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.
Why is ammonium sulfate specifically used?
Ammonium sulfate is highly soluble in water which causes the more hydrophobic proteins to associate —> precipitate.
What is the purpose of precipitation in this experiment?
Concentrate the enzyme rather than to remove the proteins.
After the centrifugation in the ammonium sulfate precipitation step, where is the enzyme?
It is in the pellet. Pellet contains protein.
After the centrifugation in the ammonium sulfate precipitation step, where is the enzyme?
It is in the pellet. Pellet contains protein.
Why do we dialyze after the ammonium sulfate precipitation?
to remove ammonium sulfate.
What is ion exchange chromatography?
separate molecules based on their charged groups by electrostatic interactions with a stationary phase with opposite charge.
What is cation exchange chromatography?
the stationary phase is negatively charged, the cations exchange.
What is anion exchange chromatography?
the stationary phase is positively charged, the anions exchange.
How is the elution done in ion exchange chromatography?
by increasing ions or changing the pH.
At what pH is ion exchange chromatography performed
at 1 pH unit away from pI of protein of interest to assure that it is charged.
pH < pI
proteins (+) charged use CEC
pH > pI
proteins (-) charged use AEC.
What assay was done to measure the enzymatic activity of alkaline phosphatase?
AP catalyzes the reaction PNPP + H2O —> PNP + Pi
What is enzyme activity?
micromoles of product formed per min = 1U
How do we calculate enzyme activity?
using the slope, divide by extinction coefficient. This will give value in M. Convert M to microM. Determine micromole for reaction volume
What is the total activity?
(U/mL in enzyme prep) x (volume of prep in mL).
What is protein concentration?
by Bradford (mg/mL)
What is total protein?
protein conc (mg/mL) x volume of prep in mL
What is specific activity?
total activity/total protein
What is yield (%)?
% yield = total activity/total activity at stage 1
What is purification?
specific activity/specific activity at stage 1
