An enzyme sample contains 24 mg protein/mL. Of this sample, 20
microliters in a standard incubation volume of 0.1 mL catalyzed the
incorporation of glucose into glcogen at a rate of 1.6 nmol/min. Calculate the
velocity of the reaction in terms of the following:
A)micromoles/minute
B)micromoles/liter/minute
C)micromoles/miligrams of protein/minute
D)Units/milliliter
E)Units/miligrams of protein
Of this sample, 50 mL were fractionated by ammonium sulfate
precipitation. The fraction precipitating between 30 and 50% saturation was
redissolved in a total volume of 10 mL and dialyzed. The solution after
dialysis had 12 mL and contained 30 mg protein/mL. Of the purified fraction,
20 microliters catalyzed the reaction rate of 5.9 nmol/min under the
standard assay conditions. calculate the following:
A. The recovery of enzyme after the ammonium sulfate step.
B. The fold purification after the ammonium sulfate step.
note: for recovery: divide total units of he sample by the total units of the crude homogenate and multiply by 100.
for fold purification: it is the ratio of the specific activity of a given fraction divided by the specific activity of the crude homogenate.
Note: a Unit is ((ΔA/Δmin)/(6220 M -1 cm -1)) X 106 μM/M X 3 X 10-3 L
To calculate the velocity of the reaction, we need to convert the given values to different units.
Given:
- Enzyme sample concentration = 24 mg protein/mL
- Volume of enzyme sample used = 20 μL
- Incubation volume = 0.1 mL
- Reaction rate = 1.6 nmol/min
Let's calculate the velocities in terms of the given units:
A) micromoles/minute:
Since 1 μmol = 1000 nmol, the velocity in micromoles/minute can be calculated as follows:
Velocity = (Reaction rate x Volume of enzyme sample used) / Incubation volume
Velocity = (1.6 nmol/min x 20 μL) / 0.1 mL
Velocity = 320 μmol/min
B) micromoles/liter/minute:
Using the same formula as above, we need to convert the volume of enzyme sample used to liters:
Volume of enzyme sample used (in liters) = 20 μL / 1000
Velocity = (1.6 nmol/min x 20 μL / 1000) / 0.1 mL
Velocity = 32 μmol/L/min
C) micromoles/milligrams of protein/minute:
To calculate the velocity in micromoles/milligrams of protein/minute, we need to consider the concentration of the enzyme sample:
Velocity = (Reaction rate x Volume of enzyme sample used) / (Incubation volume x Concentration of enzyme sample)
Velocity = (1.6 nmol/min x 20 μL) / (0.1 mL x 24 mg protein/mL)
Velocity = 1.333 μmol/mg/min
D) Units/milliliter:
Units are often specific to each enzyme assay and can vary. Without a specific conversion factor, we cannot calculate this value.
E) Units/milligrams of protein:
Units are specific to each enzyme assay, so this calculation cannot be performed without a specific conversion factor.
Now, let's move on to the calculations for the purified fraction after ammonium sulfate precipitation and dialysis:
A. The recovery of enzyme after the ammonium sulfate step:
To calculate the recovery, we need to divide the total units of the sample by the total units of the crude homogenate and multiply by 100.
Recovery = (Units of purified fraction / Units of the crude homogenate) x 100
Note: We do not have the information about the units, so this calculation cannot be performed.
B. The fold purification after the ammonium sulfate step:
To calculate the fold purification, we need to find the ratio of the specific activity of the purified fraction to the specific activity of the crude homogenate.
Fold Purification = Specific activity of purified fraction / Specific activity of crude homogenate
Note: We do not have the information about the specific activity, so this calculation cannot be performed.