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; *d.craig@uwinnipeg.ca

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 can be calculated using the known concentrations of both
objective of undergraduate biochemistry courses. It is cen- ferro- and ferricyanide and the Nernst equation. This mea-
tral to the study of the respiratory chain. In this particular surement is made at several ratios of ferro-兾ferricyanide. The
experiment the redox potential of cytochrome c, a protein ratio of the ferro-/ferricyanide half-reaction that corresponds
involved in the respiratory chain, is measured spectrophoto- to when the ratio of ferro-兾ferricytochrome c is unity is de-
metrically using the “methods of mixtures” developed by Dav- termined graphically and the midpoint redox potential of cy-
enport and Hill (1) and simplified by Wada and Okunuki tochrome c is calculated.
(2).
The spectrum of cytochrome c differs in its two redox Materials
states. Ferrocytochrome c has an absorption peak centered
at 550 nm whereas ferricytochrome c does not. The fraction Stock solutions of 100 mM potassium hexacyano-
of cytochrome c that is in each state can be determined spec- ferrate(II) trihydrate (ferrocyanide), potassium hexacyano-
trophotometrically by comparing the absorbance of the peak ferrate(III) (ferricyanide), KH 2 PO 4 , and bovine heart
in a mixture to that of the completely oxidized and reduced cytochrome c were obtained from Sigma. Sodium dithionite
forms of the protein. In this experiment three solutions of was from Fluka.
identical concentrations of approximately 0.5 mg兾mL cyto-
chrome c were prepared in 10 mM ferrocyanide, 10 mM fer- Methods
ricyanide, or 10 mM dithionite. In place of dithionite,
ascorbic acid can be used. The cytochrome c in the ferricya- Stock solutions of 100 mM ferrocyanide, ferricyanide,
nide solution is fully oxidized and that in the dithionite so- and dithionite in deionized water were made fresh prior to
lution is fully reduced. use and degassed by vacuum. A stock solution of ∼0.5
Aliquots of the ferricyanide兾cytochrome c solution are mg兾mL cytochrome c in phosphate buffer (pH 7.0) was pre-
added to the ferrocyanide兾cytochrome c solution. Equilib- pared. Working solutions, all with identical cytochrome c
rium is reached rapidly and results in the redox potential of concentrations, were prepared from 2.7 mL of the stock cy-
the ferro-兾ferricytochrome c half-reaction being equal to that tochrome c solution and 0.3 mL of the stock ferrocyanide,
of the ferro-兾ferricyanide half-reaction. Measurement of the ferricyanide, and dithionite. Absorbances at 550 nm were
A550 of the cytochrome c and comparison to that of the fully measured of all three solutions using a Hewlett Packard
oxidized and reduced protein allows the determination of the 8452A diode array spectrophotometer. A 7.5 µL aliquot from
ratio of the ferro-兾ferricytochrome c ratio when its redox the ferricyanide兾cytochrome c solution was added to the
potential is equal to that of the ferro-兾ferricyanide, which ferrocyanide兾cytochrome c solution and the absorbance mea-
sured. This was repeated for an additional five 7.5 µL addi-
tions and a further three 15 µL additions, where the
absorbance was measured after each addition.
Table 1. Data Obtained in the Measurement
of the Redox Potential of Cytochrome c
Hazards
log([ferro-]/ log([ferro-]/
NSample A5 5 0
[ferricyt c]) [ferriCN]) Mixing of solutions of ferro- and ferricyanide with ac-
NferriCN and cyt c 0.264 ids liberates hydrogen cyanide, which is a very toxic gas.
Ndithionite and cyt c 0.738
N(ferroCN and cyt c) 0.433 ᎑0.256 2.603 Results and Discussion
N+7.5 µL (ferriCN and cyt c)
N+7.5 µL (ferriCN and cyt c) 0.374 ᎑0.520 2.300 The experiment was designed to be completed by third-
N+7.5 µL (ferriCN and cyt c) 0.342 ᎑0.706 2.125 year undergraduates in a three-hour period. The experiment
N+7.5 µL (ferriCN and cyt c) 0.326 ᎑0.823 2.000 takes less than half the allotted time allowing the students to
N+7.5 µL (ferriCN and cyt c) 0.315 ᎑0.919 1.903
analyze the data and calculate the quantitative result by the
᎑1.001
end of the laboratory period. It was designed to complement
N+7.5 µL (ferriCN and cyt c) 0.307 1.824
classroom discussions on basic redox chemistry and the use
N+15 µL (ferriCN and cyt c) 0.302 ᎑1.060 1.699
of the Nernst equation. The data obtained by one of the stu-
N+15 µL (ferriCN and cyt c) 0.296 ᎑1.140 1.602 dents performing this experiment in the course are shown in
N+15 µL (ferriCN and cyt c) 0.289 ᎑1.254 1.523 Table 1.

www.JCE.DivCHED.org • Vol. 83 No. 9 September 2006 • Journal of Chemical Education 1325

 In the Laboratory

−0.2 concentrations of the working solutions and the dilutions

y = −2.63 + 0.909x used. The graph of log([ferro-]兾[ferricytochrome c]) versus
x-intercept = 2.89 log([ferro-]兾[ferricyanide]) is shown in Figure 1.
R 2 = 0.996
log([ferrocyt c] / [ferricyt c])
−0.4
The x intercept is the log([ferro-]兾[ferricyanide]) that
corresponds to when the log([ferro-]兾[ferricytochrome c]) is
−0.6 unity. The redox potential of the ferri-兾ferrocyanide half-re-
action at this point is equal to that of the midpoint redox
potential of cytochrome c. It is given by the Nernst equation,
−0.8 where in this case E⬚ = 430 mV and log([ferro-]兾[ferricyanide]
is the x intercept of Figure 1:
−1.0
RT [ferroCN]
E = E ° − 2 .303 log
−1.2
nF [ferriCN]
1.6 1.8 2.0 2.2 2.4 2.6 The calculated potential for cytochrome c is 259 mV. The
log([ferroCN]/[ferriCN]) reported midpoint redox potential for native cytochrome c
ranges in the literature from 256–266 mV (3).
Figure 1. Log of the ferro-/ferricytochrome c reaction quotient vs
log of the ferro-/ferricyanide reaction quotient. Final Remarks
This laboratory exercise has been used for one year in
the third-year metabolism course, which has an enrollment
The fraction of the cytochrome c in the ferro- form is of approximately 110 students. The exercise is relatively in-
obtained by expensive to run, ran without any notable problems from the
onset, and students obtained good data. The experiment does
fraction ferro-
≡
[ferrocyt c] not require a full three-hour period to run, allowing for time
cytochrome c for calculation of the final result in the laboratory.
[ferrocyt c] + [ferricyt c]
A550,sample − A550,ferriCN&cyt c W
Supplemental Material
=
A550,dithionite &cyt c − A550,ferriCN&cyt Instructions for the students and notes for the instruc-
tor are available in this issue of JCE Online.
where A550,sample is the absorbance measured on the sample
after the addition of each aliquot of the ferricyanide兾 Literature Cited
cytochrome solution. The fraction of the cytochrome c in
the ferri-form is obtained by: 1. Davenport, H. E.; Hill, R. Proc. R. Soc. London Ser. B 1952,
fraction ferri- fraction ferro- 139, 327–347.
cytochrome c = 1 − cytochrome c 2. Wada, K.; Okunuki, K. J. Biochem. (Tokyo) 1969, 66, 249–
254.
From these values the log([ferro-]兾[ferricytochrome c]) is ob- 3. Wallace, C. J. A.; Proudfoot, A. E. I. Biochem. J. 1987, 245,
tained. The log([ferro-]兾[ferricyanide]) is obtained from the 773–779.

1326 Journal of Chemical Education • Vol. 83 No. 9 September 2006 • www.JCE.DivCHED.org