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In: Chemistry

How can I prepare a 150 U/mL alpha-amylase solution The alpha-amylase from porcine pancreas (25MG, A4268...

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"

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Expert Solution

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.


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