Running head: RELATIVE REDOX ACTIVITY OF LABORATORY ENRICHED

CHLOROPLAST 1

Laboratory Experiment to Measure the Relative Redox Activity of Laboratory Enriched

Chloroplast.

Name

Institution
RELATIVE REDOX ACTIVITY OF LABORATORY ENRICHED CHLOROPLAST 2

Laboratory Experiment to Measure the Relative Redox Activity of Laboratory Made

Enriched Chloroplast

Introduction

An indication of redox reactions, DCPIP is reduced by the addition of two electrons

and two hydrogen ions to each of its molecules. DCPIP, in either of its forms, is hydrophobic

and has the ability to insinuate itself into cellular membranes. It does this by taking electrons

and hydrogen ions from plastoquinone, which is one of the components of the chloroplastic

electron transfer chain, which is located within the thylakoid membranes of chloroplasts.

Since DCPIP can also be reduced by high-energy electrons coming from other sources, it is

necessary for the calculations that give a measure of photosynthetic activity to be based on

the changes in absorbance that occur between the samples that are illuminated and those that

are kept in the dark. This is because DCPIP can also be reduced by high-energy electrons.

The goal here is to rule out the possibility of a reduction in absorbance brought on by the

lowering of DCPIP brought about by electron donors that are not involved in photosynthesis.

There are two photosystems in the chloroplast thylakoid membrane, and light is what drives

electron transport through them. In vitro investigation of the process is possible if an electron

acceptor is present. For the purpose of this activity, the oxidized form of DCPIP (2,6-

dichlorophenol indophenol) will accept two electrons per molecule from photosystem II. This

will be demonstrated by the reaction below:

H20 + DCPIPox light ½ O2 + 2H+ + DCPIPred

The oxidized form of DCPIP is a dark blue color, while the reduced form has no discernible

hue. A spectrophotometer is used in this test to measure the change in the amount of light that

is absorbed at a wavelength of 590 nm. This allows the level of DCPIP decrease to be

calculated.
RELATIVE REDOX ACTIVITY OF LABORATORY ENRICHED CHLOROPLAST 3

Method

The laboratory experiment utilised the following apparatus; test tubes, test tube rack,

marker, pestle and mortar, 50ml beakers, 50 mL centrifuge, 50 mL test tube, 10mL droppers,

forceps, spatula, cheesecloth, micropipette, a microscope, microscope slides

spectrophotometer, and a stopwatch. Reagents used in the experiment included 3 spinach

leaves, cold chloroplast isolation buffer, DCPIP, reaction buffer, and distilled water. The

experiment was conducted in two exercises. The first execise was conducted to achieve

chloroplast enrichment while the second measured the chloroplast’s relative redox activity.

The first exercise was conducted as described below.

The first exercise involved encriching chloroplasts located in spinach leaves and

extracting them. Three spinach leaves were obtained and all the large central stalks and veins

were removed using forceps to ensure there was no difficulty in grinding. After this was

done, the spinach leaves were placed in a mortar. Using a dropper, 10ml of cold chloroplast

isolation buffer was measured and added to the spinach in the mortar. The spinach leaves and

chloroplast isolation buffer mixture was then ground into a paste. An additional 10ml of cold

chloroplast isolation buffer was measured using a 10 mL dropper and added to the ground

mixture in the mortar. After the buffer was added in the mortar, the mixture was ground

continuously into a paste. A drop was extracted from the resulting paste using a dropper. The

drop was then placed on the slide of a microscope and a coverslip used to cover the liquid.

Thc covered sample was then labelled as sample 1.

The remaining paste consisting of the cold chloroplast isolation buffer and the spinach

in the mortar was then extracted using a spatula and placed on a cheesecloth. Using the

cheesecloth, the paste was filtered into a cold 50ml beaker. The resulting filtered liquid was
RELATIVE REDOX ACTIVITY OF LABORATORY ENRICHED CHLOROPLAST 4

then added to a chilled 50ml centrifuge and the liquid was spun fo five minutes at a speed of

1000 times the gravitational force. After the liquid supernatant was spun, a drop of it was

extracted using a dropper . The drop was then placed on the slide of a microscope and

covered using a coverslip. After it was covered, it was labelled using a marker as sample 2.

The liquid supertanant that remained was dropped into another 50 ml tube. Extra care was

taken to ensure that the pellet located at the tube’s bottom was not disturbed during the

transfer of the liquid.

Using a dropper, 10 ml of chloroplast solution buffer was measured and added to the

pellet in the tube’s bottom. Gentle vortexing was used to suspend it in the buffer. One drop of

the resuspended pellet was extracted using a dropper and placed on the slide of a microscope.

The drop was then covered using a cover slip and then labelled using a marker as sample 3.

The resuspended pellet that remained was the stored on ice. Samples 1, 2, and 3, were then

observed under a microscope to draw comparisons. Notes were taken of how the chloroplast

numbers were affected by the process of isolation. The debris amount that was observed in

each sample was also noted. This marked the end of the first exercise.

The second exercise focused on quantifying photosynthetic activity. The first step in

this exercise was picking 10 empty test tubes from the test tube rack and labelling them from

0-9. After labelling was done, the test tubes were returned to the test tube rack. A dropper

was used to add 1.5 ml of water while 500µL of DCPIP and 500 µL of reaction buffer were

added using a micropipette, to the ten test tubes apart from the test tube labelled ‘0’. The test tube

labelled ‘0’ was chosen as the control and it was filled with 2.0 ml of water instead of 1.5ml.

Additionally, it was filled with 500 µL of reaction buffer and no 500 µL DCPIP was added.

Using a micropipette, 25 µL of choloroplasts that had been enriched and prepared in exercise

one was added to the test tubes labelled 0, 4, 5, 6, 7, 8 and 9. A vortex was then used to
RELATIVE REDOX ACTIVITY OF LABORATORY ENRICHED CHLOROPLAST 5

thoroughly mix the contents of these test tubes.Vortexing was stopped after the chloroplasts

solution had sufficiently mixed with the mixtures in the test tubes.

Immediately after mixing was done, test tube 0 was used to blank the

spectrophotometer. After blanking, the spectrometer was used to observe each tube’s

absorbance at 600nm. The test tubes were divided into four groups; test tube 0, test tubes 1 to

3, test tubes 4 to 6, and test tubes 7 to 9. Test tubes 0, 1, 2, 3,7, 8, and 9 were placed in a tube

rack that was positioned directly under sunlight but they were separated . Test tubes 4, 5, and

6 were placed in a test tube rack and the rack placed in darkness. Using a stop watch, 5

minutes were measured. After 5 minutes elapsed, the spectrophotometer was used to observe

each test tube’s asorbance at 600nm. And each test tube’s absorbance reading was recorded.

During this process the test tubes were retrieved from their racks put in the

spectrophotometer, their readings recorded and the test tubes returned to their respective

racks immediately after. This process was repeated using 10, 15, and 20 minutes as the

waiting times. The results of each of the trials was recorded in a data table.

Results

The results of the experiment were recorded in the data table below.

Table 1. Data table highlighting the test tube number, sample group, the mean absorbance,

and time in minutes.

Test Samples Abs(0 Abs(5mins) Abs(10mins) Abs(15mins) Abs(20mins)

tube min)
#
1 No 0.346 0.346 0.345 0.346 0.345
chloroplast
2 No 0.334 0.333 0.333 0.333 0.332
chloroplast
3 No 0.330 0.331 0.330 0.330 0.329
chloroplast
4 Chloroplasts 0.342 0.336 0.337 0.332 0.339
in dark
RELATIVE REDOX ACTIVITY OF LABORATORY ENRICHED CHLOROPLAST 6

5 Chloroplasts 0.329 0.328 0.328 0.329 0.330

in dark
6 Chloroplasts 0.330 0.328 0.329 0.329 0.329
in dark
7 Chloroplasts 0.331 0.280 0.240 0.213 0.176
in light
8 Chloroplasts 0.324 0.268 0.227 0.194 0.163
in light
9 Chloroplasts 0.331 0.278 0.233 0.216 0.200
in light

Figure 1. Graph of mean absorbance versus time for the group contaiing no chloroplast

No Chloroplast
Mean Absorbance vs Time
0.35

0.345
TEST TUBE 1
0.34 Logarithmic (TEST TUBE 1)
Mean Absorbance

Linear (TEST TUBE 1)

TEST TUBE 2
0.335
Polynomial (TEST TUBE 2)
TEST TUBE 3
0.33 Polynomial (TEST TUBE 3)

0.325

0.32
0 5 10 15 20 25

Time (minutes)

Figure 2. Graph of mean absorbance versus time for the group that had chloroplast in the

dark.
RELATIVE REDOX ACTIVITY OF LABORATORY ENRICHED CHLOROPLAST 7

Chloroplast in Dark
Mean Absorbance vs time
0.345 TEST TUBE 4
Polynomial (TEST
TUBE 4)
0.34

0.335
Axis Title

0.33

0.325

0.32
0 5 10 15 20 25
Axis Title

Figure 3. Graph of mean absorbance versus time for the group that had chloroplast in the

light.

Chloroplast in Light
Mean Absorbance vs time
TEST TUBE 7
0.35
Polynomial
0.33 (TEST TUBE 7)
0.31 TEST TUBE 8
0.29 Polynomial
Mean Absorbance

(TEST TUBE 8)
0.27
0.25
0.23
0.21
0.19
0.17
0.15
0 5 10 15 20 25
Time(minutes)
RELATIVE REDOX ACTIVITY OF LABORATORY ENRICHED CHLOROPLAST 8

Discussion

Which fraction from exercise 1 had the largest number of chloroplasts? What purpose did the

cheese-cloth and centrifugation step serve?

After that, the leftover paste in the mortar, which consisted of the cold chloroplast isolation

buffer and the spinach, was scraped out with a spatula and spread on some cheesecloth. The

paste was strained through the cheesecloth and placed in a cool beaker of 50 ml capacity.

Compare samples with no chloroplasts with those of samples that contained chloroplasts in

the light. What did you observe with the absorbance at 600 nm in the tubes with chloroplasts?

The mean absorbance with chloroplasts in light decreases with time while the mean

absorbance with no chloroplasts remains relatively constant over time.

RELATIVE REDOX ACTIVITY OF LABORATORY ENRICHED CHLOROPLAST 9

References

Buss, D. M., & Schmitt, D. P. (2016). Sexual strategies theory. In Encyclopedia of

evolutionary psychological science (pp. 7509-7513). Cham: Springer International

Publishing. Retrieved March 23, 2022, from

https://www.researchgate.net/publication/312133506_Sexual_Strategies_Theory