Objective
To isolate chloroplast and estimate the chlorophyll concentration from spinach leaves.
Theory
Chloroplasts are organelles 5-10 um in size in plants which perform the function of
photosynthesis. These structures are mainly located in the palisade parenchyma of the mesophyll
cells in the leaf. Chloroplasts belong to a family of specialized organelle called the plastid. The stem,
leaves and unripened fruit in all the plants contain chloroplast. But leaves are major part of plants
performing the process of photosynthesis. The green color of these structures in plants is due to the
presence of a pigment called chlorophyll which resides in the chloroplasts.
Chloroplasts like mitochondria are evolved by endosymbiosis. Both chloroplasts and mitochondria
are involved in generating metabolic energy. Both organelles have their own genetic systems. During
photosynthesis, the conversion of light energy derived from sunlight is converted into chemical
energy in the form of carbohydrates and during this process ATPs are generated. In spite of having
their own genetic system, most of the chloroplast proteins are encoded by the cell’s nuclear DNA.
These proteins are synthesized in the ribosomes of the cytosol and are then imported to the
organelle. Approximately 30 proteins are encoded by the chloroplast genome and some of these
proteins are involved in photosynthesis.
Structure
of
Chloroplast
Chloroplasts are found
in the mesophyll cells
of the leaves. The
chloroplast is divided
into
compartments
three
bounded
by
three
membrane systems:
an
intermembrane
space between the
inner
and
membranes,
stroma
thylakoid
and
outer
the
the
lumen.
Chloroplasts
have a double membrane structure called the chloroplast envelop. The chloroplast envelop has an
inner membrane and an outer membrane. A third membrane system called
membrane surrounds the thylakoids in the stroma.
the thylakoid
Chloroplasts are located inside the thylakoid membranes. Thylakoid membrane consists of the
thylakoids which are flattened discs arranged on top of the other and they are termed as grana. The
thylakoids are located inside the stroma. Photosynthesis takes place in the thylakoid membranes.The
chlorophyll molecules absorb light in the form of photons and this leads to the emission of electrons
by the chlorophyll molecules. This drives the hydrogen ions across the membrane surrounding the
thylakoid stack. This leads to the formation of an electrochemical gradient which drives the
production of ATP.
Isolation of Chloroplast
In chloroplast isolation method, the cell wall is broken mechanically using a blender or homogenizer.
Then the unbroken leaf tissue and the cellular debris are removed by filtration. The chloroplasts are
collected by centrifugation using a percoll gradient. Leaves of spinach, lettuce are commonly used
for the isolation of chloroplasts. Both intact and broken chloroplasts are formed after centrifugation.
The broken chloroplasts are removed and the intact chloroplasts are taken for further studies. Intact
chloroplasts are the best source for studying the processes like carbon assimilation, electron flow
and phosphorylation. The concentration of the chlorophyll molecules can be estimated from the
isolated chloroplasts. DNA and RNA of chloroplasts can also be extracted.
Materials Required
1.Spinach leaves 30 grams.
2.Knife and scissors.
3.Cutting board.
4.Kitchen blender.
5.Muslin cloth.
6.Glass beaker.
7.50 ml centrifuge tubes.
8.1.5 ml centrifuge tubes.
9.Micropipette.
10.Glass pipette.
11.Cooling centrifuges.
12.Spectrophotometer.
Reagents and buffers
1. 1x Chloroplast isolation buffer without BSA:- 0.33M sorbitol, 0.1M tris-Cl ph 7.8, 5mM
MgCl2, 10mM NaCl, 2mM EDTA.
2. 1x
Chloroplast
isolation
buffer
with
BSA
(0.1%w/v):
3. 40% percoll: 4ml percoll and 6 ml 1x CIB buffer with BSA to make 10 ml of 40% percoll.
(Use
10
ml
4. 80% acetone.
of
40%
percoll
for
6ml
of
chloroplast
suspension)
Procedure
1. Wash 30 gms of spinach leaves thoroughly first with tap water and then with distill water.
2. Remove the midrib veins of the leaves and cut into small pieces.
3. Add 120 ml of 1xCIB buffer with BSA to the cut leaves in a blender. Blend with 2-3 strokes.
4. Filter
the
blended
leaves
through
6
layers
of
muslin
cloth.
The filtrate is then evenly divided into four 50 ml centrifuge tubes.
5. Centrifuge the tubes for 3 minutes at 200xg. A white pellet will be obtained.
Transfer the supernatant into chilled 50 ml centrifuge tubes and centrifuge at 1000xg for 7
minutes. A green pellet will be obtained.
6. Discard the supernatant and break the green pellet gently by finger tapping.
Resuspend the pellet in 2ml of 1x CIB buffer with BSA and mix gently by pipetting up and
down.
7. Pool
the
suspended
pellet
into
one
centrifuge
tube.
Preparation of 40% percoll layer: Mix 4 ml percoll with 6 ml of 1x CIB buffer with BSA.
8. Gently overlay 6ml of the chloroplast suspension over this 40% percoll layer.
9. Centrifuge at 1700 xg for 6 minutes. The intact chloroplast will sediment to the bottom of
the tube as a green pellet and the broken chloroplast will form the upper layer.
Carefully remove the upper layer of the chloroplast suspension leaving only the pellet
containing the intact chloroplast.
10. Mix
the
pellet
with
500
ul
of
1x
CIB
buffer
without
BSA.
Estimation of chlorophyll concentration
16.Add 10 ul of chloroplast suspension to 990ul of 80% acetone solution and mix gently.
17.Centrifuge
at
3000xg
for
2
minutes.
18.Take 100ul of the supernatant and transfer into a cuvette and measure the absorbance at 650
nm.
Use
100
ul
of
80%
acetone
as
blank.
19.Take
duplicate
OD
650
values.
20.Take the average of the two values and estimate the mg/ml chlorophyll concentration using the
following
formula:
A
650
x
100/36
=
mg/ml
chlorophyll.
Where A 650 is the absorbance at 650 nm, 100 is the dilution factor and 36 is the
extinction coefficient of chlorophyll.
Difference Encountered in a Real Laboratory
In an actual laboratory setting, there are certain important steps that are not necessarily
applicable
1.
2.
in a virtual lab:
Before starting the experiment sterile the laminar air flow chamber using spirit.
Always disinfect your work area when you are finished.