophyll content, the chlorophyll was extracted l from the
chloroplasts by mixing, in a conical centrifuge tube, 0.05 mL of well-mixed chloroplast
suspension with 9.9 mL of 80% acetone in water. Spun in a tabletop centrifuge for 10
minutes. the supernatant transferred to a glass cuvette and the absorbance read at 652 nm
using 80% acetone in water as blank.

For measurement of proton up take, the experimental arrangement was done as shown in
Figure 3. the temperature of the water bath maintained at 10°C by adding ice. A trace of
CUSO4 solution added to the water bath to eliminate infrared radiation from the lamp. A test
tube that will hold the pH electrode and 10 to 15 mL of solution was obtained. The
chloroplast solution was diluted with suspension buffer to a chlorophyll concentration of
about 300 μg/mL. Adjust the pH meter with standard pH 7 buffer. Three experimental
conditions were done. Condition one, chloroplasts with no redox cofactor10 mL of
chloroplast suspension (about 3 mg chlorophyll), 1 mL of suspension buffer). Condition two,
chloroplasts with redox cofactor (10 mL of chloroplast suspension, 5 mg of phenazine
methosulfate or other redox dye, 1 mL of suspension buffer). Condition 3, chloroplasts with
redox cofactor, ADP, and phosphate (10 mL of chloroplast suspension, 5 mg of phenazine
methosulfate or other redox dye, 0.5 mL of ADP solution, 0.5 mL of phosphate buffer). For
each condition, the reaction mixtures were mixed well and transferred to the reaction tube. A
small magnetic stir bar inserted and placed the pH electrode into the reaction mixture. The
magnetic stirrer turned on to a slow rate and checked to see whether the stirrer affects the
pH meter. The lamp was then turned on to illuminate the chloroplasts. The recorder was
allowed to trace the pH reading with time for 60 seconds, and then turned off the light but
continue to record the pH. Turned the lamp on after a dark interval of 30 seconds. Continued
to record the pH for 60 seconds, again turned the lamp off, and continuously recorded the
pH. Repeat the experiment with a fresh portion of chloroplasts.
Figure 3 Experimental arrangement for measuring proton uptake by illuminated chloroplasts.

Results
The tables and graph below represent the results that were obtained in the preparation of
chloroplasts, determination of chlorophyll and the measurement of protein uptake

Table 1: Absorbance of chlorophyll at 652nm

Tube. (Solution) Absorbance at 652nm
1. BLANK (80% acetone in water) 0.00
2. Chlorophyll 0.106

c = A = A652

El 34.5 x l

0.106
= ml
35.5 cm−1 x 1 cm
mg

= 0.003 ml/mg
Table 2: pH of Chloroplasts with no redox cofactor
Chloroplasts with no redox cofactor
Time Stirrer on/ off Light on/ off pH
0 off off 7.47
30 on off 7.04
60 off on 7.02
90 off off 7.01
120 off on 7.01
150 off off 7.00

Table 2: pH of Chloroplasts with redox cofactor

Chloroplasts with redox cofactor

Time Stirrer on/ off Light on/ off pH

0 off off 6.95

30 on off 6.88

60 off on 6.86

90 off off 6.85

120 off on 6.82

150 off off 6.52

 pH of Chloroplasts without cofactor in intervals of 30
seconds
7.6
7.5
7.4
7.3
7.2
pH

7.1
7
6.9
6.8
6.7
0 20 40 60 80 100 120 140 160
Time (seconds)

pH of Chloroplasts with cofactor in intervals of 30 seconds

7

6.95

6.9
pH

6.85

6.8

6.75
0 20 40 60 80 100 120 140 160
Time (seconds)

Due the phenazine methosulfate running out. The third which is chloroplasts with redox
cofactor, ADP, and phosphate could not be done.
 Discussion
For experimental condition 1, The initial pH for chloroplast with no co factor is 7.47 and the
final is 7.00 with a change of -0.47. This experimental result was not in line with the
expected results, that is if the electron transport chain is pumping H + into the cell under
normal conditions the expected H+ concentration on the outside of the cell will decrease and
in turn raise the pH, so there should have been a rise in pH (Hogg, 2002). The plausible
reason for the result that there must have been some mistake done during the process,
because there were no additional stimuli added, this should have resulted in the cell taking 2
H+ in at a time through cytochrome complex but not expelling any via ATP synthase
mechanism (Rao, 2008).

For experimental condition 2, chloroplast with co factor, the initial pH is 6.95 and the
final pH is 6.52 with a change of – 0.43. This was not expected, if the co factor helps
increase electron transfer chain efficiency by functioning similar to plastoquinone the
result should have been a higher pH due to decreasing H+ concentration on the
outside of the cell (Bansal, 2006). Since the electron transport chain should be
functioning at a higher level with the redox co factor, in theory, the cell should be
pimping in more electrons causing a loss in H+ concentration on the outside of the
cell, and thus increasing the pH (Hogg, 2002). There can be some possible reasons
for inaccuracy of the result. The H+ concentration on the outside of the cell, electron
transport chain is taking up 2 H+ then ATP synthase is pumping out 4 H+ at a time, but
this is not possible because the ATP synthase machinery should not be functioning
due to a lack of substrates (Rao, 2008). Phenazine methosulfate is supposed to be
only work on electron transport chain, and thus only helping bring 2 H+ into the cell
(Hogg, 2002). The condition of the chloroplast cells may have contributed to the
inaccuracy of the result.

The experiment suffered from sources of error because it was carried out by a group
of people. Every individual handled a certain part of the experiment. This variety of
people handling each step introduces many unknown variables. The skills and
understanding of laboratory protocol differ from individual to individual.
 Conclusion
The aim of the experiment was to prepare chloroplast, determine chlorophyll content and
measure proton uptake. The experiment failed in its aim because the measurement of proton
uptake was not completed, and the results obtained were not expected. The experiment may
be improved by changing the number of individuals in a group to two because having many
hands takes away from individual learning opportunities. If the results are wrong, there is no
way to try and figure out why or what went wrong.
 References
.
Bansal, P. (2006). Potentials of plant biotechnology. New Delhi, India: Gene-Tech Books,
pp.345-476.

Berry, S. and Rumberg, B. (1999). Proton to electron stoichiometry in electron transport of

spinach thylakoids. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1410(3), pp.248-
261.

Fuks, B. and Homble, F. (1996). Mechanism of Proton Permeation through Chloroplast Lipid
Membranes. Plant Physiology, 112(2), pp.759-766.

Ho, J. and Po, E. (1996). Light-induced proton transport through chloroplast

membranes. Biochemical Education, 24(3), pp.179-180.

Hogg, P. (2002). Building an ER electron transport chain. Redox Report, 7(1), pp.3-4.

Rao, G. (2008). Advances in plant biotechnology. 1st ed. Houston, Tex.: Studium Press, pp
156-169.