Experiment 9: Purification of Alkaline Phosphatase Flashcards
What is the function of alkaline phosphatase enzyme?
remove phosphate groups from phosphorylated compounds facilitating transport across membranes and providing the cell with a source of inorganic phosphate.
Where is AP located in E.coli cells?
Located in the periplasmic space.
What is the periplasmic region?
region between the inner (cytoplasmic) and outer membrane.
What was the first step in the purification process?
Lysozyme Treatment.
What does lysozyme treatment involve?
digest cell wall and osmotic shock releases proteins from periplasm. Inner membrane remains sealed forming vesicles called spheroplasts.
In the lysozyme treatment, after centrifugation where is the enzyme located?
in the supernatant.
What is dialysis?
used to separate molecules on the basis of size, to add or remove salt or to change the buffer.
Why do we use dialysis after the lysozyme treatment?
to remove products of lysozyme, DNase, EDTA, etc.
What was the second step in the purification process?
Heat Denaturation
What is heat denaturation?
Involves the disruption and possible destruction of both secondary and tertiary structure but primary structure remains.
What does the heat disrupt?
hydrogen bonds and non-polar hydrophobic interactions.
After centrifugation in the heat denaturation step, where is the enzyme located?
In the supernatant.
What is the significance of the heat denaturation step?
Most cellular proteins are irreversibly denatured by heating at 80 deg C. AP from E.coli is heat stable and will remain soluble and active after such treatment.
What is the third step in the purification process?
Ammonium sulfate precipitation
Why is ammonium sulfate used?
Proteins are less soluble at high salt concentrations, “salting out”. The salt concentration at which a protein precipitates differs from one protein to another.
