Please help me fill this chart as well as show calculationsf

Please help me fill this chart as well as show calculations/formula to learn how to do this for the future. Question is from the filled chart, which fraction had most enzyme? ASKED THIS QUESTION 3 TIMES ALREADY PLEASE DO NOT COMMENT IF YOU WILL NOT ANSWER. If this helps: 40mL of 1mg/mL of cyt c, myoglobin, and glucose oxidase in 50mM Tris pH 8 was used, and 50microliters of alkaline phosphatase was used, (50U Total).

*Assume the entension coefficient of all these protein at 280 nm is 1 mg/mL/cm

*Assume alkaline phosphatases product has an extension coefficient of 0.1 umol/mL/cm

Which fraction has the most of enzyme?

First column: Absorbance 420nm: Protein mix [2.332] Fraction 1 [0.236] Fraction 2 [0.127] Fraction 3 [0.070]

Abs Abs Total Specific activity yield Fold 280 protein (mg) For alkaline phosphatase (umol/min/mg) nm nm Protein A mix Fraction 1 Fraction 2 Fraction 3 Assume the entension coefficient of all these protein at 280 nm is 1 mg/mL/cm, Assume alkaline phosphatase\'s product has an extension coefficient of 0.1 umol/mL/cm Which fraction has the most of enzyme?

Solution

To solve this, we need to know about Beer Lambert’s law.

As per Beer Lambert’s law, the concentration of the enzyme is proportional to the absorbance of the light by the solution.

The formula for Beer Lambert’s law is

Absorbance (A) = Extinction coefficient (E) * molar concentration (C) * pathlength (L)   

A = E C L

Here we have to find concentration of the enzyme alkaline phosphatase in each fraction using the absorbance values given in first and second columns.

To find concentration using the above formula

C =   A/E L

Given l (path length) = 1 cm

Now, solving for Protein mix A = 2.332 (observe that Absorbance has no units!)

C = 2.332 / (0.1) (1) = 23.32 µmol/mL

To convert µmol/mL of protein to ‘mg’ of the enzyme we need to know the molecular weight or molar mass of alkaline phosphatase.

Let’s go with Fraction 1 of column 1 for more understanding.

A = 0.236

Now, calculating for concentration (C)

We know that, C = A/E L

C = 0.236/ (0.1) (1) = 2.36 µmol/mL

It is obvious from the given absorbance values that Protein mix has more amount of protein as per Beer Lambert’s law as explained above.

Hope my solution helped you!