CRAM SESHHHHHHHHHHHHH Flashcards
How to do calculations (what are the units)
- Protein concentration: by Bradford (mg/mL). Use Calibration Curve from
Experiment 3.1 - Specific Activity = enzyme activity per mg of protein = U/mg
Determines purity of an enzyme
definition: The higher the specific activity, the purer the enzyme
-Total protein = (U/mL in enzyme prep) x ( volume of prep in mL) = total units
Determines the loss of the enzyme during each step of the purification process
- yield %, total activity/total activity at stage 1 * 100%
- purification= specific activity/specific activity at stage 1 *100%
steps to purify alkaline phosphotase
1) Lysozyme treatment is the selectively rupture of the outer cell membrane and the cell wall. The rupture releases DNA, AP and other proteins. (DNASE) denatures the DNA.
Enzyme activity definition
μmoles of product formed per min = 1U
Conditions: Constant Tº, Vi = 0, high [S], V = Vmax
Km
Michaelis constant
at Vmax/2, [s] = Km
Michaelis mentin equation
Vo = (Vmax[s])/(Km + [s])
Line-weaver burk plot
X- Intercept = -1/km
Slope= Km/Vmax
Y-Intercept = 1/Vmax
x axis = 1/[s]
y axis = 1/v
competitive inhibition
can be outcompeted with increasing substrate concentration
no change to active site
therefore no effect on Vmax, but increase of Km
noncompetitive inhibition
cannot be outcompeted with increasing substrate concentration
Change to active site
thus Vmax changes, but no effect on Km
reagents for the experiment
[Lysosyme treatment and dialysis]
1) DNAse
2) EDTA
3) MgSO4
Cellular components: Spheroplasts, Outter membranes, protein, DNA
centrifuge (collect supernatant bc AP soluble)
dialysis (EDTA and MgSO4 leave the dialysis tube & AP stays)
[Heat denaturation]
Denaturation at 80 degrees celsius. AP is heat stable and denatured proteins are in the pellet
[Salt Precipitation] (
Just the right amount of ions (ammonia sulfate) are addded to associate with the side chains of alkaline phosphotase. This neutralizes the charge and makes the proteins precipitate.
centrifuge (AP in pellet bc it precipitated)
Dialyzed again
centrifuge (AP in supernatant)
[Anion exchange chromatography]
DEAE-celllulose used to elute the enzyme.
[SDS vs native]
SDS - want to see a band at 43kda since AP is a dimer
Native- want to see a 86 kda band
Bradford assay (of all the samples to see conc of protein)
Time sensitive assay (to see the specific activity of AP, the higher the specific activity the purer the enzyme)
Quantification of alkaline phosphotase activity
Measure PNPP —-> PNP + Pi
Plot absorption over time
