Protein and Cytochrome c quantification
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.
Note: If a sample is turbid it contains undissolved material that should be removed by centrifuging briefly at high speed before using it in any assay.
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.
Cytochrome c Determination
Reagents:
The objective here is to accurately assay the cytochrome c concentration while using as little of the protein sample as possible. Accurate determinations require that the solution have an absorbance difference DA550 (reduced - oxidized) of ~ 0.01 A, and a volume of 0.6 mL (using a semi-microcuvette). If the cytochrome c appears to have an absorbance significantly greater than 0.01 A, a small, precise volume of cytochrome c solution can be carefully diluted with a precise volume of buffer to give a final volume of 0.6 mL.
To be sure all of the heme iron of the cytochrome c is oxidized to Fe3+ add one very small crystal of potassium ferricyanide (K3Fe(CN)6), cover the cuvette with Parafilm, and gently invert it several times. Record the spectrum from 350 - 750 nm, and note the absorbance values at 550 nm and 542 nm. Reduce the heme iron from Fe3+ to Fe2+ by adding 50 - 100 mg of sodium hydrosulfite (Na2S2O4) to the sample, cover, and mix by slowly inverting. Make sure you observe a color change upon reduction of the sample. Again record the spectrum, noting the absorbance values at 550 nm and 542 nm. A reasonably good determination of the cytochrome c concentration can be made by using the second equation below and a somewhat better estimate by using the third equation. This will be further discussed in class.
Note: The spectrum of oxidized cytochrome c does not have a peak at 550 nm. If a peak is present, oxidize the cytochrome c by adding a tiny crystal of K3Fe(CN)6 and record the spectrum again.
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)
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.
Purification Step | Vol. (mL) | [Protein] (mg/mL) | Total Protein (mg) | [Cyt c] (mg/mL) | Total Cyt c (mg) | Sp. Act. (Cyt c/ Prot.) | Overall Yield (%) |
| Tissue Weight ____ | - | - | - | - | - | - | 100 |
| Step 2 - Homogenized Sample | - | - | - | ||||
| Step 7 - Combined Filtrate | |||||||
| Step 11 - Pooled Eluant Fractions | |||||||
| Step 14 - Supernatant | |||||||
| Step 16 - Pooled Eluant Fractions |
Note: If a sample is turbid it contains undissolved material that should be removed by centrifuging briefly at high speed before using it in any assay.
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.
Cytochrome c Determination
Reagents:
solid K3Fe(CN) 6 or potassium ferricyanideProtocol:
solid Na2S2O4 or sodium hydrosulfite
The objective here is to accurately assay the cytochrome c concentration while using as little of the protein sample as possible. Accurate determinations require that the solution have an absorbance difference DA550 (reduced - oxidized) of ~ 0.01 A, and a volume of 0.6 mL (using a semi-microcuvette). If the cytochrome c appears to have an absorbance significantly greater than 0.01 A, a small, precise volume of cytochrome c solution can be carefully diluted with a precise volume of buffer to give a final volume of 0.6 mL.
To be sure all of the heme iron of the cytochrome c is oxidized to Fe3+ add one very small crystal of potassium ferricyanide (K3Fe(CN)6), cover the cuvette with Parafilm, and gently invert it several times. Record the spectrum from 350 - 750 nm, and note the absorbance values at 550 nm and 542 nm. Reduce the heme iron from Fe3+ to Fe2+ by adding 50 - 100 mg of sodium hydrosulfite (Na2S2O4) to the sample, cover, and mix by slowly inverting. Make sure you observe a color change upon reduction of the sample. Again record the spectrum, noting the absorbance values at 550 nm and 542 nm. A reasonably good determination of the cytochrome c concentration can be made by using the second equation below and a somewhat better estimate by using the third equation. This will be further discussed in class.
Note: The spectrum of oxidized cytochrome c does not have a peak at 550 nm. If a peak is present, oxidize the cytochrome c by adding a tiny crystal of K3Fe(CN)6 and record the spectrum again.
Equation 1 for Determining Cyt c Concentration:
(least accurate)
[Cytochrome c ] mM = _______________________ x Dilution
‚¬550(reduced )
Equation 2 for Determining Cyt c Concentration:
(more accurate)
[Cytochrome c ] mM = _______________________ x Dilution
D‚¬550(reduced - oxidized)
Equation 3 for Determining Cyt c Concentration:
(most accurate)
[Cytochrome c] mM = ________________________________________________ x Dilution
D‚¬550(reduced - oxidized)
| Wavelength (nm) | Reduced (mM-1cm-1) | Oxidized (mM-1cm-1) | Difference (D‚¬) (mM-1cm-1) |
| 410 | 106.1 | 106.1 | 0 |
| 416 | 129.1 | 88.8 | 40.3 |
| 504 | 7.4 | 7.4 | 0 |
| 520 | 17.3 | 11.7 | 5.6 |
| 526 | 12.1 | 12.1 | 0 |
| 534 | 8.3 | 11.8 | -3.6 |
| 542 | 9.8 | 9.8 | 0 |
| 550 | 29.4 | 9.8 | 19.6 |
| 556 | 9.1 | 9.1 | 0 |
| 564 | 3.5 | 8.1 | -4.6 |
Comments
Post a Comment