In: Chemistry
How can I prepare a 150 U/mL alpha-amylase solution
The alpha-amylase from porcine pancreas (25MG, A4268 from Sigma) label reads "0.84 mL, 27.1 mg protein/ml (UV); 1157.643 units/mg proteins"
Ans. Given,
I. Total volume of standard enzyme solution = 0.84 mL
II. Total [protein] = 27.1 mg/ mL
III. Enzyme activity = 1157.643 U/ mg
Now,
Total protein content in solution = [Protein] x Volume of enzyme solution
= (27.1 mg/ mL) x 0.84 mL
= 22.764 mg
Total enzyme activity = mg of protein x enzyme activity per mg protein
= 22.764 mg x (1157.643 U/ mg)
= 26352.585252 U
Therefore, total enzyme activity in enzyme solution = 26352.585252 U
Total enzyme volume = 0.84 mL
Concertation of enzyme activity = 26352.585252 U / 0.84 mL = 31372.1253 U/ mL
# Now, using C1V1 = C2V2
C1= Concertation of enzyme activity, and V1= volume of standard enzyme solution
C2= Concertation of enzyme activity, and V2 = Volume of working enzyme solution.
Let’s the volume of working solution to be made = 1.0 mL
Putting the values in equation 1-
31372.1253 U/ mL x V1 = 150 U/ mL x 1.0 mL
Or, V1 = (150 U/ mL x 1.0 mL) / (31372.1253 U/ mL) = 0.00478 mL
Or, V1 = 0.00478 mL = 4.78 uL
Preparation: Transfer 4.78 uL of standard enzyme solution to a sterile Eppendorf tube. Add sufficient diluent (say, phosphate buffer or as instructed) to make the final volume to 1000.0 uL. It is the desired solution.
Note: You need to make larger volumes of working enzyme solution if taking 4.78 uL enzyme solution in NOT feasible.