In the Laboratory
Spectroscopic Measurement of the Redox Potential of W Cytochrome c for the Undergraduate Biochemistry Laboratory Douglas B. Craig* Department of Chemistry, University of Winnipeg, Winnipeg, Manitoba, Canada R3B 2E9; *
[email protected] Ellert R. Nichols Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3
An understanding of redox chemistry is an important objective of undergraduate biochemistry courses. It is central to the study of the respiratory chain. In this particular experiment the redox potential of cytochrome c, a protein involved in the respiratory chain, is measured spectrophotometrically using the “methods of mixtures” developed by Davenport and Hill (1) and simplified by Wada and Okunuki (2). The spectrum of cytochrome c differs in its two redox states. Ferrocytochrome c has an absorption peak centered at 550 nm whereas ferricytochrome c does not. The fraction of cytochrome c that is in each state can be determined spectrophotometrically by comparing the absorbance of the peak in a mixture to that of the completely oxidized and reduced forms of the protein. In this experiment three solutions of identical concentrations of approximately 0.5 mg兾mL cytochrome c were prepared in 10 mM ferrocyanide, 10 mM ferricyanide, or 10 mM dithionite. In place of dithionite, ascorbic acid can be used. The cytochrome c in the ferricyanide solution is fully oxidized and that in the dithionite solution is fully reduced. Aliquots of the ferricyanide兾cytochrome c solution are added to the ferrocyanide兾cytochrome c solution. Equilibrium is reached rapidly and results in the redox potential of the ferro-兾ferricytochrome c half-reaction being equal to that of the ferro-兾ferricyanide half-reaction. Measurement of the A550 of the cytochrome c and comparison to that of the fully oxidized and reduced protein allows the determination of the ratio of the ferro-兾ferricytochrome c ratio when its redox potential is equal to that of the ferro-兾ferricyanide, which
Table 1. Data Obtained in the Measurement of the Redox Potential of Cytochrome c NSample
A5 5 0
NferriCN and cyt c
0.264
Ndithionite and cyt c
0.738
N(ferroCN and cyt c) 0.433 N+7.5 µL (ferriCN and cyt c)
log([ferro-]/ log([ferro-]/ [ferricyt c]) [ferriCN])
᎑0.256
Stock solutions of 100 mM potassium hexacyanoferrate(II) trihydrate (ferrocyanide), potassium hexacyanoferrate(III) (ferricyanide), KH 2 PO 4 , and bovine heart cytochrome c were obtained from Sigma. Sodium dithionite was from Fluka. Methods Stock solutions of 100 mM ferrocyanide, ferricyanide, and dithionite in deionized water were made fresh prior to use and degassed by vacuum. A stock solution of ∼0.5 mg兾mL cytochrome c in phosphate buffer (pH 7.0) was prepared. Working solutions, all with identical cytochrome c concentrations, were prepared from 2.7 mL of the stock cytochrome c solution and 0.3 mL of the stock ferrocyanide, ferricyanide, and dithionite. Absorbances at 550 nm were measured of all three solutions using a Hewlett Packard 8452A diode array spectrophotometer. A 7.5 µL aliquot from the ferricyanide兾cytochrome c solution was added to the ferrocyanide兾cytochrome c solution and the absorbance measured. This was repeated for an additional five 7.5 µL additions and a further three 15 µL additions, where the absorbance was measured after each addition. Hazards Mixing of solutions of ferro- and ferricyanide with acids liberates hydrogen cyanide, which is a very toxic gas. Results and Discussion The experiment was designed to be completed by thirdyear undergraduates in a three-hour period. The experiment takes less than half the allotted time allowing the students to analyze the data and calculate the quantitative result by the end of the laboratory period. It was designed to complement classroom discussions on basic redox chemistry and the use of the Nernst equation. The data obtained by one of the students performing this experiment in the course are shown in Table 1.
N+7.5 µL (ferriCN and cyt c) 0.374
᎑0.520
2.300
᎑0.706
2.125
N+7.5 µL (ferriCN and cyt c) 0.326
᎑0.823
2.000
N+7.5 µL (ferriCN and cyt c) 0.315
᎑0.919
1.903
N+7.5 µL (ferriCN and cyt c) 0.307
᎑1.001
1.824
N+15 µL (ferriCN and cyt c)
0.302
᎑1.060
1.699
N+15 µL (ferriCN and cyt c)
0.296
᎑1.140
1.602
N+15 µL (ferriCN and cyt c)
0.289
᎑1.254
1.523
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Materials
2.603
N+7.5 µL (ferriCN and cyt c) 0.342
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can be calculated using the known concentrations of both ferro- and ferricyanide and the Nernst equation. This measurement is made at several ratios of ferro-兾ferricyanide. The ratio of the ferro-/ferricyanide half-reaction that corresponds to when the ratio of ferro-兾ferricytochrome c is unity is determined graphically and the midpoint redox potential of cytochrome c is calculated.
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Journal of Chemical Education
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In the Laboratory
concentrations of the working solutions and the dilutions used. The graph of log([ferro-]兾[ferricytochrome c]) versus log([ferro-]兾[ferricyanide]) is shown in Figure 1. The x intercept is the log([ferro-]兾[ferricyanide]) that corresponds to when the log([ferro-]兾[ferricytochrome c]) is unity. The redox potential of the ferri-兾ferrocyanide half-reaction at this point is equal to that of the midpoint redox potential of cytochrome c. It is given by the Nernst equation, where in this case E⬚ = 430 mV and log([ferro-]兾[ferricyanide] is the x intercept of Figure 1:
log([ferrocyt c] / [ferricyt c])
−0.2
y = −2.63 + 0.909x x-intercept = 2.89 R 2 = 0.996
−0.4
−0.6
−0.8
−1.0
E = E ° − 2 .303 −1.2 1.6
1.8
2.0
2.2
2.4
2.6
log([ferroCN]/[ferriCN]) Figure 1. Log of the ferro-/ferricytochrome c reaction quotient vs log of the ferro-/ferricyanide reaction quotient.
The fraction of the cytochrome c in the ferro- form is obtained by fraction ferro≡ cytochrome c =
[ferrocyt c] [ferrocyt c] + [ferricyt c]
A550,sample − A550,ferriCN&cyt c A550,dithionite &cyt c − A550,ferriCN&cyt
where A550,sample is the absorbance measured on the sample after the addition of each aliquot of the ferricyanide兾 cytochrome solution. The fraction of the cytochrome c in the ferri-form is obtained by: fraction ferrifraction ferrocytochrome c = 1 − cytochrome c
From these values the log([ferro-]兾[ferricytochrome c]) is obtained. The log([ferro-]兾[ferricyanide]) is obtained from the
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RT log nF
[ferroCN]
[ferriCN]
The calculated potential for cytochrome c is 259 mV. The reported midpoint redox potential for native cytochrome c ranges in the literature from 256–266 mV (3). Final Remarks This laboratory exercise has been used for one year in the third-year metabolism course, which has an enrollment of approximately 110 students. The exercise is relatively inexpensive to run, ran without any notable problems from the onset, and students obtained good data. The experiment does not require a full three-hour period to run, allowing for time for calculation of the final result in the laboratory. W
Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Davenport, H. E.; Hill, R. Proc. R. Soc. London Ser. B 1952, 139, 327–347. 2. Wada, K.; Okunuki, K. J. Biochem. (Tokyo) 1969, 66, 249– 254. 3. Wallace, C. J. A.; Proudfoot, A. E. I. Biochem. J. 1987, 245, 773–779.
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