U2P6 and 7
Separation of Macromolecules
Your Task
• Each pair will be given a mixture of three
proteins. This mixture will be unique to your
group. Your mixture will contain between 4 and
8 mg of any three of the following proteins:
Myoglobin, Haemoglobin, Cytochrome c,
a-lactalbumin, Ribonuclease, Bovine Serum
Albumin, Ferritin and Catalase. It is your task to
separate and identify these three proteins.
Clues to help you
• Your mixture of three proteins will either contain
2 heavy and 1 light protein or 1 heavy and 2 light
proteins. For the purposes of this experiment, a
heavy protein is defined as one with a molecular
weight of over 50,000 and a light protein is one
with a molecular weight of less than 50,000.
The two smaller/heavier proteins in the mixture
have pI’s that differ by at least 2 pH units. These
can be separated by ion exchange
chromatography at pH 7.5.
Hint…..
• Ion Exchange Chromatography involves a
charged column, in our case a positively
charged matrix, DEAE cellulose.
• If you can separate 2 proteins by ion exchange
chromatography at pH 7.5 then the 2 proteins
must have pIs on either side of 7.5 so they are
opposite charges at pH 7.5. In this way one
protein sticks to the column and the other is
eluted.
Estimating the charge of a protein
[H+]
pI ~5
[OH-]
Protein becomes
increasingly +ve
[H+]
[OH-]
Protein becomes increasingly -ve
Estimating the charge of a protein
Protein 2
Protein 1
pI ~5
[H+]
pI ~9
[OH-]
Protein 1 +ve
[H+]
[OH-]
Protein 1 -ve
Protein 2 +ve
Protein 2 -ve
Estimating the charge of a protein
pH of column
buffer
Protein 2
Protein 1
pI ~5
[H+]
pI ~9
[OH-]
Protein 1 +ve
[H+]
[OH-]
Protein 1 -ve
Protein 2 +ve
Protein 2 -ve
Before coming to class…
• Set up a table similar to the one on
page 2 of the lab notes. Use the
information at the back of these prac
notes to fill in the details. For example:
Bovine Serum Albumin: This is the most abundant serum protein. It
is involved in osmotic regulation and in the transport of metabolites
(usually fairly insoluble ones such as fatty acids). It has a molecular
weight of 69,000 and an isoelectric point of 4.7. At the pH of blood
(7.4) it has a net negative charge of 18. It does not have a visible
spectrum and it has an E1% of 6.9 at 280 nm. A410/A280 = 0
Before coming to class…
Bovine Serum Albumin: This is the most abundant serum protein. It
is involved in osmotic regulation and in the transport of metabolites
(usually fairly insoluble ones such as fatty acids). It has a molecular
weight of 69,000 and an isoelectric point of 4.7. At the pH of blood
(7.4) it has a net negative charge of 18. It does not have a visible
spectrum and it has an E1% of 6.9 at 280 nm. A410/A280 = 0
Protein
Mol. Wt.
Native/
subunit
pI
Charge at Charge
pH 7.5
at pH 8.8
Absorbance
410:A280
BSA
69 000
1 subunit
4.7
negative
Colourless
so no A410
negative
Before coming to class…
• Group the heavy proteins together (those
with mol. wts > 50,000) in your table then
the light proteins.
• From this information you should be able
to work out whether you have 2 heavy and
1 light protein or 2 light and 1 heavy
protein.