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Complete 2 pages APA formatted article: Studies on Photosynthesis Using the Hill Reaction. HILL REACTION LAB REPORT pc [Pick the STUDIES ON PHOTOSYNTHESIS USING THE HILL REACTION LAB REPORT Introduction Life on earth is largely based on the photochemical process of food synthesis carried out by chlorophyll carrying plants and cyanobacteria. This cellular process known as photosynthesis is carried out within the chloroplasts of these organisms and involves a series of sequential chemical reactions culminating in the conversion of sun’s energy and resulting in the synthesis of carbohydrates from carbon dioxide (CO2). Photosynthesis is represented by the following reaction:

6H12O6+6CO2+6H2O

The complex process of photosynthesis is classified in to two stages based on the need of light. The primary ‘light reaction’ stage requires light and involves the excitation of electrons of the chlorophyll. These electrons then proceed along a series of electron carriers embedded in the thylakoid of the chloroplast resulting in synthesis of ATP in the stroma and the reduction of NADP. The dark reactions of photosynthesis do not directly require light involve the use of ATP and NADPH formed during the light reaction (figure 1) (Berg et al., 553).

Figure 1: z-scheme: pathway of electron flow from H2O to NADP+ in photosynthesis (Berg et al., ch. 19)

Hill reaction named after its discoverer Robert Hill (1937) involves the photoreduction of an electron acceptor by the hydrogen atoms of water, along with evolution of oxygen. In laboratory, Hill reaction is demonstrated when isolated and illuminated ‘chloroplasts’ reduce an electron acceptor present with it, and release molecular oxygen:

The electron acceptors used in laboratory conditions are not the natural electron acceptors or NADP but instead are artificial electron acceptors (Walker, 109). These artificial electron acceptors intercept the electrons before they reach PS1700. Further they are generally dyes that change color when reduced e.g. DCIP (2,6-dichlorophenolindophenol). Thus Hill reaction can be written as:

Thus using the above equation, rate of Hill reaction in isolated protoplasts can be experimentally estimated. The change in absorbance of the dye at 600nm measured under varied conditions of experiment gives the rate of Hill reaction occurring under the preselected set of conditions. Several of the detergents are also known to be inhibitors of photosynthesis such as DCMU [3-(3, 4-dichlorophenyl)-1, 1-dimethylurea], DNP [dinitrophenol] and triton-X etc.

Hypothesis

The reagents DCMU, DNP and triton-X, and the absence of light. disrupt the Hill reaction.

Procedure

Chloroplast was extracted from deveined, washed and blot dried fresh baby spinach leaves. The standard fractionation protocol for extraction of chloroplasts was followed. 50g spinach leaves were homogenized in a pre-chilled blender jar along with buffered isotonic salt solution and antioxidant solution. The homogenate was then squeezed through cheesecloth to ensure only smaller pieces are utilized for centrifugation. The filtrate was next centrifuged at 1600rpm for 90seconds. The supernatant was then centrifuged at 6000rpm for 10 min, leading to sedimentation of most chloroplasts and nuclei. All solutions and containers were maintained at 0◦C during the fractionation procedure.

Chlorophyll concentration was determined by recording absorbance of 10µl suspension along with acetone. 5 tubes are prepared with their corresponding blanks as shown in table 1 and 2. Tube 1 is placed in dark, tube 2 in light, and to tube 3, 4 and 5. DCMU, DNP and Triton-X are added respectively. Absorbance is recorded at 600nm and the graph is plotted to make a comparative assessment of rate of Hill reaction under the 5 conditions.

Table 1: Reagents Blanks

Tubes

Components

Volume (mL)

A

Isolation Buffer

3.5

Distilled water

1.0

Chloroplast suspension

0.5

B

Isolation Buffer

3.5

Distilled water

0.5

0.8 mg chl/mL

0.5

Chloroplast suspension

0.5

C

Isolation Buffer

3.5

Distilled water

0.5

0.15 mM DCMU

0.5

Chloroplast suspension

0.5

D

Isolation Buffer

3.5

Distilled water

0.5

0.2% (v/v) triton X-100

0.5

Chloroplast suspension

0.5

Table 2: Preparation of Reaction Tubes

Volume (mL)

Tube no.

Isolation Buffer

Distilled Water

1 mM DCPIP

0.8 mM DCMU

0.5 mM DNP

0.2% (v/v) Triton

Chloroplast suspension

Treatment

Reagents Blank

1

3.5

0.5

0.5

–

–

–

0.5

Dark

A

2

3.5

0.5

0.5

–

–

–

0.5

Light

A

3

3.5

–

0.5

0.5

–

–

0.5

Light

B

4

3.5

–

0.5

–

0.5

–

0.5

Light

C

5

3.5

–

0.5

–

–

0.5

0.5

Light

D

Results

Table 3: Data for Absorbance and rate of Hill reaction

Time point

Tube 1

Tube 2

Tube 3

Tube 4

Tube 5

Dark Control

Light Control

DCMU

DNP

Triton X-100

A600

A600

A600

A600

A600

A600

A600

A600

A600

A600

0

0.297

0.000

0.300

0.000

0.302

0.000

0.298

0.000

0.351

0.000

1

0.297

0.000

0.266

-0.034

0.302

0.000

0.278

-0.020

0.314

-0.037

2

0.295

-0.002

0.251

-0.049

0.304

0.002

0.260

-0.038

0.291

-0.060

3

0.294

-0.003

0.245

-0.055

0.303

0.001

0.245

-0.053

0.279

-0.072

5

0.295

-0.002

0.240

-0.060

0.303

0.001

0.231

-0.067

0.275

-0.076

6

0.294

-0.003

0.189

-0.111

0.303

0.001

0.205

-0.093

0.266

-0.085

7

0.294

-0.003

0.166

-0.134

0.302

0.000

0.195

-0.103

0.260

-0.091

8

0.294

-0.003

0.144

-0.156

0.302

0.000

0.182

-0.116

0.257

-0.094

9

0.294

-0.003

0.123

-0.177

0.302

0.000

0.169

-0.129

0.245

-0.106

10

0.293

-0.004

0.118

-0.182

0.302

0.000

0.156

-0.142

0.233

-0.118

11

0.293

-0.004

0.111

-0.189

0.302

0.000

0.143

-0.155

0.220

-0.131

12

0.293

-0.004

0.105

-0.195

0.301

-0.001

0.129

-0.169

0.211

-0.140

Figure 2: Graphical representation of rates of Hill Reaction under varied experimental conditions

Discussion

Data collected during the experiment clearly showed marked variation under the different set of conditions as is evident both from table 3 and figure 2. In the dark conditions, the rate of Hill reaction is negligible indicating light to be an essential prerequisite for Hill reaction to occur. On the contrary in the light conditions, the decrease in absorbance over time is high, indicating the lack of Hill reaction.

In the presence of DCMU, DNP and triton-X, the change in absorbance as indicated by table 3 is high. It can be interpreted from this that the rate of Hill reaction is found to be significantly low in presence of these reagents. This supports the initial hypothesis that the three reagents used are effective inhibitors of Hill reaction. This also correlates with the evidences that these three reagents disrupt the Hill reaction by disturbing the sequential electron flow.

Conclusion

The experiment studied photosynthesis using the rates of Hill reaction under the conditions of light, light control, and in presence of the three chemicals DCMU, DNP and triton-X. The experiment was based on the hypothesis that the three reagents and absence of light inhibit the process of photosynthesis by disrupting the Hill reaction. Therefore, the experiment was designed to enable the estimation of the rates of Hill reaction in presence and absence of the three individual reagents. The experimental set up effectively proves the hypothesis that light is essential for and DCMU, DNP and Triton-X act as inhibitors of photosynthesis.

References

Berg, J. M, J. L Tymoczko and L. Stryer. Biochemistry. New York: W. H. Freeman, 2002.

Walker, D. A. “And whose bright presence-an appreciation of Robert Hill and his recation.” Govindjee, J. T, B. H Gest and J. F. Allen. Discoveries in photosynthesis. Springer, 2005. 109-12.