Isolation and characterization of erythrocyte membranes
Objective:
·
To be familiar with protein and lipid isolation
protocols.
·
To study about the composition of human red blood
cell membrane.
Introduction:
In this experiment the membrane
constituent of the red blood cells (erythrocytes) membrane was isolated. Protein
and lipid isolation form biological systems are important process in
biochemical researches. Generally proteins are made with amino acid monomers
which have some polarity so these proteins can dissolve in water or ethanol
like solvents. Although lipids are somewhat different than proteins which are
consist of more hydrophobic properties than proteins therefore lipids are more
favorable to dissolve in nonpolar solvents. So using those qualities the whole
extraction procedure is conducted.
Erythrocytes
are commonly use cell type in this practical because it can be obtained easily
and also it has not organelles so it would not contain RNA and DNA other than
that this cells conduct relatively little metabolism and the offer a
homogeneous source of easily obtainable membranes.
Erythrocytes
consist of three basic components. Those are lipid bilayer, trans-membrane
proteins and a cytoskeletal network. The lipid bilayer is a semipermeable layer
which is asymmetric in composition and separates the cytoplasm from the extracellular
medium. Phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylethanolamine
and the sterol cholesterol are the dominant component of it.
Transmembrane
proteins are immerged in the bilayer, and those proteins have great mobility in
the plane of the membrane. The major transmembrane proteins are glygoproteins,
band 3 and glygophorin. Band 3 is a multispanning ion transport channel. Other
than that Ankyrin, Glycophorins , Aquaporins (water channel proteins) and Rh
(is a protein complex) are present in the membrane as proteins. The
cytoskeleton is an irregular hexagonal lattice of fibrous polypeptide called
spectrin, which are tied together by actin. The skeleton makes a two
dimensional network which is very flexible and compressible.
Centrifugation
technique was highly used throughout this experiment. Centrifugation is a
separation of a heterogeneous mixture to different layers by spinning it. A
centrifuge is the equipment that use in this technique. This equipment is generally
driven by an electric motor, which puts an object in rotation around a fixed
axis, applying a force perpendicular to the axis to separate substances of
different densities. Using this phenomena blood samples can be separated to
layers as the cells and the plasma.
Throughout
the extraction procedure well buffered system should be maintained. This buffer
maintained the isotonic solution for erythrocytes and it prevents the lysis of
the cells. This is done because in this experiment the erythrocyte membrane
should be isolated not the whole proteins in blood. so the to do that the
plasma should be removed from the samples to isolate the erythrocytes, plasma
also containing some proteins like Albumins, Globulins, Fibrinogen so those
proteins must not be in the erythrocyte membrane extract. But after the
isolation of erythrocytes has been done the cells would be lysis using a
hypotonic solution.
While this experiment is conducting samples
should be maintained at 4 ºC temperature because to reduce the enzymatic
activities. All the cells have compartments which contains hydrolytic enzymes
that are capable of breaking down all kinds of biomolecules, including
proteins, nucleic acids, carbohydrates and lipids. But in this experiment the
whole membrane should be isolated without getting denatured or disrupted of the
protein/lipid constituents of it.
Methodology
Materials: Micro
centrifuge tubes, centrifuge, vortex mixture micro pipettes, test tubes,
droppers
Reagents: blood sample,
310 mM and 20 mM sodium phosphate buffer, chloroform, chloroform: methanol
mixture (1:2), methanol, 0.1 M HCl, distilled water.
a. Isolation of erythrocyte membranes from human blood.
ü Maintain 4 ºC temperatures throughout this protocol.
To 10 ml of blood sample,
↓
Add 1.78 ml of 310 mM sodium phosphate buffer and
EDTA,
↓
Mix and centrifuge at 1000g for 10 min,
↓
Remove the buffer coat using micro pipette,
↓
Resuspend
the pallet in 6.67 ml of buffer,
↓
Mix and centrifuge (at 4 ºC) at 1000g for 10 min
(above 3 steps repeat 4 times),
↓
Resuspend the above pallet with buffer to 1.67 ml of
final volume,
↓
Add
0.278 ml of above suspension to 1.39 ml of 10mM (hypotonic) phosphate buffer,
↓
Invert several times to mix,
↓
Centrifuge (at 4 ºC) at 14000g for 40 min,
↓
Remove the supernatant,
↓
Resuspend the pallet in 1.39 ml of 10mM (hypotonic)
phosphate buffer,
↓
Centrifuge (at 4 ºC) at 14000g for 20 min,
↓
Repeat the above 3 steps for 2 more times,
↓
Remove the supernatant and get the loosely packed
membrane pallet.
↓
The residue is saved for further experiments.
b. Lipid extraction
Get 0.044 ml of above final extraction,
↓
Add 0.167 ml of chloroform: methanol (1:2) mixture,
↓
Mix vigorously with vortex mixture for 30 sec,
↓
Add 0.055m ml of chloroform,
↓
Mix vigorously with vortex mixture for 30 sec,
↓
Add 0.055 ml of distilled water,
↓
Mix vigorously with vortex mixture for 30 sec,
↓
Centrifuge at 500g for 5 min,
↓
(Denatured proteins stuck in the chloroform; water
interface. If the protein layer extends to the bottom layer add 0.0055 ml of
0.1 M HCl and mix and centrifuge again)
↓
Remove as much as upper methanol/water layer,
↓
Using a syringe carefully puncture thought the
protein layer,
↓
Then get the bottom chloroform layer to clean
weighted tube,
↓
Evaporate the Separated chloroform layer (use hot
water bath).
↓
The residue is saved for further experiments.
Discussion
Blood samples were obtained in different
donors who are in different genders. All the samples were mixed with
anticoagulant because blood can be clotted outside of the body and if the blood
was clotted the cell isolation process should be very difficult. Usually
Heparin sulfate will use as an anticoagulant but in this lab session EDTA was
used. The EDTA can make complexes with metal ions as a chelator. Ca2+
ions are needed to coagulation process so when EDTA was used the Ca2+
ions can be removed from the solution and prevented the coagulation.
After the first centrifugation step the
expected color of the supernatant was pale yellow color because after
centrifugation the cells and plasma would be separated. But the observed color
was reddish yellow color this happens because of some red blood cells has been
lysed when the buffer was added, the Tonicity of the blood
cells was not known exactly. The isotonic buffer was prepared as it was given
according the previous experiment results in lab manuals. The tonicity of blood
can be differ with the genetic trait as well as the donors feeding habits
(usual water intake).
Throughout
the membrane extraction procedure all the mixing or dissolutions were conduct
in gently manner and the vortex mixer was not used in this procedure. Because
the vigorous swirls can damage and destruct the protein molecules. So this can
cause false observation in the SDS-PAGE separation experiment.
In the lipid extraction procedure
chloroform methanol water system was used, because most of lipids have non
polar properties as it was mentioned earlier. But membrane-associated lipids
are more polar and require polar solvents such as methanol to disrupt hydrogen
bondings or electrostatic forces. So using a methanol chloroform mixture is
more efficient to extract lipids, and chloroform can denature proteins as well.
