Question

You will be determining the concentration of cytochrome c based on the reduced Fe content. The calculation in the lab will determine mM of cytochrome c from your absorbance values. How will you convert this to mg/ml for the protein purification table? Show two sample calculations.

Answer 1

To determine the amount and purity of the cytochrome c you have isolated, two independent methods are required. First, the visible absorbance difference spectra, which is dependent on the difference in absorbance between reduced and oxidized heme iron, is used to determine the cytochrome c concentration of the solution, in units of millimoles/Liter (mM). From this, using the volume of the solution and the molecular weight of cytochrome c, (12,327 g/mol) we can calculate the amount of cytochrome c (in grams). Then, by using the Bradford protein assay we can determine the total concentration of protein in the solution, in units of milligrams/milliliter (mg/mL) and using the volume of the solution, the total amount of protein (in grams). By comparing the amount of cytochrome c (in grams) and the total amount of protein (in grams) we can determine the purity of our preparation.
These two methods should be used to also determine the concentration of cytochrome c and of total protein for each step of the purification . Using the solution volumes, you can then determine how much total protein and cytochrome c is present at each step (obviously, it is very important to keep track of the units during these calculations!). This allows to follow precisely the purification process, that will be summarized by recording all the results in the table below.

Total Protein Determination
For a detailed explanation of the process see Bradford protein assay.
Prepare a standard curve using either BSA or cytochrome c (this will be discussed in class) and assay your samples of solution collected during the purification using the Bradford protein assay.

1. Bovine Serum Abumin (BSA) (Sigma-Aldrich)
2. Coomassie Brilliant Blue G-250 (Sigma-Aldrich, catalog number: 27815 )
3. Methanol
4. Phosphoric acid (H3PO4)
5. Bradford reagent

Bradford reagent
Dissolve 50 mg of Coomassie Brilliant Blue G-250 in 50 ml of methanol and add 100 ml 85% (w/v) phosphoric acid (H3PO4).
Add the acid solution mixture slowly into 850 ml of H2O and let the dye dissolve completely (note: Do not add H2O into the acid solution).
Filter using Whatman #1 paper to remove the precipitates just before use.
Store in a dark bottle at 4 °C.

Equation 1 for Determining Cyt c Concentration:
(least accurate)

A550(reduced)
[Cytochrome c ] mM = _______________________ x Dilution
‚¬550(reduced )

Equation 2 for Determining Cyt c Concentration:
(more accurate)

A550(reduced) - A550(oxidized)
[Cytochrome c ] mM = _______________________ x Dilution
D‚¬550(reduced - oxidized)

Equation 3 for Determining Cyt c Concentration:
(most accurate)

A550(reduced) - A542(reduced) + A542(oxidized) - A550(oxidized)
[Cytochrome c] mM = ________________________________________________ x Dilution
D‚¬550(reduced - oxidized)