Jose Hernandez and Laura Constantine

Photosynthesis, part 2

When light hits the leaves of plants, the light energy gets converted in the
photosystems II to boost the electrons to a higher energy state. At this time the
energy is used to produce ATP and reduce NADP to NADPH then in the carbon
fixation cycle ATP and NADPH are used to incorporate CO2 into organic molecules.
Will blocking the light of the unboiled chloroplasts reduce the amount of
photosynthesis compared to boiled chloroplasts that receive light and how does this
compare to the cold, undamaged normal light absorbing chloroplasts?

Procedure

Keep all equipment cold for the chloroplasts and have a bucket of ice to keep them
chilled at all times.

Extraction of Chloroplasts from spinach leaves

1. Gather around 35mg of spinach leaves and cut into 1cm^2 skipping the
central vein.
2. Add 70ml ice-cold grinding buffer 20mM Tris-HCL pH7.5 and 0.4 N sucrose.
Place in a precooled blender jar add leaves slowly while grinding them at slow
speed.
3. When all the leaves are added grind for approximately 15 seconds. It should
be a homogenous green suspension.
4. Filter the suspension through 4 layer cheese cloth into a 250-ml flask on ice.
5. Divide the filtrate into two pre-chilled 50-ml plastic centrifuge tubes, balance
the tubes and spin at 3500rpm for 2 minutes. It should leave a dark green
pellet.
6. Carefully discard the supernatant and place the tubes with remaining pellets
on ice.
7. Add 4-ml of ice cold grinding buffer to each tube and resuspend the pellet
gently. Do not use vigorous agitation as this can damage the chloroplasts.
When they are evenly resuspended combine in one tube and cover with foil
to exclude light and keep it on ice in the dark. This is the stock solution of
chloroplasts.
8. Pipet 1µL of chloroplasts and 9µL of grinding buffer on to a microscope slide,
cover with a cover slip and view.

Part II

Use a Bradford Assay like before to determine the concentration of protein in your
chloroplast preparation. Run a new standard curve. Find a cell lysate that falls
within the standard curve, once found measure it in triplicate.

Part III
We will measure the rate at which Photosystems II is working in our chloroplasts by
boiling 1 batch, one batch will never be exposed to light (well we will do our best to
prevent light from hitting the cuvette and another will be a control with normal
chloroplasts, with light hitting them.

1. Gather tubes that contain the boiled chloroplasts, dark covered chloroplasts,
and normal control chloroplasts, set spectrophotometer at absorbance of
600nm.
2. Make three cuvettes that have 500µL reaction buffer (20mM Tris-HCL pH7.5,
10mM NH4Cl and 0.13M sucrose). To all three cuvettes add enough dH20 so
that final volume is 895µL. Keep the cuvettes on ice to keep them as cold as
possible until we begin.
3. Add 5µL of normal chloroplast to one cuvette and cover with parafilm to mix
it gently by inverting it once or twice. Place in the spectrophotometer and
zero the instrument. This will be our control.
4. Add 400µM DCIP to the cuvette and quickly cover it with a little piece of
parafilm and mix it gently inverting it once or twice, measure absorbance and
then place the sample under the lamp for bright illumination. Take out of
spectrophotometer after every reading, read absorbance every 30 seconds
for a total of three minutes plus the initial “time zero” reading.
5. Add 5µL of boiled chloroplast to one cuvette and cover with parafilm to mix it
gently by inverting it once or twice. Place in the spectrophotometer and zero
the instrument.
6. Add 400µM DCIP to the cuvette and quickly cover it with a little piece of
parafilm and mix it gently inverting it once or twice, measure absorbance and
then place the sample under the lamp for bright illumination. Take out of
spectrophotometer after every reading, read absorbance every 30 seconds
for a total of three minutes plus the initial “time zero” reading.
7. Add 5µL of dark chloroplast to one cuvette and cover with parafilm to mix it
gently by inverting it once or twice. Place in the spectrophotometer and zero
the instrument.
8. Add 400µM DCIP to the cuvette and quickly cover it with a little piece of
parafilm and mix it gently inverting it once or twice, measure absorbance and
keep the sample in the spectrophotometer after every reading, read
absorbance every 30 seconds for a total of three minutes plus the initial
“time zero” reading.
9. Sketch a rough graph absorbance vs. time in the notebook.