Jonathan Khan November 14, 2010

Ms. Crowley AP Biology Lab Four: Plant Pigments and Photosynthesis

1. The rate of photosynthesis may vary with changes that occur in environmental

temperature, wavelength of light and light intensity. Using a photosynthetic organism of

your choice, choose only ONE of the three variables (temperature, wavelength of light, or

light intensity) and for this variable.

a. Design a scientific experiment to determine the effect of the variable on the rate

of photosynthesis for the organism;

i. Temperature

b. Explain how you would measure the rate of photosynthesis in your experiment;

c. Describe the results you would expect.

d. Explain why you would expect these results

In order to effectively test the effects of temperature on the rate of photosynthesis in live

chloroplasts, a dye reduction technique will be implements over a series of chloroplast

suspensions exposed to different temperatures. Extracted chloroplasts from spinach leaves are

incubated with DPIP in the presence of light. As the DPIP is reduced by the electrons from

Photosystem I, it turns from blue to colorless. The resultant increase in light transmittance is

measured over a period of time using a spectrophotometer.

Procedure:
1. Turn the spectrophotometer, and let the instrument warm up with a wavelength setting of

605 nm.

2. Set up 5 separate controlled environments of 10°C, 20°C, 30°C, 40°C, and 50°C

3. In each environment, set up an incubation area that includes a light, Erlenmeyer flask

filled with water. And a test tube rack. The water acts as a heat sink by absorbing most of

the infrared radiation given off by the lamp, without affecting the visible radiation.

4. Prepare a chloroplast suspension from spinach leaves by blending the leaves in a blender

with some water, and then extracting the solids from the solution as a waste. What will be

left will be a suspension of chloroplasts and water.

5. Pour the suspension into a beaker, and keep the beaker on ice at all times.

6. Place one cuvette in each rack with the following in each, 1mL of phosphate buffer, 3 mL

of distilled water, and 1 mL of DPIP. Also set up one extra cuvette setup to act as a

recalibration method between readings.

7. Add 3 drops of the chloroplast suspension to five of the six cuvette setup.

8. Insert the control cuvette into the spectrophotometer, and by using the light control knob,

set the instrument to 100 % transmittance. Repeat this procedure with the other five

cuvettes measuring and recording the respective percent transmittance of each.

9. Place the each five non control setups into a different environment that was previously set

up, and turn on the light in each.

10. Record the percent transmittance of each of these five at five minute intervals for the next

twenty minutes. Occasionally insert the control cuvette to make sure that the readings are

accurate.
11. In order to determine the rate of photosynthesis, the change in percent transmittance from

the zero time reading to the 20 minute time reading is indicative of the rate oh

photosynthesis, because more electrons are free to reduce molecules of DPIP.

Results & Explanation of Results
1. The results of this experiment are the following

a. 10°C

i. At this temperature, the rate of photosynthesis will be the lowest of those

sample which do produce a result

ii. This is due to the low average kinetic energy [temperature] of the

environment produces little effective collisions between the cytochromes

[enzymes] and their respective substrates.

b. 20°C

i. At this temperature, there will be the median rate of photosynthesis of

those samples which do produce a result.

ii. This is due to the average kinetic energy [temperature] of the environment

being at an acceptable range for somewhat normal metabolic function. It

will not be the highest, because a temperature of 20°C is still a significant

amount away from optimal operating temperature of enzymes which is

37°C.

c. 30°C

i. At this temperature, the rate of photosynthesis will be the highest of those

sample which do produce a result

ii. This is due to the average kinetic energy [temperature] of the environment

being at an acceptable temperature for normal metabolic activity. 30°C is

close to the optimal operating temperature of enzymes which is 37°C.

Thus producing effective collisions between the cytochromes [enzymes]

 and their respective substrates. And electrons being able to leave the

photosystems, and reduce DPIP

d. 40°C

i. At this temperature, there would be no photosynthesis

ii. At this temperature, the enzymes [cytochromes] will have denatured due

to the cytochromes being outside of their acceptable operating

environment of approximately 37°C.

e. 50°C

i. At this temperature, there would be no photosynthesis

ii. At this temperature, the cytochromes [enzymes] will have denatured due

to the cytochromes being outside of their acceptable operating

environment of approximately 37°C.