8: Purification of protein based pharmaceuticals

Question 0

Describe differential centrifugation

Answer 0

Differential centrifugation involves tissue homogenization at different speeds of the centrifuge.

1) At low speed, the tissue homogenate is centrifuges to form a pellet supernatant consisting of whole cells, nuclei, cytoskeletons and plasma membranes
2) Then, at medium speed, pellets are formed containing mitochondria, lysosomes and peroxisomes
3) Then, at high speed, pellets are formed containing microsomes (fragments of ER) and vesicles.
4) Finally, at very high speed, soluble proteins are formed.

Question 1

Explain the uses and information behind protein purification

Answer 1

Proteins are purified from complex mixtures to study their characteristics for therapeutic uses. They are typically separated from each other based on their size/charge. This requires a source of proteins usually from a tissue/microbe to prepare a crude extract to be centrifuged, causing cells, organelles and macromolecules to be separated. The Svedberg unit of sedimentation coefficient can then be calculated.
This sedimentation is dependent on; Size, shape and density of particle as well as the density of the solution being centrifuged.

Question 2

Explain density gradient centrifugation

Answer 2

(also known as sucrose-density centrifugation)
Involves the centrifugation to produce a sucrose density gradient that can be fractionated to produce samples of different densities.

Question 3

Explain 3 other preliminary fractionation and concentration methods used to remove small particles and ions from a protein sample

Answer 3

Precipitation - ‘Salting out’ is done by increasing the concentration of salt, the globular protein solubility increases, hence reducing the electrostatic attraction between proteins that would otherwise lead to protein precipitation, however, if the salt concentration is greater than 1M, the salt competes for the water molecules so the protein ‘precipitates’ out as well as the other molecules. (Only want the molecules to precipitate out; NOT the protein)
Dialysis - Proteins are placed in a semipermeable membrane in a bathing solution allowing small molecules and ions to pass through while the proteins remain.
Ultrafiltration- Proteins in solution are centrifuged through a porous membrane; depending on the size of the pores, different sized molecules/ions/proteins will pass through/remain.

Question 4

What characteristics of a protein are utilised during protein purification?

Answer 4

• Size and shape
• Overall charge
• Presence of surface hydrophobic groups
• Ability to bind to various ligands.
  (These characteristics are dependent on the sequence of amino acids within the protein)

Question 5

Explain the ionisation state of amino acids in an aqueous solution

Answer 5

At neutral pH, amino acids exists as zwitterions; Therefore they can act as an acid (proton donor) or as a base (proton acceptor). They are amphoteric and are therefore referred to as ampholytes.
The ionisation state of an amino acid varies with pH.
- at pH 0-4 (both groups are protonated) - the concentration decreases as the pH increases.
- at pH 7-14(both groups are deprotonated) - the concentration increases as the pH increases.
- at pH 0-12 the amino acid can be in zwitterion form; It’s concentration increases till it hits a peak between pH 4-9, after which point the concentration decreases until pH 12.

Question 6

Explain the 2 main stages of titration curve data using Glycine as an example

Answer 6

There are two main stages that correspond the the deprotonation of two different groups of glycine.
pK - A measure of tendency of a group to give up a proton. This indicates the region at which glycine possesses the greatest buffering power. It is equal to the pH at which it is half dissociated.
pI - Occurs when the first proton has been removed and the 2nd proton removal has commences. It is the pH at which glycine is fully ionise but possess no net charge (Isoelectric point). Above the pI glycine is negative, below the pI glycine is positive.

Amino acids that have an ‘R’ group that ionises possess a more complex titration curve; consisting of 3 x pK values that are dependent on temperature, ionic strength and the microenvironment. The pI reflects the nature of the ‘R’ group.

Question 7

How do amino acids interact with water?

Answer 7

Highly hydrophobic amino acids are non-polar (isoleucine, valine, leucine and phenylalanine)
Moderately hydrophobic amino acids are mostly non-polar (methionine, alanine, glycine, tryptophan) and a polar (Cysteine)
Mildly hydrophobic amino acids are mostly polar (Tyrosine, threonine and serine) and a non-polar (proline)
Mildly hydrophilic amino acids include polar (asparagine and glutamine), acidic (Histidine) and basic (Glutamate)
Highly hydrophilic amino acids include basic (aspartate) and acidic (lysine and arginine)

Question 8

Explain the components of a protein

Answer 8

Proteins are polymers of amino acids that are covalently linked by peptide bonds. They contain an amino terminal reside and a carboxy terminal residue, both of which ionise. Non-terminal residues are bonded. Some amino acids consist of ‘R’ groups that can ionise and contribute to acid-base properties. Each protein possesses a characteristic titration curve with pK and pI values.