Corwin Mancuso

Abstract

Introduction

Cytochrome c is a metalloprotein, featuring a heme group as both the redox active site

and a vital structural support bonded to cysteine amino acids via thioester bonds (Kang & Carey,

1999). The central iron ion can exist in a reduced ferrous and oxidized ferric state, the different

ions contributing to differing structural and bonding capabilities of the protein as a whole; the

reduced form is noticeably more stable than the oxidized form (Trewhalla et al., 1988).

The cytochrome c heme can be oxidized with use of ferricyanide ion with the

mechanism:

𝑐𝑦𝑡 𝐼𝐼 + 𝐹𝑒 𝐼𝐼𝐼 ⇌ 𝑐𝑦𝑡 𝐼𝐼 − 𝐹𝑒 𝐼𝐼𝐼 ⇌ 𝑐𝑦𝑡 𝐼𝐼𝐼 − 𝐹𝑒 𝐼𝐼 ⇌ 𝑐𝑦𝑡 𝐼𝐼𝐼 + 𝐹𝑒 𝐼𝐼 ,

while hydrosulfite reduces heme by splitting into two molecules of 𝑆𝑂2− (Fig. 1) and following

the mechanism:

𝑆𝑂2− + 𝑐𝑦𝑡 𝐼𝐼𝐼 → 𝑆𝑂2 + 𝑐𝑦𝑡 𝐼𝐼 ,

only small amounts of each salt required for each redox reaction (Ohno & Cusanovich, 1981;

Lambeth & Palmer, 1973).

When put into a spectrometer, these proteins and complexes have characteristic peaks at

certain wavelengths. Cytochrome c will generate emissions ranging from midrange ultraviolet

light (~200 nm) to the high end of red light (~750 nm). One important wavelength is at around

280 nm, correlating to the aromatic rings in amino acids such as tryptophan or tyrosine. The area

around 550 nm corresponds to the changes in structure as the hemeprotein forms complexes with

the redox salts. There are paired peaks approximately 30 nm apart in the 550-nm area for the
reduced form, while the oxidized form had a singular hump in the same general area (Ohta &

Tobari, 1981). The 416-nm band is known as the Soret peak which appears when hemeproteins

are observed.

Materials and Methods

The procedure was followed from “Cytochrome c Concentration Determination,”

Results

The key absorptions for Fraction 2 (280, 416, and 550 nm), were consistently higher than

the absorptions for Fraction 3 of isolated cytochrome c. While the reduced spectra around 550

nm shows the characteristic double peaks, the oxidized spectra did not have a consistent single

hill, but rather a smaller hill followed by a peak around 550 nm. This peak was most likely

remaining reduced Fe2+ in solution and was significantly smaller than reduced spectra. For the

F2 graph, the oxidized spectrum was lost due to formatting errors, so it was not included.

However, the maximum absorbances were roughly recorded: 280-nm = 1.095 A.U., 416-nm =

1.147 A.U., and 550-nm = 0.26

Spectrum Max abs. Max abs. Max abs. Max abs. Max abs. Max abs.

~280-nm wavelength ~416-nm wavelength ~550-nm wavelength

(A.U.) (nm) (A.U.) (nm) (A.U.) (nm)

Raw 1.2273 270.644 1.3259 409.063 0.26760; 520.787;

oxidized 0.26874 548.352

Reduced 1.1898 269.463 1.3328 412.557 0.26744; 520.212;

0.32451 549.498

𝐴𝑏𝑠550(𝑟𝑒𝑑.) 0.32451 𝐴. 𝑈.
[𝑐𝑦𝑡𝑜𝑐ℎ𝑟𝑜𝑚𝑒 𝑐] = ∙ 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛; [𝑐𝑦𝑡 𝑐𝐹2 ] = ∙ 1: 50
𝜀550(𝑟𝑒𝑑.) 29.4
𝑚𝑀 ∙ 𝑐𝑚

0.0002208 𝑚𝑚𝑜𝑙 1𝐿 1 𝑚𝑜𝑙 884.893 𝑔 1000 𝑚𝑔

= ∙ ∙ ∙ ∙
𝐿 1000 𝑚𝐿 1000 𝑚𝑚𝑜𝑙 𝑚𝑜𝑙 1𝑔

= 0.0001953 𝑚𝑔/𝑚𝐿

𝐴𝑏𝑠550(𝑟𝑒𝑑.) − 𝐴𝑏𝑠550(𝑜𝑥.)
[𝑐𝑦𝑡𝑜𝑐ℎ𝑟𝑜𝑚𝑒 𝑐] = ∙ 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛
∆𝜀550(𝑟𝑒𝑑.−𝑜𝑥.)

0.32451 − 0.26874 𝐴. 𝑈.
= [𝑐𝑦𝑡 𝑐𝐹2 ] = ∙ 1: 50 = 0.0000
19.6
𝑚𝑀 ∙ 𝑐𝑚
Spectrum Max abs. Max abs. Max abs. Max abs. Max abs. Max abs.

~280-nm wavelength ~416-nm wavelength ~550-nm wavelength

(A.U.) (nm) (A.U.) (nm) (A.U.) (nm)

Raw 0.69735 268.282 0.99579 412.557 0.14741; 520.787;

oxidized 0.21651 548.925

Oxidized 0.62726 267.101 0.82013 408.480 0.11609; 520.212;

0.11726 546.632
 Reduced 0.59236 266.510 0.91696 416.049 0.10510; 519.637;

0.20456 548.925

Purification step Total vol. of [cytochrome c] Total Relative purity

each fraction (mg/mL) cytochrome c (Raw abs416nm /

(mL) (mg) raw abs280nm)

Fraction 1 1,750 - - -

Fraction 2 7.7

Fraction 3 6.7

Discussion

References

Kang, Xinshan & Carey, J. (1999). Role of heme in structural organization of cytochrome c

probed by semisynthesis. Biochemistry, 38(48), pp. 15944-51.

Lambeth, D.O. & Palmer, G. (1973). The kinetics and mechanism of reduction of electron

transfer proteins and other compounds of biological interest by dithionite. Journal of

Biological Chemistry, 248(17), pp. 6095-103.

Ohno, N. & Cusanovich, M.A. (1981). Reaction of c-type cytochromes with the iron

hexacyanides. Biophysical Journal, 36, pp. 589-605.

Ohta, S. & Tobari, J. (1981). Two cytochromes c of Methylomonas j. Journal of Biochemistry,

90(1), pp. 215-24.

Trewhalla, J., Carlson, V.A.P., Curtis, E.H., & Heidorn, D.B. (1988). Differences in the solution
structures of oxidized and reduced cytochrome c measured by small-angle x-ray

scattering. Biochemistry, 27, pp. 1121-5.