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.
To calculate the velocity of the reaction in different terms, we need to convert the given information and use appropriate formulas.
Given:
Enzyme concentration (protein) in sample = 24 mg/mL
Incubation volume = 0.1 mL
Amount of enzyme used = 20 μL (microliters)
Rate of reaction = 1.6 nmol/min
To calculate the velocity of the reaction in different terms:
A) To calculate micromoles/minute:
We need to convert nmol to μmol.
1 nmol = 0.001 μmol
Velocity = (Reaction rate in nmol/min) * 0.001
Velocity = 1.6 * 0.001 = 0.0016 μmol/min
B) To calculate micromoles/liter/minute:
First, we need to convert the volume from mL to L.
1 mL = 0.001 L
Velocity = (Reaction rate in μmol/min) / (Incubation volume in L)
Velocity = 0.0016 / 0.1 = 0.016 μmol/L/min
C) To calculate micromoles/milligrams of protein/minute:
We need to divide the velocity by the concentration of protein in the sample.
Velocity = (Reaction rate in μmol/min) / (Protein concentration in mg/mL)
Velocity = 0.0016 / 24 = 0.000067 μmol/mg/min
D) To calculate units/milliliter:
Units refer to the specific activity of the enzyme.
We need additional information to calculate this. Units represent the number of substrate molecules transformed per minute per milliliter of the enzyme solution. The specific activity depends on the particular enzyme and the substrate being used. Once we have this information, we can calculate the units.
E) To calculate units/milligrams of protein:
Similar to D), we need additional information about the specific activity of the enzyme.
Now, moving on to the second part of the question:
Given:
Dialyzed solution volume = 12 mL
Protein concentration in the dialyzed solution = 30 mg/mL
Amount of purified enzyme used = 20 μL (microliters)
Rate of reaction under standard assay = 5.9 nmol/min
A) To calculate the recovery of enzyme after the ammonium sulfate step:
We need the total units of the original sample (before ammonium sulfate step) and the total units of the crude homogenate.
Recovery = (Total units of purified fraction) / (Total units of crude homogenate) * 100
B) To calculate the fold purification after the ammonium sulfate step:
We need the specific activity of the purified fraction and the specific activity of the crude homogenate.
Fold Purification = (Specific activity of purified fraction) / (Specific activity of crude homogenate)
Note: The specific activity can be calculated by dividing the total activity (units) by the total protein concentration (mg) in each case.
Since we do not have the information about the specific activities, we cannot calculate the units/milliliter and units/milligrams of protein, or the recovery and fold purification in this case.
Please provide the specific activity information to calculate these values.