Lesson 6C Protein Analysis Flashcards
Protein Analysis
The amino acid composition of a protein can be determined
through chromatography and electrophoresis.
In order to use either method the peptide bonds must be
hydrolyzed to produce individual amino acids which can be
identified.
Acid Hydrolysis
The peptide bond is a strong resonance stabilised bond.
Acid hydrolysis breaks these bonds.
A 6 mol dm-3 HCl solution is used at 110◦C for 1-3 days is required.
The resulting amino acids can be identified and amounts determined using
high performance liquid chromatography.
We can identify the individual amino acids using paper chromatography or
electrophoresis.
Paper Chromatography
Chromatography is used to separate mixtures of substances into their
components.
Chromatography works on the principle that different amino acids have
different relative solubilities in two solvents.
One solvent is the water in the paper (stationary phase) and the other is the
eluting solvent (mobile phase), usually a mixture of 1-butanol and ethanoic
acid.
Paper Chromatography
Procedure
A tiny sample of an amino acid
mixture is spotted on the paper at
the origin (a line drawn on the
paper in pencil).
The paper is suspended in the
eluting solvent with the origin
above the solvent.
The solvent rises up the paper by
capillary action.
As the solvent passes the spot, the amino acids in
the mixture will distribute themselves between the
two phases.
The extent to which they are soluble in each phase
will determine how far up the paper they will travel.
An amino acid that is more soluble in the eluting
solvent will travel farther than one that is more
soluble in the paper.
When the solvent reaches the near the top of the
paper, it is marked as a line known as the solvent
front and removed.
The paper must than be sprayed with ninhydrin in
order to make the amino acids visible as purple
spots.
Electrophoresis
A method of separating similarly sized
molecules based on their electric
charge.
The presence of acidic and basic R
groups produces positively and
negatively charged ions in amino
acids.
How an amino acid behaves in an
electric field depends on the number
of charged groups and the pH of the
solution used.
The Isoelectric Point
The isoelectric point (pHi) is the pH at which the positive and negative
charges are exactly balanced, the molecule shows no net charge and it show
no net migration in the electric field.
Every amino acid has a different isoelectric point.
Electrophoresis Procedure
Electrophoresis can be carried out using paper, cellulose acetate or other solid
supports that have been saturated with a buffer solution of known pH.
The amino acid mixture is applied to the center of the paper, the electrodes and
ends of the paper are placed in the buffered solution and an electric potential is
applied to the electrodes.
Any amino acid at its isoelectric point will not move.
Amino acids with positive charges will move towards the cathode while those with
negative charges will move towards the anode.
The paper is removed and dried then sprayed with ninhydrin to make the amino
acids visible.
Interpreting Electrophoresis Results
When pH > pHi the amino acid bears a negative total charge: it migrates
towards the positive electrode (anode).
When pH < pHi the amino acid bears a positive total charge: it migrates
towards the negative electrode (cathode).
When pH = pHi the amino acid is in its zwitterionic, neutral form: it does
not migrate and remains at the starting point.
