WATER SPLITTING IN

PHOTOSYSTEM II

Long Vo Pham

Degree Thesis in Chemistry (15 ECTS)

Bachelor’s level

Report passed: June 9th

, 2010

Supervisor: Prof. Johannes Messinger

Examiner: Wolfgang Schröder

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Table of content

1. Introduction ........................................................................................ 3

2. Experiments, results and discussions ................................................. 5

1. Preparation of thylakoid and “BBY” samples from spinach .................. 5

2. Measuring oxygen evolving activity with Clark-type electrode ............. 8

1. The oxygen evolving activity of BBY sample ............................ 9

2. The oxygen evolving activity of thylakoid sample .................. 12

3. Protein gel electrophoresis .................................................................... 16

4. Performing oxygen flash-yield measurement with Joliot electrode ..... 20

3. Conclusions ...................................................................................... 28

4. Acknowledgements .......................................................................... 29

5. References ........................................................................................ 30

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1. Introduction The oxygen molecule is essential for the life of all higher organisms. It plays a crucial role

in energy metabolism, functioning as the oxidizing agent in the end of the glycolysis-citric acid

cycle-electron transport-pathway, yielding the energy-rich ATP molecules. Oxygen is produced by

plants and some specific bacteria, which can utilize CO2, solar energy and H2O for the

photosynthetic reaction. The generation of the present day oxygen-rich atmosphere with the ozone

layer (Kasting and Seifert 2002) started 2-3 billion years ago. At that time, prokaryotic

cyanobacteria were able to split water into molecular oxygen and metabolically bound hydrogen,

using light as energy source (Buick 1992; David 2000; Xiong and Bauer 2002).

Based on a lot of research about biochemical structure inside chloroplast (fig. 1.1), the

interior compositions and their basic function were identified more clearly with every passing day.

Figure 1.1: the amplification of thylakoid membrane from a plant cell

The series of above pictures illustrated sufficiently the proteins that played important roles

in photosynthetic process with their names. In addition, this project concentrated on photosystem II

proteins, which produce oxygen by oxidizing water molecules. One of the interesting investigations

around the photosynthetic process of natural photosynthetic system is the measurement of oxygen

evolution. There are many techniques and methods were found and developed to be able to analyze

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oxygen concentration at trace contents. Some modern methods, which are still used in present, are

mass spectrometry and polarography (Clark and Joliot electrode). (Gernot Renger, Bertram

Hanssum 2009)

Although Clark and Joliot electrode were also create based on the basic principles of

polarography, these dissimilar kind of electrode still have some fixed differences. Therefore, this

means each of the electrodes has still some their specific points. In addition, the signals, which

were recorded from Joliot electrode, are one of important differences between two types of

electrodes. In 1969, Joliot Pierre and his co-workers created Joliot electrode and measure oxygen

evolution with single-turnover light flashes. And they are aware that oxygen yield in plot looked

like a function of the flash number and expressed a periodicity of four (fig. 1.2)

Figure 1.2: The plot of oxygen yield from PSII as a function of flash number (left figure) and one

of the current Kok model with five oxidation states (right figure)

The periodicity of four readily disappeared after several cycles and the maximum oxygen

yield occurred on the third flash rather than fourth flash. All of the observations indicated for

complexity in the mechanism of water oxidation and it is not easy to explain. According to the

assessments and observations, Kok and his co-workers were suggested an elegant model (also

called the “Kok model”) to explain for these problems of Joliot group. In the Kok model, the

oxygen-evolving complex (PSII) could exist in one of the five oxygen oxidation states, labeled S0,

S1, S2, S3 and S4 (see figure 1.2). Moreover, each photochemical reaction removes a single electron

from PSII to advance the next higher S state until enough four electrons were released from PSII to

leading the oxidation of two water molecules and one molecular dioxygen was formed. Of course,

the mechanisms of the break O-H covalent bonds and connect together two oxygen atoms are really

challenging problems.

The aim of this project is to investigate the activity with a Clark-type electrode of

thylakoid- and BBY-samples, isolated from spinach leaves. The presence of major protein

constituents in these samples will be examined using SDS-PAGE. In addition, oxygen flash-yield

measurements will be performed using a Joliot-type electrode, to compare the flash pattern of

thylakoids and BBY, and to determine the lifetime of S2 in thylakoids. The results of this project

could supply hopefully some hints for the next investigation.

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2. Experiments, results and discussion 1. Preparation of Thylakoid and BBY samples from spinach

Before preparing the thylakoid and BBY samples, the buffers, which are marked from 1 to

4, were prepared (following the Berkeley protocol). These comprise buffer 1 (grinding buffer, pH =

7.5), buffer 2 (washing buffer, pH = 6.0), buffer 3 (resuspension/incubation buffer, pH = 6.0) and

buffer 4 (sucrose buffer, pH = 6.0).

Table 1.1: The composition of all buffers used in this projects

Name of buffer Composition pH

Buffer 1

(Grinding buffer)

1mM EDTA

50mM HEPES

4mM MgCl2

400mM NaCl

5mM Sodium Ascorbate

2mg/ml BSA

7.5

Buffer 2

(Washing buffer)

50mM MES

8mM MgCl2

150mM NaCl 6.0

Buffer 3

(Resuspension or

incubation buffer)

50mM MES

10mM MgCl2

5mM CaCl2

15mM NaCl

(Triton X-100)

6.0

Buffer 4

(Sucrose buffer)

50mM MES

5mM MgCl2

5mM CaCl2

15mM NaCl

400mM Sucrose

6.0

Buffer SMN 50mM MES

400mM Sucrose

15mM NaCl

6.5

Phosphate buffer 2.5mM NaH2PO4 2.5mM Na2HPO4 7.8

Buffer B 50mM MES

10mM MgCl2

5mM CaCl2

15mM NaCl

250mM NH4Cl

6.0

Buffer C 50mM MES

400mM Sucrose

15mM NaCl

10mM NH4Cl

6.5

Joliot electrode 20mM NaCl

5mM MgCl2

50mM MES 6.5

In addition, one more solution was prepared to measure the chlorophyll concentration in

thylakoid or BBY samples. This was the mixture of phosphate buffer (2.5mM NaH2PO4 and

2.5mM Na2HPO4, pH = 7.8) and technical acetone (purity > 99%) with volumetric ratio 1:4,

respectively. This mixture is called the 80% buffered acetone solution (or buffer A).

Then, the preparation of thylakoid and BBY samples was performed (following Messinger’s

group protocol) at +4ºC and in dim green light.

Footstalks of fresh spinach leaves were carefully removed to avoid destroying the

remaining parts of the leaves, since they contain small amount of chloroplasts and in

addition are hard to grind. Then, these leaves were washed by distilled water (at

least 5 times) to eliminate most of soil and chemicals that cling to the surface of the

spinach leaves.

Cleaned leaves were ground in grinding buffer (buffer 1) by washed blender

(Waring Blender, USA) to damage the cell membrane (cellulose membrane) and

supply advantageous condition for extraction of chloroplasts molecules.

Then, the mixture of ground leaves and buffer was filtered by several layers of

cheesecloth (about 20 μm pore length) to exclude the unnecessary plant materials

and get most of the chloroplast molecules in the extracted solution.

The extraction was centrifuged (Avanti centrifuge, Beckmann CoulterTM

) for 10

minutes at 8.000 rpm using a JA-10 rotor. The pellet was collected and resuspended

with washing buffer and centrifuged once more. The supernatant was discarded and

the pellet was resuspended in buffer 3. The aim of a two-step centrifugation was to

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not only collect and concentrate chloroplast molecules and thylakoid membranes but

also to remove a part of other protein complexes from the stroma and of proteases

that can destroy photosystem II.

Chlorophyll concentration was measured by UV-VIS spectrophotometer. Three

replicates of thylakoid sample were prepared to measure chlorophyll concentration.

Each of these samples consisted of 4.99 ml of 80% buffered acetone solution and 10

μl thylakoid sample that were thoroughly mixed carefully by vortex in order to

extract all chlorophyll. The result is presented in Table 1.1.

Table 1.2: Absorbance of chlorophyll in thylakoid samples at different wavelengths.

646.6nm 663.6nm 750nm

Replicate 1 0.180 0.436 0.000

Replicate 2 0.179 0.435 0.000

Replicate 3 0.179 0.433 0.000

Average value 0.179 0.435 0.000

For calculation of the chlorophyll concentration the following mathematical

equation (Robert J. Porra 2002) was used:

646.6 750 663.6 75017.75 7.34 * totalA A A A K Chlorophyll

5000

: 50010

solution

sample

VWith K diluted parameter times

V

total thylakoid membrane3185 / 3.185 /Chlorophyll g ml mg ml

Thylakoid membrane solution was diluted to lower the chlorophyll concentration

from 3.2 mg/ml to 2.0 mg/ml. After that, 25% Triton X-100 solution was slowly

added (drop by drop) to this thylakoid membrane solution, to a final weight ratio of

25 mg (pure Triton X-100) : 1 mg (Chlorophyll total). Triton X-100 is a surfactant

that engages and disorganizes lipid membrane units, which in this case facilitates the

release of photosystem II (BBY). The stirred thylakoid/Triton X-100 solution was

incubated in dark cold room for 20 minutes.

Then, the solution of disrupted thylakoid membranes and Triton X-100 was

centrifuged for 15 minutes at 15.000 rpm (JA-20 rotor) to remove starch

contamination and non-solubilized plant materials.

The pellet was re-suspended and homogenized in buffer 3, followd by a second

centrifugation for 2 minutes at 2.500 rpm (JA-20 rotor). This centrifugal step at low

speed and short time was performed to get rid of heavy compounds, for example

starch and all membranes.

Following resuspension of the pellet with buffer 4, a number of centrifugal steps at

high velocity (15.000 rpm and 20.000 rpm) and long time (30 minutes and 12

minutes, respectively) was carried out to remove more residual detergents and

concentrate BBY content also more starch is removed if resuspension is done

carefully.

The measurement of chlorophyll concentration in the BBY was carried out in the

same way as for the thylakoid sample. The result is summarized in Table 1.2.

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Table 1.3: Absorbance of chlorophyll in BBY samples at different wavelengths.

646.6nm 663.6nm 750nm

Replicate 1 0.363 0.768 0.003

Replicate 2 0.332 0.707 0.002

Replicate 3 0.351 0.746 0.002

Replicate 4 0.326 0.689 0.003

Average value 0.343 0.728 0.003

Using again mathematical equation (Robert J. Porra 2002):

total "BBY"5678 / 5.678 /Chlorophyll g ml mg ml

The thylakoid and BBY samples were flash frozen in N2 (l) to preserve the original

activity of these samples, and then stored at -80 ºC.

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2. Measuring oxygen evolving activity with Clark-type electrode The measurement of oxygen evolving activity by using Clark electrode is a useful and

commonly used method, which is employed in the research of many photosynthetic systems. Clark

electrode is based on the principles of polarography for quantitative determination. Thus, this

system can detect and measure oxygen concentration at trace content level. The figures below show

the exterior shape (Figure 2.1) and structure of the Clark electrode with detailed compositions and

names (Figure 2.2).

Figure 2.1: The exterior shape of Clark

electrode.

Figure 2.2: The structure and composition of

the Clark electrode.

The Clark electrode, which was used in the experiments in this study, comes from Qubit

System Inc. Company (Ontario Canada), its model is K7M3X9 and the code is OX1LP-4ml. The

working principles of Clark electrode, which have the aim to measure oxygen concentration at trace

content (ppm), are followed almost basic principles of polarography. Thus, this electrochemical

system consists of two electrodes, which are connected each other by low voltage direct current

(usually 700mV) and salt bridge. The anode of the system is often made of Ag metal, this electrode

is immersed by a saturated solution of electrolyte (salt bridge) which is often KCl or KOH to give

rise to a constant potential with insoluble compounds AgCl or Ag2O, respectively. Our Clark

electrode used KCl standard solution (1M) that was attached with whole this system following

producer’s delivery (Qubit System Inc.). Thus, the electrochemical reaction takes place at Ag-

anode (David Walker, 1990; Gernot Renger, Bertram Hanssum, 2009).

Ag-anode: 4 4 4

4Ag 4 4

or 4Ag 4 4

Totally: 4Ag 4 4 4

4Ag 4 4 4

Ag Ag e

Cl AgCl

OH AgOH

Cl AgCl e

or OH AgOH e

Four electrons, which were formed from Ag-anode, were supplied for Pt-cathode to reduce

dissolved oxygen molecules. The surface of cathode usually contacts with dissolved oxygen, which

was diffused through semi-permeable membrane (Teflon membrane) to take place oxygenic

reduction reaction. Thus, a noble metal must be used to manufacture this cathode with the aim to

avoid oxidizing this metal by oxygen molecules and form metal oxide (basic oxide). On the other

exact hand, this metal is only intermediate place to transfer electrons that were supplied from Ag-

anode and it must not exchange any its own ions or electrons with above electrolytic environment

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(KCl solution). In addition, this metal has to be sensitive with oxygenic reduction reaction occurred

in its surface to show exactly the chance of dissolved oxygen concentration. Therefore, there are so

a little bit kinds of metal could be satisfied all of strict conditions, so, Platinum is the most optimal

choice. And reactions which occur on surface of Pt metal are represented below (David Walker,

1990; Gernot Renger, Bertram Hanssum, 2009).

2 2 2 2

2 2

2 2

Pt-cathode: O 2 2 2

H 2 2

Totally: O 2 4 4

H O e H O OH

O e OH

H O e OH

Small amount of hydroxide ions that were formed on the surface of Pt-cathode do not affect

so much to pH of electrolyte solution (KCl). The samples were illuminated by light source of

projector (Leitz Prado brand, Germany) and there are not any light filters that were used for

measurements. The temperature of sample was kept at defined value (200C) with cryostat. The

samples (BBY or thylakoid membrane) were kept under solid form (in deep frozen fridge, -800C)

and were melted in the dark ice bath on approximately one hour before performing measurement

with them. The data, which was collected from the change of dissolved oxygen concentration, was

recorded with computer (HP brand) and specific software (Logger Pro 3 version).

In these experiments, it is necessary to add more one of artificial electron acceptors (or

combination of them with an optimal concentration ratio) such as phenyl-parabenzoquinone

(PPBQ), di-chloro-benzoquinone (DCBQ) or K3[Fe(CN)6] (potassium ferricyanide or potassium

hexacyanoferrate(III) – IUPAC name) (Gernot Renger, Bertram Hanssum, 2009). By cause of

hydrophobic residues, DCBQ and PPBQ must be dissolved in organic solvents (often dimethyl

sulfoxide - DMSO or ethanol). When a natural photosynthetic system is investigated by Clark

electrode, there are a lot of concerning variables appear in this investigation. For example, the

concentration of photosynthetic system (thylakoid membrane or BBY), types of electron acceptor,

concentration of each kind electron acceptor, the kinds of buffer, etc. This research was planed by

changing only one variable and fixing all of the rest variables to optimize and find out the best

condition for this variable.

1. The oxygen evolving activity of BBY sample: The type of photosynthetic system that was taken to investigate firstly is BBY and the

concentration of BBY is the first variable (from 10μg/ml to 20μg/ml) with three different kinds of

electron acceptor (EA) - PPBQ, DCBQ and K3[Fe(CN)6]. Each of the below experiment was done

at least three times, then, all collected values were calculated to get an average value and total error.

Table 2.1: Measuring and calculating oxygen-evolution activity of BBY samples as a function of

Chlorophyll concentration (pH = 6.0, temperature = 20ºC)

No Kind of EA [EA]

(μM)

Slope SD [BBY]

(μg/ml)

Activity SD Buffer

Number

of repeat (μmol*l-1

*s-1

) (μmol*mg-1

*h-1

)

1 PPBQ 500 1.0370 ± 0.1678 10 370 ± 60 Buffer 4 6

2 DCBQ 500 0.6964 ± 0.0603 10 250 ± 20 Buffer 4 3

3 K3[Fe(CN)6] 500 0.0843 ± 0.0075 10 30 ± 3 Buffer 4 3

4 PPBQ 500 1.9142 ± 0.0187 20 345 ± 13 Buffer 4 3

5 DCBQ 500 1.3994 ± 0.1888 20 250 ± 30 Buffer 4 3

6 K3[Fe(CN)6] 500 0.2065 ± 0.0366 20 37 ± 7 Buffer 4 3

Explanation: No = number PPBQ = phenyl parabenzoquinone

EA = electron acceptor DCBQ = 2,5-dichloro-1,4-benzoquinone

SD = standard deviation

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Note: the mathematical equation to calculate oxygen-releasing activity

2 O

The unit of the activity is X*

mol

mg Chlorophyll h

(David Walker, 1990, Eqn.5.1)

2 O

With: m and * final

g Chl mg Chlmolslope Chlorophyll

l s ml l

2*3600 O 3600

* * .1*

total

m slopeX

Chlorophyl

mol s lX Eqn

l s h mg Chl l

Based on the result of oxygen evolving measurements that were conducted on BBY samples

(table 1), some evaluation was showed as follow:

Activity of BBY in presence of DCBQ and K3[Fe(CN)6] has a similar value at the two

different BBY concentration (10μg/ml and 20μg/ml). This indicted that the light is

saturating. Also, in the presence of PPBQ (at the same concentration of electron

acceptor), two values of two different BBY concentration (10μg/ml and 20μg/ml) do not

have big difference. In addition, the value at 10μg/ml has a big error (SD is standard

deviation, total of systematic error and random error), it is difficult to compare two

values with the large difference of SD. Thus, the best BBY concentration has not chosen

with six experiments yet.

The assistance of many kinds electron acceptor makes the reduced reaction of oxygen

gas on Platinum-cathode’s surface, which is produced in photosynthetic process of

BBY, occurs easier and the rate of oxygen evolving rate is faster than this absence of

electron acceptors. Moreover, the activity of BBY achieved highest value with the

presence of PPBQ in both of two cases that are different about concentration of BBY.

Therefore, PPBQ is supposed to be the best type of electron acceptor.

Due to the final evaluation (in three above evaluations), two experiments (1 and 4) were

chosen to be repeated with great care.

Table 2.2: Measuring again oxygen-evolution activity of BBY samples as a function of

Chlorophyll concentration with only PPBQ (pH = 6.0, temperature = 20ºC)

No Kind of EA [EA]

(μM)

Slope ± SD [BBY]

(μg/ml)

Activity ± SD Buffer

Number

of repeat (μmol*l-1

*s-1

) (μmol*mg-1

*h-1

)

1 PPBQ 500 1.3029 ± 0.0290 10.58 443 ± 10 Buffer 4 6

4 PPBQ 500 2.3191 ± 0.0350 21.16 395 ± 6 Buffer 4 6

Standard deviation of experiment 1 was gone down to approximate equal value compared

with standard deviation of experiment 4. With these results, the comparison about BBY

concentration between experiment 1 and experiment 4 becomes easier. The activity of BBY at low

BBY concentration is larger than this activity at high BBY concentration. As a result, the best BBY

concentration is 10μg/ml.

On the other hand, the higher BBY concentration is not good for measuring oxygen-

evolving activity. The reason, which is given to explain for this phenomenon, could be the great

density of BBY in a fixed volume. There are only photosystem II (PSII) complexes, which are

received and trapping light directly from projector, could do photosynthetic process, evolve oxygen

gas. Differences between values in table 1 and table 2 are likely due to the more precise chlorophyll

concentration determination during the experiments from table 2.

After that, the best BBY concentration (10μg/ml) was used for all further experiments. In

which other factors were varied, concentration of electron acceptor is the next factor.

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Table 2.3: Displays the results of varying the electron acceptor concentration with three of

different electron acceptors. (pH = 6.0, temperature = 20ºC)

No Kind of EA [EA]

(μM)

Slope SD [BBY]

(μg/ml)

Activity SD Buffer

Number of

repeat (μmol*l-1

*s-1

) (μmol*mg-1

*h-1

)

1 PPBQ 500 1.0370 ± 0.1678 10 370 ± 60 Buffer 4 6

2 DCBQ 500 0.6964 ± 0.0603 10 250 ± 22 Buffer 4 3

3 K3[Fe(CN)6] 500 0.0843 ± 0.0075 10 30 ± 3 Buffer 4 3

7 PPBQ 200 1.0003 ± 0.0855 10 360 ± 15 Buffer 4 6

8 DCBQ 200 0.8974 ± 0.0227 10 323 ± 4 Buffer 4 3

9 K3[Fe(CN)6] 200 0.0927 ± 0.0054 10 33 ± 1 Buffer 4 3

The results of six above experiments show that decrease of electron acceptor concentration

does not affect so much on the BBY activity. These numbers of BBY activity are approximately

equal with the same kinds of electron acceptor and the same concentration of electron acceptor

except for DCBQ. It is difficult to get perfect explanations for the difference between DCPQ and

two rest kinds of electron acceptor. Perhaps, at the high concentration, DCPQ is not only an

electron acceptor but also an inhibitor of PSII.

The next variable, which was chosen to explore continuously, is a different type of buffer.

So far, all the experiments (1 to 9) were performed with buffer 4. This new buffer is called buffer

SMN (400mM Sucrose, 20mM MES and 35mM NaCl, pH = 6.5). Buffer SMN has enough

components compared with buffer 4 (without MgCl2 and CaCl2). The best BBY concentration and

the best concentration of electron acceptor are used for all of below experiments.

Table 2.4: Measuring and calculating oxygen-evolution activity of BBY samples with the change

of two different buffers (temperature = 20ºC)

No Kind of EA

[EA]

(μM)

Slope SD [BBY]

(μg/ml)

Activity SD Buffer

Repeat

(times) (μmol*l-1

*s-1

) (μmol*mg-1

*h-1

)

1 PPBQ 500 1.0370 ± 0.1678 10 373 ± 60 Buffer 4 6

2 DCBQ 500 0.6964 ± 0.0603 10 251 ± 22 Buffer 4 3

3 K3[Fe(CN)6] 500 0.0843 ± 0.0075 10 30 ± 3 Buffer 4 3

Table 2.2 1 PPBQ 500 1.3029 ± 0.0290 10.58 443 ± 10 Buffer 4 6

10 PPBQ 500 1.2846 ± 0.0471 10.58 437 ± 16 Buffer SMN 6

11 DCBQ 500 0.9567 ± 0.0326 10.58 326 ± 11 Buffer SMN 3

Following the results were collected in above table, the using buffer SMN for measurement

of BBY activity decreased lightly dissolved oxygen concentration in BBY solution, it means

oxygen evolving activity of BBY sample reduced a little bit than these experiments which do not

use buffer SMN. This phenomenon means the presence of cations (e.g. Ca2+

and Mg2+

) does not

have a bad effect on the BBY activity. Thus, buffer 4 and buffer SMN are also used for measuring

BBY activity.

As we know, the best kind of electron acceptor is PPBQ. The next variable is trial with

mixture of two different types of electron acceptor (PPBQ + K3[Fe(CN)6]) which concentration

ratio of two kinds is varied many times. Of course, all of the best conditions, which were optimized

above, are used for these current experiments.

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Table 2.5: Displays the BBY activity of varying the volumetric ratio (or concentration ratio) with

two of fixed electron acceptors. (pH = 6.5, temperature = 20ºC)

No

PPBQ

(μM)

K3[Fe(CN)6]

(μM)

Slope SD [BBY]

(μg/ml)

Activity SD Buffer

Repeat

(times) (μmol*l-1

*s-1

) (μmol*mg-1

*h-1

)

12 100 1000 1.0922 ± 0.0246 10.58 372 ± 8 Buffer SMN 3

14 500 1000 1.1395 ± 0.0248 10.58 388 ± 8 Buffer SMN 3

Table 2.4 10 500 0 1.2846 ± 0.0471 10.58 437 ± 16 Buffer SMN 6

13 200 1000 1.2497 ± 0.0154 10.58 425 ± 5 Buffer SMN 3

The BBY activity of experiment 13 is the largest value in three above experiment. This

means the best concentration ratio of PPBQ and K3[Fe(CN)6] is 1:5, respectively. In series of three

above experiments, there are some opinions are needed to discuss as follow:

There has ever had a statement, which discusses about the good effect of types

electron acceptor, is PPBQ could be supposed to be the best electron acceptor. If the

comparison about this good effect between using only PPBQ (experiment 10 from

table 4) and using mixture of PPBQ and K3[Fe(CN)6] (concentration ratio of PPBQ :

K3[Fe(CN)6] = 1:5) (experiment 14 of table 5) is showed, the using this mixture of

PPBQ and K3[Fe(CN)6] is similar the using only PPBQ.

Having one more other effect appears on these experiments. As we know, two

compounds PPBQ and DCBQ are the derivatives of benzoquinone which their

conformational structure contain some hydrophobic residues. Thus, they cannot be

dissolved in water and it is necessary to use an organic solvent to dissolve these

compounds. DMSO (abbreviation of Dimethyl Sulfoxide) is the most usual organic

solvent beside ethanol and methanol. All of three intermediate solvents could

dissolve many organic compounds and dissolve themselves into water to form a

homogenous mixture (with any volumetric ratios) very well. Therefore, one more

other effect, which is mentioned above, is the effect of solvent on photosynthetic

process. When the experiment was practiced at the high concentration of PPBQ, it is

the same meaning with adding a large amount of DMSO into this mixture. Perhaps,

DMSO became a inhibitor at its high concentration and is a partial cause of the

decrease BBY activity in experiment 14 (compared with experiment 13). In my

opinions, some experiments are necessary to investigate the effects of organic

solvents (with concentration gradient).

2. The oxygen evolution of thylakoid membrane samples: The thylakoid membrane samples, which are prepared at step 7 of Messinger’s group

protocol, are used for all of the next measurable experiments. The kind of buffer is the first

variable, which was chosen to investigate and find out the best buffer for oxygen evolving

measurements. In the photosynthetic process, oxygen gas is produced simultaneously with the

production of proton (H+) inside thylakoid membrane at Manganese cluster.

2 2Chemical reaction: 2 4 4 *sunlightH O O H e

With purpose to avoid the significant increase the concentration of protons (cause of the

decrease pH) which were formed from reaction (*) have a bad effect for BBY activity (Rumberg

and Siggel 1969), a trapping proton compound is needed to add into the buffer (for example,

NH4Cl). Thus, two more buffers were prepared with following components:

Buffer B: consist of buffer 4 and NH4Cl (250mM), pH = 6.0

Buffer C: include buffer SMN and NH4Cl (10mM), pH = 6.5

Totally, four buffers were used to study the oxygen-evolving activity of thylakoid

membrane samples. These are buffer 4, buffer SMN, buffer B and buffer C. The optimized

condition of BBY concentration, which was achieved from previous measurable experiments of

13

BBY sample, is used for all the farther experiments of thylakoid membrane samples. It means the

concentration of thylakoid membrane samples are kept at a fixed value.

Table 2.6: Measuring and calculating oxygen-evolution activity of Thykakoid samples with the

change of four different buffers (temperature = 20ºC)

No Kind of EA [EA]

(μM) Buffer

Slope SD [Thylakoid]

(μg/ml)

Activity SD Repeat

(times) (μmol*l-1

*s-1

) (μmol*mg-1

*h-1

)

1 PPBQ 500 Buffer 4 0.12500 ± 0.00681 3.33 135 ± 7 3

2 DCBQ 500 Buffer 4 0.11221 ± 0.01278 3.33 121 ± 14 3

3 K3[Fe(CN)6] 500 Buffer 4 0.07167 ± 0.00847 3.33 77 ± 9 3

4 PPBQ 500 buffer B 0.23835 ± 0.02809 3.33 260 ± 30 3

5 DCBQ 500 buffer B 0.19501 ± 0.03618 3.33 210 ± 40 3

6 K3[Fe(CN)6] 500 buffer B 0.19344 ± 0.03588 3.33 210 ± 40 3

7 PPBQ 500 buffer SMN 0.27590 ± 0.05555 3.33 300 ± 60 3

8 DCBQ 500 buffer SMN 0.17822 ± 0.03485 3.33 190 ± 40 3

9 K3[Fe(CN)6] 500 buffer SMN 0.12563 ± 0.03725 3.33 140 ± 40 3

10 PPBQ 500 buffer C 0.78852 ± 0.02576 14.15 201 ± 7 3

11 DCBQ 500 buffer C 0.40915 ± 0.07660 14.15 104 ± 19 3

12 K3[Fe(CN)6] 500 buffer C 0.46977 ± 0.03501 14.15 120 ± 9 3

The same phenomenon occurs with oxygen evolving experiments of thylakoid membrane.

The results of series of experiments 1-3 and experiments 7-9 show that buffer SMN is still better

than buffer 4 through the significant increase of oxygen-evolving activity in the experiments 7-9.

This means the presence of cations Mg2+

and Ca2+

look like two inhibitors for the production of

oxygen in photosynthetic process. The effects of two these cations on photosynthetic process had

ever mentioned in some articles (from 1970 to 1990)

The effect of Mg2+

as a significant inhibitor for photosynthesis of spinach (Mordhay

and Martin 1974; Steven C. Huber April 20, 1978; Miyao and Murata 1986) or

barley (Steven C. Huber November 29, 1978; Steven C. Huber April 20, 1978)

The protective effect of Ca2+

as a substance which could restore oxygen evolution

activity of photosystem II (Miyao and Murata 1986; Charlene et al. 1989)

Moreover, some of monovalent cations (Na+, K

+, Cs

+) also have bad effect for

photosynthetic oxygen evolution (Miyao and Murata 1986)

The best electron acceptor is still PPBQ with many biggest numbers of BBY activities from

experiment 1 to experiment 12. There should be a division of two below group:

Group 1 (from experiment 1 to 6): A comparison of two buffers has the same pH,

the same components apart from NH4Cl (250mM). The oxygen evolving activity of

thylakoid membrane in series of experiments 4-6 become better than this activity in

the rest experiments. Thus, the presence of NH4Cl has a good effect on the

photosynthetic process of thylakoid membrane samples.

Group 2 (from experiment 7 to 12): this is still a comparison with the aim to

research the effect of NH4Cl on thylakoid membrane activity. There is a contrast

compared with result of group 1, the absence of NH4Cl has a good effect for

thylakoid membrane activity.

14

The evaluation between two above group is so different. It is not difficult to understand why

this reverse occurs at here. Because there are not some the same conditions between two series of

experiments 4-6 and 10-12. These two experimental series is practiced in two different days, so, the

concentration of thylakoid membrane samples is so big different (3.33μg/ml and 14.15μg/ml). In

addition, buffer B contains NH4Cl at high concentration (250mM) and pH = 6.0. Besides, the

concentration of NH4Cl in buffer C is lower 25 times than buffer B (10mM) and pH of buffer C is

higher than buffer B. Due to all of these reasons, it is difficult to have any conclusions about the

effect of NH4Cl on the oxygen production of photosynthetic process. Thus, a new series of

experiments are decided to check with NH4Cl concentration gradient.

Table 2.7: Measuring and calculating oxygen-evolution activity of BBY samples as a function of

ammonium chloride concentration (pH = 6.5, temperature = 20ºC)

No [NH4Cl]

(mM)

PPBQ

(μM)

K3[Fe(CN)6]

(μM)

Slope SD [Thylakoid]

(μg/ml)

Activity SD Buffer

Repeat

(times) (μmol*l-1

*s-1

) (μmol*mg-1

*h-1

)

1 0 200 1000 0.5957 ± 0.0553 11.13 193 ± 18 buffer SMN 6

2 10 200 1000 0.5708 ± 0.0195 11.13 185 ± 6 buffer SMN 6

3 100 200 1000 0.5137 ± 0.1396 11.13 170 ± 50 buffer SMN 9

4 300 200 1000 0.3815 ± 0.0931 11.13 120 ± 30 buffer SMN 6

5 500 200 1000 0.2496 ± 0.0089 11.13 81 ± 3 buffer SMN 3

A steady decrease of thylakoid membrane activity showed that thylakoid membrane

samples achieve the best activity with the absence of NH4Cl. The increase of NH4Cl concentration

is the same meaning with adding a large amount of inhibitor, which result bad effect on oxygen

evolving activity.

As we know, adding NH4Cl solution into photosynthetic reaction system has the aim to

prevent the enlargement of proton concentration inside thylakoid membrane (lumen). When NH4Cl

is dissolved in water, it is dissociated electrolytically to form NH4+ and Cl

-. Moreover, ammonium

ion always exist a chemical equilibrium to form proton and ammoniac molecules, which can be

dissolved very well in water.

4 4

4 3

Electrolytic dissociation: NH

Chemical equilibrium: NH

Cl NH Cl

NH H

Actually, thylakoid membranes were injured in whole process of the thylakoid membrane’s

preparation. Therefore, this formed a significant amount of holes, which play a role as channels for

the movement of protons, on the surface of thylakoid membrane. The using NH4Cl at high

concentration does not only increase proton concentration but also increase ammonia concentration

at stroma. If there is a difference of ammonia concentration between stroma and lumen, ammonia

diffusion will be occurred to transfer ammonia molecules from high ammoniac concentration to

low ammonia concentration (according to diffusible law). Dissolved ammonia molecules could be

diffused from stroma to lumen. Due to having a free electron pair on nitrogen atom, ammonia

molecule is a good ligand (or strong nucleophile) which can bind with most of transition metal ions.

Thus, the existence with high concentration of ammonia inside thylakoid membrane can compete

strongly and significantly with water molecules to attach successfully with four manganese atoms.

This means conformational structure of manganese cluster will be changed and prevent (or stop) all

of redox reactions which occur in manganese cluster. Furthermore, the water splitting occurs more

difficult and results lower oxygen yield.

Five experiments (table 7) were done with using the mixture of PPBQ and K3[Fe(CN)6] at

the fixed concentration’s ratio. These experiments are the application result of three below

experiments from number 16-18.

15

Table 2.8: Displays the results of varying the electron acceptor concentration with three of

different electron acceptors and the mixture of PPBQ and K3[Fe(CN)6] with different volumetric

ratio. (pH = 6.5, temperature = 20ºC)

No Kind of EA [EA] Slope SD [Thylakoid]

(μg/ml)

Activity SD Buffer

Repeat

(times) (μmol*l-1

*s-1

) (μmol*mg-1

*h-1

)

10 PPBQ 500μM 0.78852 ± 0.02576 14.152 200 ± 7 buffer SMN 3

11 DCBQ 500μM 0.40915 ± 0.07660 14.152 104 ± 19 buffer SMN 3

12 K3[Fe(CN)6] 500μM 0.46977 ± 0.03501 14.152 120 ± 9 buffer SMN 3

13 PPBQ 200μM 0.89551 ± 0.07828 14.152 230 ± 20 buffer SMN 3

14 DCBQ 200μM 0.48165 ± 0.04480 14.152 123 ± 11 buffer SMN 3

15 K3[Fe(CN)6] 200μM 0.42025 ± 0.00172 14.152 107 ± 0 buffer SMN 3

No PPBQ K3[Fe(CN)6] Slope SD [Thylakoid]

(μg/ml)

Activity SD Buffer

Repeat

(times) (μmol*l-1

*s-1

) (μmol*mg-1

*h-1

)

16 100μM 1000μM 0.79716 ± 0.04748 14.152 203 ± 12 buffer SMN 3

17 200μM 1000μM 0.90998 ± 0.16549 14.152 230 ± 40 buffer SMN 3

18 500μM 1000μM 0.79440 ± 0.13260 14.152 200 ± 30 buffer SMN 3

In general, the effect of electron acceptor concentration on oxygen evolving yield does not have big

difference between high concentration and low concentration of electron acceptor. The practical

results, which were achieved from experiments 10-15, are clear evidence for this statement.

16

3. Protein gel electrophoresis: The aim of this experiment was to test the purity of the BBY and thylakoid membrane

samples and the method that was chosen for this purpose was sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE). In this method SDS, an anionic detergent, is

added to the sample, which efficiently binds to proteins based on their mass and also unfolds the

protein giving rise to similar denatured structures. The SDS-bound proteins have the same charge-

to-mass ratio, and the different proteins can be separated based on their molecular weights. By

using a protein standard containing proteins with known molecular weight, it is possible to identify

the proteins in the sample mixture.

The protein gel is composed of two separate gels, the separation and stacking gels:

Separating gel (80%-85% total area of gel) was used to separate proteins which were

showed under coloured bands on the gel.

Stacking gel (15%-20%) was covered on the separating gel with the aim to load the

protein samples into fixed wells.

Depending on the purpose of detailed investigations (raw investigation or pure

investigation), the volume of gel solution (liquid form), which has not polymerized to become solid

form, will be estimated and calculated suitably. In addition, this volume is also depended on the

thickness and largeness of gel. The big, wide gels were used for pure investigations with the high

protein concentration. In contrary, the small, narrow gels were used for raw investigations and slow

protein concentration.

As the mentioned above, the main component of this gel, which largely effects the

separating resolution, is acrylamide. Hence, a correct choice of acrylamide concentration results in

a good separation for defined mixtures of proteins. High acrylamide concentration (>10%) is

suitable for separating low molecular proteins (<45kDa) (Nack-Shick Choi et al. 2002) and vice

versa (Nack-Shick Choi et al. 2002; Abhijit Banerjee et al. 2010; Fernando Henriques et al. 1975).

The mixture of proteins in this study, which were extracted from the spinach leaves, have molecular

weights in a rather narrow range (20-70 kDa) (Fernando Henriques et al. 1975), which makes the

determination of specific proteins more difficult.

Based on some results and figures from other research groups (Juan B. Arellano et al. 1994;

Terri G. Dunahay and Staehelin L. Andrew 1986), light harvesting chlorophyll a/b (LHC) II

proteins, which are major components of light harvesting antennae of PSII in higher plants and

green algae, were shown to be the dominant species (see Fig. 3.1 and 3.2). Thus, this means the

thylakoid membrane and BBY samples have a high content of the multi-subunit PSII. With the aim

to investigate low molecular weight proteins before investigating high molecular proteins, high

acrylamide concentration (16%) of the separating gel was chosen for the first experiment. The

separating (16%) and stacking (5%) gels were prepared according to the following protocol:

Table 3.1: The protocol for preparation of separating gel (16% acrylamide) and stacking gel (5%

acrylamide).

Separating gel (16%) Stacking gel (5%)

2 mini gels

(μl)

6 mini gels

(μl)

2 mini gels

(μl)

6 mini gels

(μl)

Water 0.8 3.4 Water 2.8 17.5

Tris (2M), pH=8.8 3.75 16 Tris (1M), pH=6.8 0.5 3.125

10% SDS 0.1 0.45 10% SDS 0.04 0.25

30% AA 5.4 23 30% AA 0.65 0.4

10% APS 0.06 0.25 10% APS 0.04 0.25

TEMED 0.006 0.025 TEMED 0.004 0.025

Total volume 10.116 43.125 Total volume 4.034 25.150

17

Note: - Tris (Tris(hydroxymethyl)aminomethane)

- 10% SDS: sodium dodecyl sulfate solution (10%)

- 30% AA: acryl amide solution (30%) (acryl amide under monomer, not polymer)

- 10% APS: ammonium persulfate solution (10%) (solider compound)

- TEMED: tetramethylenediamide solution (great than 99%) (catalyst for polymerization)

Initially, all of the components of separating gel were mixed together in 2 minutes. After

that, this mixture was poured by Pasteur pipette between two glass plates (Bio-rad Laboratories,

Inc.). The height of separating gel is approximate 85% whole height of the complete gel. Ethanol

(or isopropanol) was added on top of the separating gel with the purpose to form a gel surface.

After one hour the acryl amide had completely polymerized and then alcohol solution was

discarded and the surface of separating gel was cleaned with distilled water 3-4 times before

preparation and addition stacking gel. The stacking gel was loaded on top of the separating gel

followed by the insertion of a comb. The stacking gel was polymerized for 20 minutes, and then the

comb was removed. The wells which were formed by the comb were cleaned several times with

distilled water to remove all air bubbles.

Thylakoid and BBY samples were diluted from stock solutions to 100μg/ml. A mixture of

the diluted samples and loading buffer (containing SDS) with volumetric ratio 4:1 was prepared.

The samples were heated at 75 ºC for 5 minutes to facilitate the unfolding of all the proteins in the

sample. Following that, the samples were loaded into the wells according the table below.

Table 3.2: Display of the loading of thylakoid samples (or BBY samples) into the wells.

Number 1 2 3 4 5 6 7 8 9 10

Sample marker thy thy thy thy thy thy thy thy thy

Volume (μl) 10 6.25 10 12.5 18.75 25 31.25 37.5 43.75 50

Chl mass (μg) 0 0.5 0.8 1 1.5 2 2.5 3 3.5 4

Note: Marker = protein standard 10-250 kDa

Thy = thylakoid membrane sample

Chl mass = total mass of chlorophyll in the well

Another gel was prepared for loading the BBY sample and these two gels were immersed in

running buffer (1X Tris Glycine-SDS buffer). The electrophoresis was run with DC (direct current)

source at 100V for 4 hours in cold room (4-5ºC). After running in 4 hours, the gels were stained

with hot coomassie brilliant blue (CBB) staining solution over night. The next day, the staining

solution was discarded and replaced with destaining solution (10% acetic acid), repeated 4-5 times.

Finally, these gels were scanned to evaluate the purity of the samples (see Fig. 3.1 and 3.2).

Figure 3.1: The acrylamide (16%) gel of

thylakoid sample.

Figure 3.2: The acrylamide (16%) gel of BBY

sample.

18

As observed in Fig. 3.1 and 3.2 three dark bands (indication of high concentration) of the

mixture of LHC II (24, 26 and 28kDa) appear at the same spot for both thylakoid and BBY

preparations, whereas a clear difference is the presence of some of the subunits in ATP synthase.

The high content of alpha-CF1 (59kDa) and beta-CF1 (56kDa) can easily be seen in the thylakoid

gel (Fig. 3.1). In addition, two protein bands corresponding to D1 and D2 proteins (32-33kDa) of

PSII complexes appear at the same spot in both gels, though these bands are a little bit diffuse in

the thylakoid gel. Moreover, the other proteins in the gel of the BBY sample (Fig. 3.2) appear as

weak bands. This means that these proteins, in contrary to the major componential proteins, do not

significantly contribute to the total protein content in the thylakoid and BBY samples.

To be able observe the bands that corresponds to the three protein subunits in LHC II, the

concentration of acrylamide was increased along with the addition of 4 M urea. Urea is used to

increase the resolution, especially for membrane proteins (such as the LHC proteins).

Figure 3.3: The gel (16% acrylamide + 4 M

urea) of thylakoid sample.

Figure 3.4: The gel (16% acrylamide + 4 M

urea) of BBY sample.

As can be seen in Fig. 3.5, two new bands appear in the thylakoid gel (16% acrylamide + 4

M urea). These corresponds to Cyt b6 (in Cyt b6/f complex) (23kDa) and a mixture of plastocyanin

(protein core III in PSI complex) and Fe2S2 (in Cyt b6/f complex) (20kDa). Besides, there are two

protein bands corresponding to PsbP (23kDa) in the gel for BBY and central protein core in PSII

(43, 47kDa) (see Fig 3.6). Thus, the addition of urea in the gel was able to increase the separation

of these two proteins, even though it is a very small difference in molecular weight.

Figure 3.5: The gel (17.5% acrylamide + 4 M

urea) of thylakoid sample. Figure 3.6: The gel (17.5% acrylamide + 4 M

urea) of thylakoid sample.

19

Figure 3.7: The gel (17.5% acrylamide) of

thylakoid sample.

Figure 3.8: The gel (17.5% acrylamide) of

thylakoid sample.

The four gels in figure 3.5-3.8, the protein bands are almost identical to the gels in figure

3.1-3.4, with the exception of the light-colored protein band (16kDa) and PsbU (12kDa) in figure

3.6. Therefore it is not difficult to exactly identify the protein that corresponds to this position.

Moreover, the increase of acrylamide concentration did not attain higher resolution for BBY

and thylakoid samples significantly. Therefore, it is necessary to perform more experiments with

lower concentration of acrylamide (< 16%) to compare with the experiments in this study. The low

resolution could partially be explained of the use of low voltage to run the gel and also the size of

the gel. Hence, the experiments with high concentration of acrylamide should be run using high

voltage and a larger gel.

20

4. Performing oxygen flash-yield measurement with Joliot electrode: With the aim to investigate the oxygen flash-yield, Joliot electrode is a useful instrument,

though specifically for natural photosynthetic system. This means that this method is not suitable

for artificial photosynthetic systems, which do not work according to the Kok-cycle. Closely

resembling the basic working principles of Clark electrode, Joliot electrode is also based on the

principles of polarography for quantitative analysis. Therefore, this type of electrode can utilized to

detect and measure oxygen evolution at the trace concentration. In figure 4.1 and 4.2 the exterior

and the interior of the Joliot electrode are depicted, respectively.

Figure 4.1: The exterior shape

of Joliot electrode.

Figure 4.2: The interior structure of Joliot electrode (Gernot

Renger, Bertram Hanssum, 2009).

The Joliot electrode, which was used in this project, was designed and manufactured

handmade. Thus, it does not have the brand of producer (company or commercial corporation), its

model, its code, etc. There are some basic differences between the Clark and Joliot electrodes:

The Joliot electrode does not use a projector, which continuously illuminates the

sample compartment like the light source of Clark electrode. Also, the Joliot

electrode uses a Xenon discharge lamp. This lamp can be set to a certain frequency

(often 2Hz) to release single-turnover light flashes.

Another difference between Clark electrode and Joliot electrode is the state and

composition of the buffer. The buffer used for Joliot electrode does not contain

sugar (D-sucrose); a compound which is usually used in buffer preparations for all

experiments with Clark electrode. In addition, the state of the buffer is not

stationary; it is always mobile (see figure 4.2) on the surface of dialysis membrane,

in contrast to the stirred state for the Clark electrode.

Joliot electrode cannot be calibrated like the calibration of the Clark electrode.

Moreover, this could be a disadvantage and shortcoming of this electrode to

compare exactly the measurable results of the same sample at the different times.

Besides, due to the high sensitivity and good detection of this method, it is not

essential to add artificial electron acceptors.

The membrane (usually Teflon membrane) which was used in experiments with

Clark electrode covered on the surface of Pt-cathode to prevent the contact of water

and Pt-cathode surface, but, it allow for oxygen diffusion very well. In contrast, in

the experiments with Joliot electrode, the samples were applied directly on the Pf-

cathode surface. This means the sensitivity of Joliot electrode is much greater than

Clark electrode.

Unfortunately, the thermostat of the Joliot electrode used in this study was not functioning,

therefore all experiments were performed at room temperature (22-23 °C). Of course, the instability

21

of temperature in the whole experimental process affect the measurable results. Fortunately, the

small variation of temperature resulted in only a minor effect to the results. In all the experiments,

the thylakoid and BBY samples were diluted to contain 1 mg/ml chlorophyll. The effect of artifacts

on the result had to be ruled out. Therefore, the raw data was processed for both thylakoid and BBY

sample, using Origin software (version 8). In figure 4.3-4.6 comparison between the raw and

processed data are depicted, showing the removal of artifacts.

Figure 4.3: Plot of the thylakoid sample, based

on raw data.

Figure 4.4: The processed plot of the thylakoid

samole, to remove all artifacts (spikes).

Figure 4.5: Plot based of the BBY sample, based

on raw data. Figure 4.6: The processed plot of the BBY

sample, to clear away all artifacts (spikes).

Compared to using the Clark electrode, the Joliot electrode has fewer variables to take into

consideration. The protein sample (thylakoid or BBY sample) and experimental condition (with

preflash or not, pH of buffer, temperature, etc) comprise the important variables. With the aim to

compare the flash pattern between the thylakoid and BBY samples, at first classification of the

sample was determined without preflash, while the rest of the experimental conditions were fixed

such as the pH of the buffer (6.5) and the temperature (22 °C).

22

Figure 4.7: The processed plot of thylakoid

sample without preflash. Figure 4.8: The processed plot of BBY sample

without preflash.

Some basic differences and resemblances can be seen between the plots of BBY and

thylakoid samples without preflash. According to the Kok model, the oxygen yield is a function of

the flash number exhibiting a periodicity of four. The maximum oxygen yield occurs on the third

flash rather than fourth flash and this agrees with the first cycle of BBY and thylakoid plots (Fig.

4.7 and 4.8). In addition, the periodic property of the four flashes disappears after several cycles in

these figures, which also agrees with the Kok model.

Besides, for BBY sample (Fig. 4.8) the signal disappears faster than for the thylakoid

sample after the second cycle. Moreover, the deeper peaks (higher intensitiy) of the thylakoid

sample correspond to the smaller oxygenic production of the BBY sample than the thylakoid

sample. Compared to the investigation with the Clark electrode this finding is completely opposite.

To explain for this problem, the protein composition of BBY and thylakoid samples could be a

logical explanation. Due to the loss of PSI, cytochrome b6f and ATP synthase, BBY sample does

not have anywhere to release electrons. Thus, it could be that electron would come back to

manganese cluster and lock the central core of photosynthetic reaction. On the other hand, the

oxygen production must be stopped.

Figure 4.9: The processed plot of thylakoid

sample with preflash

Figure 4.10: The processed plot of BBY sample

with preflash

Experiments were performed with an additional preflash for 5 minutes, with the aim to

make comparison between the experiments with or without preflash. In general, there are no

differences between the preflash and non-preflash experiments of the thylakoid sample (Fig. 4.7

and 4.9) and BBY samples (Fig 4.8 and 4.10). The time between preflash and normal flash, which

23

was set up with a fixed frequency (2Hz), could be an explanation for this resemblance. When

samples are illuminated by preflash, the oxidation state S1 transfer to S2. This time (5 minutes) is

long enough for one electron to come back completely to the manganese cluster; it means S2 gets

back S1.

In order to account for the loss of the periodicity (four flashes cycle), Kok and his co-

worker assumed that in some PSII complexes, a momentary saturating light flash may not succeed

to advance to a the higher state (called misses or alpha parameter). In reverse, in some other

complex core, the flash could promote a two-state advance (resulting in so called double hits or

beta parameter). The Kok model was able to not only explain the flash dependence of oxygen

evolution but also to create an important foundation for continuous research of the mechanism of

water oxidation and oxygen release by PSII (Govindjee et al. 2010).

All of the experiments of the thylakoid and BBY samples (with preflash or non-preflash)

were attained three times and the recorded data were plotted using the Origin software (version 8).

After being printed, the length of the peaks were measured manually by ruler. Then, all measured

distances were typed into a specific Excel file, which had been designed by Johannes Messinger to

calculate α, β and damping parameters. Due to the faster loss of periodicity (four flashes cycle),

only the first ten flashes were used to calculate these parameters for the BBY sample and 16 flashes

for the thylakoid sample.

Table 4.1: Calculation of the three parameters (alpha, beta and deviation) of thylakoid and BBY

samples (with preflash or not).

alpha beta damping alpha beta damping

Thy-1 12.19 2.01 98.35 BBY-1 18.40 2.16 91.43

Thy-2 12.29 2.29 98.26 BBY-2 18.12 2.06 91.11

Thy-3 12.24 2.29 98.30 BBY-3 18.30 2.46 90.87

Average 1 = 12.24 β1 = 2.20 d1 = 98.30 Average α2 = 18.27 β2 = 2.23 d2 = 91.14

Pre-thy-1 10.71 2.88 98.67 Pre-BBY-1 17.38 3.10 92.59

Pre-thy-2 10.87 2.56 98.40 Pre-BBY-2 16.47 2.75 92.38

Pre-thy-3 10.35 3.05 98.52 Pre-BBY-3 16.82 2.97 92.15

Average 3 = 10.64 β3 = 2.83 d3 = 98.53 Average α4 = 16.89 β4 = 2.94 d4 = 92.37

Note: Thy-1 = thylakoid sample, first time (without preflash)

Pre-thy-1 = thylakoid sample, first time (with preflash)

Similar meaning for BBY-1 and pre-BBY-1

Two mechanisms were suggested for the reaction S2 to S1:

2 1

2 1

mechanism: S

Slow mechanism: S

D D

B B

Fast Y S Y very stable

Q S Q

Based on these two mechanisms of reaction S2 to S1 and the rate of these mechanisms (will

be mentioned later on), a suitable explanation could be given to answer for the difference about

misses parameter between experiments with preflash or not.

Without preflash: Normal flashes were set up with frequency 2Hz, it means there are

two flashes in one minute or the time between two flashes is 500ms. When one flash

was released to sample, PSII complexes absorb this flash and advance from tyrosine

S1 (S1YD) to tyrosine S2 (S2YD). S2YD will be transferred to S1YD radical so fast

(three seconds). In the short time (between two flashes, 500ms), this radical cannot

have enough time to stabilize completely and the loss of S2YD is significant. Thus,

S2YD also advanced continuously to next higher state (S3YD) with slow content. In

24

addition, the series of these experiments without preflash almost follow the fast

mechanism (fig. 4.11)

Figure 4.11: the plot illustrates for the major mechanism of the experiments without

preflash.

With preflash: the time (5 minutes) between preflash and normal flashes is long

enough to stabilize the S1YD radical completely. So, fast mechanism is not more

continuous (or stop completely) and slow mechanism will be continuous with the

next normal flashes. When one flash was released to sample, PSII complexes absorb

this flash and advance from plastoquinone S1 (S1QD) to plastoquinone S2 (S2QD-).

S2QD- will be transferred to S1QD (50 seconds) so slower than fast mechanism (fig.

4.12). Thus, most of S2QD- was reserved to absorb the next flash and advance to

S3QD. That is why the misses parameter of preflash-experiments is lower than these

experiments without preflash.

Figure 4.12: the plot illustrates for major mechanism of the experiments with

preflash.

The dissimilarity between these values α1 and α2, α3 and α4 indicate that the percentage of

PSII complexes, which might be failed to advance the next state, of BBY sample is greater than for

the thylakoid sample. However, these values β1 and β2, β3 and β4 are similar and this means the

other PSII complexes that may promote two-state advance is approximately equal between the

BBY and thylakoid samples.

Moreover, the differences between α1 and α3, α2 and α4 suggest there is a small effect of the

addition of the preflash before continuous normal flashes. 5 minutes is long enough for S2 get back

to S1 almost completely, so it can not be 100%. Additionally, β1 and β3, β2 and β4 are not identical

to β1 and β2, β3 and β4. Though, the dissimilarity is small which means the percentage of the core

(which could advance to a two-state (double hits)) in the oxygen evolving complex, was not

significantly larger for the experiments with preflash than for the others without preflash.

To measure the lifetime of S2, an experimental series were performed with a gradient of the

time between first flash and second flash. This gradient corresponds to the increase of time between

the first and second flash. These experiments were performed for only the thylakoid sample (not

BBY). Furthermore, the different experimental condition (with preflash for 3 minutes or not) was

also applied in these measurements.

Similar to the optimal process and calculation of the three parameters (alpha, beta and

deviation), the specific excel file (designed by Johannes Messinger) were used again to calculate

25

the other parameters (S1 and S2). Then, two plots of without preflash and with preflash were made

with S2 as a function of time (s). These plots were optimized many times to find out the specific

kinetics for the primary photochemical reaction S2 to S1.

The integrated first order-rate law (for first order reaction):

0 0* ln ln . 4.1ktA A e A kt A eq

Note: A0 = initial concentration of reagent (t=0)

k = the first order rate constant

t = the time

A = the remaining concentration or percent of reagent at a fixed time (t)

The half-life of a first-order reaction: 1/2

ln 2. 4.2t eq

k

Based on equation 4.1 and equation 4.2, all the experimental values, which were collected

from these series of experiments, were optimized with the smallest error and to calculate the half-

life of S2 (see Fig. 4.11). In the first version (version 0), the reaction from S2 to S1 is assumed to

only follow one mechanism (fast mechanism or slow mechanism).

Figure 4.11: Display of S2 as a function of time

(s) (without preflash) (version 0)

Figure 4.12: Display of S2 as a function of time

(s) (with preflash) (version 0)

The great difference between the experimental curve and calculated curve indicated that one

mechanism (slow or fast mechanism) could not be illustrated exactly the reaction of S2 to S1.

Therefore, the related calculated number (first order rate constant k, S2 at t=0 and the half-life t1/2)

are also not reliable. The reaction (S2 to S1) could be represented better with a kinetic equation,

which consists of two first order reactions looks like, the below equation:

1 2

01 02* *k t k tA A e A e or

1 2

2 2.1 0 2.2 0* *k t k t

t tS S e S e

26

Figure 4.13: Display of S2 as a function of time

(s) (without preflash) (version 1)

Figure 4.14: Display of S2 as a function of time

(s) (with preflash) (version 1)

On the other hand, S2 depended on four parameters (not two parameters as version 0). These

are k1, k2, S2.1 (t=0) and S2.2 (t=0). Based on the fig. 4.13 and 4.14, these two curves of experiment and

calculation were approximately identical (except for some different points). The identicalness

expressed correctly the mechanism of reaction S2 to S1 and prove that this reaction consist of two

major these mechanisms (slow and fast mechanism). However, four concerning parameters and the

half-life that were calculated from k1 and k2 are not so resemble. The experimental series with

preflash or not have just represented for one reaction. Thus, it is so curious if the rate (or half-time)

of this reaction in two situations (with preflash or not) is so different. The reason of this

dissimilarity could be the time (3 minutes) between preflash and normal flashes was not long

enough to stabilize completely S1YD.

With the ambition to create the similarity between two situations (with preflash or not), k1

and k2 of one situation which were trusted that more reliable will be kept constantly. These other

parameters would perform to be equal with those constants. Then, the rest parameters were

optimized again to form more version 2 (fig. 4.15 and fig. 4.16) and version 3 (fig. 4.17 and fig.

4.18).

Figure 4.15: Display of S2 as a function of time

(s) (without preflash) (version 2)

Figure 4.16: Display of S2 as a function of time

(s) (with preflash) (version 2)

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Figure 4.17: Display of S2 as a function of time

(s) (without preflash) (version 3) Figure 4.18: Display of S2 as a function of time

(s) (with preflash) (version 3)

The optimized method of version 2 and 3 are not better than version 1 because the

identicalness as version 1 cannot achieve with two these versions (see fig. 4.15 and 4.17). But, it is

so unlogical when these are the same process and gave two different results. Thus, the version 2

(following our assessment) could be the best version in four versions with the half-life of low

mechanism is 50 ± 10 (s) and fast mechanism is 3 ± 2(s).

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3. Conclusion Based on the experiments were performed with Clark electrode, the best experimental conditions

for BBY sample and thylakoid sample is summarized as following:

BBY sample: BBY concentration is 10μg/ml. The kind of electron acceptor is PPBQ

or mixture of PPBQ and K3[Fe(CN)6] with fixed concentration ratio 1:5,

respectively. The concentration of electron acceptor does not affect so much to

activity of BBY sample and the best concentration of electron accepter was

suggested to be 500μM. Buffer 4 or buffer SMN is also good; there are not any large

differences about oxygen evolving activity between these buffers.

Thylakoid sample: thylakoid concentration is 10μg/ml. The kind of electron

acceptor and the concentration of electron acceptor are the same results with BBY

samples. But, the best buffer is buffer SMN, the bad effect of magnesium ion was

aware clearly in the experiments with thylakoid sample.

Some suggestions for the continuous experiments: the mixture of DCPQ and

K3[Fe(CN)6] at the dissimilar concentration ratio will be investigated to compare

with the results of the mixture of PPBQ and K3[Fe(CN)6]; the effect of organic

solvent which were used to dissolve electron acceptors; the buffer will not contain

Mg2+

or Na+ or the other mono-valence ions that have the bad effects for oxygen

evolution activity of thylakoid and BBY samples

The results, which were collected from the SDS gel, had given us much information about the

purity of these initial samples:

Many major proteins, which exist in the thylakoid membrane, appeared under

coloured protein bands. This means the purity of initial samples (BBY and thylakoid

sample) is good and the existence of contaminant proteins is not significant.

Suggestion for future: low concentration of acrylamide should be applied to

investigate high molecular weight proteins. Besides, the higher voltage and bigger

size gel will be also used to increase the resolution of this method.

Finally, the figures and information from Joliot electrode measurements were given us more hints

about the mechanism of photochemical reaction inside PSII core protein:

The oxygen flash yield of BBY and thylakoid samples are also according correctly

to Kok model; BBY sample lose completely the periodic property (after second

cycle) faster than thylakoid sample. The preflash in long enough time (5 minutes)

did not affect so much to flash pattern of BBY and thylakoid samples.

These two mechanisms (slow and fast mechanisms) are so specific to express for the

reaction (S2 to S1) and the half-life of slow mechanism is 50 ± 10 (s) and fast

mechanism is 3 ± 2(s).

Future plans: The life-time of S2 could be measured again with great care and the

life-time of S3 will be measured with suitable experiments and explained by specific

mechanisms. Some of the experimental conditions will be changed (for example pH

of buffer or experimental temperature, . . .)

29

4. Acknowledgements Firstly, I would like to thank all the staff at the Department of Chemistry, Umeå University

and University of Natural Sciences of Hochiminh City who paved the way for me to come to Umeå

for study.

A special thank goes to my supervisors, Han Guangye and Sergey Koroidov who offered

me about lab technique in this project. Your helps, stimulating suggestions and important

comments help me finish this project as well as write this report.

I would like to thank Prof. Johannes Messinger so much for your corrections and comments

on my report. I greatly appreciate your practical lessons, useful guidance lab.

I also want to thank all my friends (especially Nguyen Le Ninh Thai and Marcus Wallgren)

for their help and encouragements during the time of the project and when writing this report.

At last, but not least, I would like to thank my family (especially my father – Vo Thanh Hai,

my mother – Pham Cuu Huyen and my younger brother – Vo Pham Lan) who always stand by me

and be my strong supports.

30

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