Learning outcome 11.

2:
(a) Describe simply the process of electrophoresis and the effect of pH, using
peptides and amino acids as examples
The background to electrophoresis
A simple inorganic example
Suppose you put a single crystal of potassium manganate (VII) (potassium
permanganate) onto some damp filter paper supported on a microscope slide. The
crystal would dissolve in the water in the filter paper, and the deep purple colour
would diffuse out to make a small circle around the original crystal.



Now let's modify this by connecting the filter paper into a simple electric circuit.
This time, when you put the potassium manganate (VII) crystal onto the paper, the
colour doesn't spread into a circle. Instead, the purple colour starts to move towards
the positively charged crocodile clip.



There isn't anything very surprising about this. The colour of the potassium
manganate (VII) is due to the manganate (VII) ions present. These are negatively
charged, MnO
4
-
, and move towards the positive electrode.
This movement of ions in an electric field is the basis of a separation technique
known as electrophoresis. Let's apply this to the more interesting (and more
complicated) case of amino acids.

Electrophoresis involving amino acids
Results of the electrophoresis of a mixture of simple amino acids
In practice, paper isn't used for serious electrophoresis. Instead, a gel is used.
Control of the pH is important, and so the gel is soaked in a buffer solution.
Little troughs are made in the gel to hold the solutions being tested. In an exam, you
may find that you have to interpret diagrams based on bits of paper or slabs of gel.
It makes no difference whatsoever to what you would need to say.
Unlike potassium manganate (VII), amino acids are colourless. To find out where they
have got to, they are sprayed with ninhydrin which shows up the amino acids as
brown or purplish stains.
The diagram shows the effect of electrophoresis on a mixture of four amino acids
at a known pH. This is just on a strip of paper, and the mixture was originally a single
drop on the start line.

We need to explain why the four amino acids have moved in this way.
Spot C
The amino acid responsible for spot C hasn't moved. Why not?
Remember that an amino acid has both a basic amine group and an acidic carboxylic
acid group.

There is an internal transfer of a hydrogen ion from the -COOH group to the -
NH
2
group to leave an ion with both a negative charge and a positive charge.
This is called a zwitterion.

At a particular pH, known as the isoelectric point, this is how the amino acid exists in
solution. The amino acid won't move during electrophoresis, because the two charges
cancel each other out, and there won't be any attraction either to the positive or
the negative electrode.
At any other pH, there will be some movement one way or the other - and we will
explore that later.
So amino acid C doesn't move because the pH of the solution happens to be exactly
the same as the isoelectric point.
Spot D
This amino acid has moved towards the negative electrode, and so must be positively
charged.
This positive charge results from having an extra -NH
2
group in the amino acid, like
this:

In a buffer solution with a pH around neutral, this will be present as the ion:

The extra -NH
2
group in the amino acid picks up a hydrogen ion from the water.
Amino acids like this carry a net 1+ charge in a solution which is around neutral.
The amino acid lysine is an example of this. You don't need to remember this formula
(or the formulae of the other named amino acids mentioned below).

During electrophoresis at a pH about neutral, amino acids like this will travel towards
the negative electrode.
Spots A and B
These travel towards the positive electrode and so must be negatively charged at
this pH. This happens in amino acids which carry an extra -COOH group, for
example:

In a buffer solution with a pH around neutral, this will be present as the ion:

The extra -COOH group loses a hydrogen ion to the water, to leave an ion which
has an overall charge of 1-, and which therefore moves towards the positive
electrode.
Two examples of this are aspartic acid and glutamic acid.

The question remains as to why the two amino acids in our electrophoresis example
have travelled different distances. One of the factors which affects this is the size
of the ions.
The ions have to find their way through the fibres in the paper or the pores in the
gel. Smaller ions will travel faster than bigger ones. So, for example, the smaller
aspartic acid will travel faster than the larger glutamic acid.
What do I mean by "smaller" or "larger"? This could be in terms of the masses of the
ions, or their shapes. Ions with bulky groups (such as benzene or other rings) will
travel more slowly than ones with, say, unbranched chains.
Since there are only two naturally occurring amino acids with an extra -COOH
group, spot A must be due to aspartic acid and spot B to glutamic acid.
Changing the pH of electrophoresis experiments with amino acids
What happens if the buffer solution you use has a low pH?
In the presence of a sufficiently acidic solution, all the -COOH groups present will
exist as -COOH groups, and not as ions.
All the -NH
2
groups will have picked up hydrogen ions to form -NH
3
+
.
That means that all of the amino acids will carry a positive charge, and all of them
will move towards the negative electrode.
The three different sorts of amino acid that we have been looking at will have ions
like this at a low enough pH:

All of them will move to the negative electrode, but they will move at different
rates because, for example, one of them carries 2+ charges whereas the others
carry only 1+. And obviously they will be different sizes as well - both in mass and
shape.
What happens if the buffer solution you use has a high pH?
In the presence of a sufficiently alkaline solution, all the -COOH groups present will
exist as -COO
-
ions.
All the -NH
2
groups will exist as simple -NH
2
groups - not ions.
That means that all of the amino acids will carry a negative charge, and all of them
will move towards the positive electrode.
The three different sorts of amino acid that we have been looking at will have ions
like this at a high enough pH:

All of them will move to the positive electrode, but they will move at different rates
because, for example, one of them carries 2- charges whereas the others carry only
1-. And obviously they will be different sizes as well - both in mass and shape.
Other factors affecting the movement of the amino acids
Voltage matters. The higher the voltage, the greater the speed at which the amino
acids move through the paper or gel.
Temperature also matters. An increase in temperature can speed up electrophoresis.
Electrophoresis involving proteins
Electrophoresis can be used to estimate the relative molecular mass of a protein, or
to sort a mixture of proteins into relative molecular mass order.
One technique for doing this is known as SDS-PAGE. SDS-PAGE stands for Sodium
Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis.
The sodium dodecyl sulfate is a constituent of many detergents and cleaning
products. It is also known as sodium lauryl sulfate. If a protein is treated with SDS
and then heated, the protein is denatured.
That means that its secondary and tertiary structures are lost. It becomes covered in
SDS molecules, and this turns the protein molecule into a long tube covered with
negative charges on the outside. The negative charges come from the SDS
molecules.
A protein which has more than one sub-unit is split into these separate bits, each of
which is coated in SDS molecules.
If these treated protein molecules undergo electrophoresis, all the molecules will
move towards the positive electrode, but the smaller molecules will move through
the gel faster than the bigger ones.
Obviously, protein molecules have to be stained in some way in order to find out
how far they have travelled. The intensity of the colour is a measure of how much
of any particular protein is in the mixture.
The apparatus for doing this can be calibrated by using proteins of known relative
molecular mass, and finding out how far they travel.
This technique is used to detect the presence or absence of proteins which might be
an indicator of disease. For example, kidney disease can be recognised by the unusual
presence of proteins in urine. Lots of diseases including liver disease and some
cancers produce abnormal levels of some proteins in the blood.