NAME: Merve MURAT 26.04.

2017
ID: 2004141
SECTION / GROUP: 3 / 1
SUBMITTED TO Baak KAYAOLU

PHOTOSYNTHESIS

AIM

The purposes of this experiment are to isolate thylakoid membrane from leaf chloroplast, then
separation of leaf pigments by using paper chromatography. In addition to these, the other
purposes are to generate absorption spectrum for these pigments, to determine chlorophyll
content of isolated leaf chloroplast and to determine PSII activity of chloroplast (the rate of
photosynthesis) by using spectrophotometer.

INTRODUCTION

In photosynthesis, there are two types of phosphorylation: cyclic and non-cyclic. Cyclic
phosphorylation includes only photosystem I (PSI) whereas non-cyclic phosphorylation
contains both photosystem I and II (PSII). ATP is produced by electron flow between electron
carriers found in these systems. Firstly, light comes to PSII and water hydrolysis occurs for
formation of O2 gas and proton (H+) formation. Then, generated electrons are transferred by
carriers (light absorbing pigments) in PSII: Pheophytin (Ph), Plastoquinones (Qa and Qb),
Cytochrome bf complex (Cyt bf) and Plastocyanin (PC) respectively. Then electrons pass to
PSI. Phylloquinone (A), Iron-Sulphur Protein (Fe-S), Ferrodoxin (Fd) and Flavoprotein (FP)
carry the electrons in PSI. at the final step, NADP+ is reduced to NADPH. The Hills Reaction
can be described as the transfer of electrons in the presence of light from H2O to Hills Reagent
(non-physiological oxidants) against chemical potential gradient (by using PSI and PSII). As a
Hills Reagent/Dye, 2-6, dichlorophenolindophenol (DCPIP) can be used as the indicator of
rate of photosynthesis by replacing plastocyanin with DCPIP. By the combination of thylakoids
with DCPIP, the rate of photosynthesis can be measured. The oxidized form of DCPIP is blue
and the reduced form is colorless. After addition of DCPIP and light exposure, the
photosynthesis starts and DCPIP become reduced (colorless) via electron transport in PSII. By
spectrophotometer measurement, the rate of color change is measured and the rate of electron
transport (photosynthesis) is determined. (Govindjee, Beatty, Gest, & Allen, 2005; Pentz, 1989)
PROCEDURE

Isolation of Thylakoid Membrane

1. Weigh 0.5 g of leaf and cut it into small pieces via scissors in beaker.
2. Put it in homogenization tube and homogenize it three times in polytron homogenizer
on ice. The important point is to work on ice to prevent excess heat formation because
excess heat causes autolysis and disruption of enzymatic reactions.
3. The first homogenization takes 1 minutes after addition of 2 ml of grinding medium
with 0.33 M sorbitol to leaf pieces. The role of sorbitol is to provide isotonic
environment and to maintain osmotic pressure required for plant cells (to prevent cell
damage). The role of grinding medium is to disrupt cell wall and tissues.
4. After wait 30 seconds, the second homogenization takes 30 seconds after addition of 2
ml of grinding medium with 0.33 M sorbitol to homogenate.
5. After wait 30 seconds, the third homogenization takes 30 seconds after addition of 1 ml
of grinding medium with 0.33 M sorbitol to homogenate. The reason of waiting 30
seconds between two homogenization is to allow homogenate to cool.
6. Filter homogenate by using 4-fold cheesecloth and clean beaker to separate cells without
cell wall.
7. Centrifuge it at 2000g for 6 minutes at 4oC to get cells without cell wall in pellet and to
get cell wall components and sorbitol in supernatant to prevent cell lysis caused by
sorbitol. The reason of centrifugation at 4oC is for enzyme activity.
8. Remove supernatant and resuspend the pellet in 2 ml of suspension medium with 0.1M
Sorbitol. The role of suspension medium is to provide hypotonic environment for
release cell content and the role of Sorbitol is to improve cell lysis.
9. Centrifuge it at 9690g for 10 minutes at 4oC to get organelles in pellet and to get cell
debris in supernatant.
10. Remove supernatant and resuspend the pellet in 150 l of suspension medium.
11. Store the tubes at dark on ice to prevent initiation of photosynthesis.

Paper Chromatography of the Pigments

1. Slowly place a drop of leaf extract to labeled point on the cellulose paper.
 2. Place the cellulose paper into a tank containing acetone and petroleumether (10:90).
Acetone and petroleumether is used to dissolve pigments (for movement of pigments).
3. Place tank into dark to prevent light absorption by pigments.

Absorption Spectrum of Photosynthetic Pigments

1. Put 1 ml of acetone into a quartz cuvette and add a few drops of leaf extract then mix.
Acetone helps dissolution of pigments. However, plastic cuvettes cannot be used for
measurement because acetone causes corrosion in plastic cuvettes, so quartz cuvette is
used for spectrophotometric measurement.
2. Get absorption spectrum of the pigments by scanning the range 320nm to 700nm by
using acetone as a blank.
3. Draw the Graph of OD vs , then observe the peaks caused by pigments.

Chlorophyll Content Determination

1. Mix 990 l of 80% acetone and 10 l of sample, and vortex it to mix well. Work in
duplicates.
2. Record measurements at 645nm and 663nm via quartz cuvettes due to acetone.
3. Calculate chlorophyll content of chloroplasts.
g/ml = [(A663 x 8.02) + (A645 x 20.02)] x DF
4. Find the amount of extract that contains 40 g of chlorophyll.

PS II Activity Assay

1. Put 1 ml of assay medium includes DCPIP in quartz cuvettes and use it as blank.
2. Add calculated amount of extract in assay medium and measure it at 590nm.
3. Place the cuvette to the light to initiate electron transfer and measure the absorbance at
590nm for every 15 second for about 3 minutes.
4. Draw the graph of OD vs Time and find the rate of photosynthesis from slope of graph.
RESULTS

Fig 1. The results of separation of chlorophyll pigments by paper chromatography.

Rf = Distance travelled by pigment / distance travelled by solvent front

Rf1 = 1.75 / 13 = 0.135
Rf2 = 2.7 / 13 = 0.208
Rf3 = 4.8 / 13 = 0.36
Rf4 = 12.8 / 13 = 0.985
Abs

(nm)
Graph 1. Absorption Spectrum of Photosynthetic Pigments

Chlorophyll content calculations:

A663 = 0.328 A645 = 0.192
g/ml = [(A663 x 8.02) + (A645 x 20.02)] x DF
g/ml = [(0.328 * 8.02) + (0.192 * 20.02)] * [(990+10) l / 1000 l] = 647.5
There is 647.5 g of chlorophyll in 1 ml of extract.

1ml of extract contains 647.5 g of chlorophyll

X ml of extract contains 40 g of chlorophyll X = (1ml * 40g) / 647.5 g = 0.062 ml
0.062 ml = 62 l of extract contains 40 g of chlorophyll

Table 1. Shows Abs values for PSII activity during 3 minutes.

Time (sec) Abs

0 0.820
15 0.518
30 0.493
45 0.672
60 0.637
75 0.635
 90 0.629
105 0.628
120 0.623
135 0.619
150 0.609
165 0.609
180 0.609

Time vs Abs
0.9

0.8
y = -0.0002x + 0.641
0.7 R = 0.0229

0.6
Abs590

0.5

0.4

0.3

0.2

0.1

0
0 15 30 45 60 75 90 105 120 135 150 165 180 195

Time (sec)

Graph 2. Absorbance data for PSII activity during 3 minutes.

The rate of photosynthesis is equal to the slope of Graph 2 which is 0.0002.

DISCUSSION
The most usable result for paper chromatography is the result of Group 5. The experimental
error took place during experiment. The tank should have been placed in dark to prevent
absorption of light by pigments. However, it was not placed in dark, thus it can cause this result.

According to the result of Group 5, 1 can be Chlorophyll b, 2 can be Chlorophyll a, 3 can be

Xanthophyll and 4 can be Carotene. These predictions is done by compared with Fig 2.
Chlorophyll b has six polar group, Chlorophyll a has five polar group, Xanthophyll has two
polar group and Carotene is non-polar. In other words, Chlorophyll b is the most polar, and
Chlorophyll a comes, then Xanthophyll comes and Carotene is the least polar (non-polar).
 (Morgan & Carter, 2002; Tian, Han,
Zhang, & Skibsted, 2008) Stationary phase
is polar and mobile phase is non-polar.
Thus, the solubility order in mobile phase
is Carotene (the most soluble),
Xanthophyll, Chlorophyll a and
Chlorophyll b (the least soluble). The
molecular weight of Chlorophyll b is
907.492 g/mol (CHLOROPHYLL B |
C55H70MgN4O6 - PubChem, n.d.), the
molecular weight of Chlorophyll a is
893.509 g/mol (UNII-YF5Q9EJC8Y |
C55H72MgN4O5 - PubChem, n.d.), the

Fig 2. Standard Rf values for chlorophyll pigments molecular weight of Xanthophyll is

http://mrhalverson.com/2010B_chromatogram.png 568.886 g/mol (XANTHOPHYLL |
C40H56O2 - PubChem, n.d.) and the molecular weight of Carotene is 536.888 g/mol (beta-
carotene | C40H56 - PubChem, n.d.). The size order is Carotene (the smallest), Xanthophyll,
Chlorophyll a and Chlorophyll b (the higher). Due to having high polarity, Chlorophyll b is not
dissolved well in mobile phase, so it is prone to stay in stationary phase and it is left behind.
Due to having high non-polarity, Carotene is dissolved well in mobile phase, so it is prone to
stay in mobile phase and moves faster.
According to absorption spectrum of photosynthetic pigments, it can be said that indicates
Chlorophyll a, indicates Chlorophyll b and indicates Carotenoids by comparing standard
values in Graph 3.

Graph 3. Standard absorption spectrum for chlorophyll pigments.

http://www.bbc.co.uk/education/guides/z23ggk7/revision/2
The results of PS II activity assay is not exactly the expected result although there is a decrease
in absorbance because there is increase between 30th and 45th seconds and there is not much
decrease in absorbance (the absorbance is almost the constant during 3 minutes). The possible
reason of it can be the low yield of isolation, the exposure of light before spectrometric
measurement (improper storage conditions for extract, or improper light exposure during
spectrophotometric measurements. Thus the calculated rate of photosynthesis is low.

RESEARCH QUESTIONS

In which types of experiments PSII activity assay would be useful?

PSII activity assays are generally used in the field of plant physiology. To determine the
mechanisms found in photosynthesis and to examine the response of plants to environmental
changes, the sensitivity of PSII activity to abiotic and biotic factors must be determined by these
assays. In addition, they can be used to understand genetic variation, and ecological diversity
of plants. For instance, PSII activity assay is recently used to improve crops to generate
desirable plant traits and to generate linkage between genomic information and phenological
responses. (Murchie et al., 2013)
Effect of CO2 absence/presence or concentration on PSII activity in Hills rxn.
It cannot be easily said that there is a direct relationship between CO2 and PSII activity because
the effects of absence of CO2 can be easily reversible and the effects of CO2 can change little
bit by other factors such as incubation of sample in the dark or light, duration of dark incubation.
Despite this little changes, this can be deduced that CO2 requirement of PSII activity is a general
phenomenon. In other words, the removal of CO2 decrease the activity of PSII especially when
ferricyanide, various indophenol dyes, triphosphopyridine nucleotide, and flavin
mononucleotide function as Hill oxidants. In addition, an hour of controlled illumination is
necessary for the chloroplasts in order to indicate the dependence of ferricyanide
photoreduction on CO2. Moreover, if CO2 concentration increases, the rate of photosynthesis
increases because CO2 is used as substrate and it diminishes the photorespiration rate.(Good,
1963; Stern & Vennesland, 1962)

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