Enzyme Purification Practical

Question 1

Initial stages in the enzyme purification process? (hint - cells)
- How are these done?
What type of proteins are these steps not necessary for?

Answer 1

Cell harvesting - Centrifugation
Cell lysis - Burst cell membrane

Not necessary for secreted proteins

Question 2

How are protein separated from the complex mixture of the cell free extract (CFE)?
What method of separation is this followed by?

Answer 2

Salt precipitation, Heat denaturation

Followed by chromatography

Question 3

What aspects of proteins are exploited during separation? (4 aspects)
- 2 of these have listed methods

Answer 3

Ion exchange
Hydrophobicity
Size - Gel filtration
Affinity - Affinity chromatography

Question 4

How does affinity chromatography work?
3 steps

Answer 4

Makes use of specific binding interactions between molecules

Sample incubated with immobilised ligand
Non bound sample is washed away
Bound sample is eluted

Question 5

Explain Pseudo-affinity dye chromatography (hint - biomimetic ligands)
How can selectivity be limited?

Answer 5

Dye ligands are biomimetic ligands - Look like a substrate or cofactor
- Can act as inhibitors of enzymes
Selectivity may be more limited because they might bind whole classes of molecule

Question 6

What method is used to monitor purification process before and after each step?

Answer 6

SDS-PAGE

Question 7

What is enzyme activity unit and definition?

Answer 7

Units/ml
1 unit of activity is the amount of enzyme needed to convert 1 μmole of substrate to product in 1 minute

Question 8

What does specific activity provide a measure of?; What happens to it during purification process?
Equation for specific activity
- Units for the 2 parts
Units for specific activity?

Answer 8

Provides a measure of enzyme prep purity; Should increase during purification process

Enzyme activity ÷ Protein Concentration (mg/ml)
Units/mg

Question 9

What does % yield indicate about protein amount?
% yield equation?

Answer 9

Indicates how much of the protein has been recovered or lost during purification

(Total activity after ÷ Total activity before) X 100

Question 10

What does purification factor indicate?
Purification factor equation?

Answer 10

Purification factor indicates how well the purification method discriminates between desired and contaminating proteins

Specific activity after ÷ Specific activity before