As part of Rose-Hulman’s CHEM291 Introduction to Chemical Research course, this group project entailed the extraction, isolation, and quantification of cytochrome c from gross bovine liver tissue, using an experimental protocol assembled from review of multiple sources of primary literature. Core biochemical laboratory techniques such as tissue extraction, cell lysis, centrifugation, and purification were practiced, along with quantitative methods for confirming experimental yield. Following the completion of the project, our group designed a scientific poster in addition to this report. The poster can be viewed at the following link: https://www.slideshare.net/MichaelDeBrota/agarwal-a-debrota-m-fenimore-l-purification-and-quantification-of-cytochrome-c-from-bovine-liver-tissue-project-poster

1. Purification and Quantification of
Cytochrome C from Bovine Liver
Tissue
Amol Agarwal [CM 1502], Michael DeBrota [CM 1612], and
Logan Fenimore [CM 1641]
CHEM291 Introduction to Chemical Research | Dr. Weatherman
February 16, 2018

2. Introduction.
Description of cytochrome c.
Cytochrome c (Cyt c) is an integral protein involved in oxidative phosphorylation, a part of
cellular respiration. It is located in the intermembrane space within mitochondria, present
in all animal cells. Cytochrome c serves to transfer electrons between transmembrane
protein complexes III (Coenzyme q - Cyt c reductase) and IV (Cyt c oxidase), ultimately
allowing oxygen to accept the electrons and form water [1].
Purification strategy.
In this experiment, the strategy used to extract the cytochrome c involved homogenization
of the liver tissue to release the mitochondria from the cells, centrifugation to separate the
mitochondria from other cell materials and proteins, and energization of the mitochondria
using a succinate solution to induce apoptosis in the mitochondria, releasing cytochrome c
for isolation and quantification. We chose to induce apoptosis so that the contents of the
mitochondria, including cytochrome c, could be released and subsequently centrifuged to
separate the cytochrome c from the other materials.
Procedure.
Preparation of buffer.
To begin the extraction process, a buffer was prepared by mixing 40 mL of 0.1 M Tris (2-
Amino-2-(hydroxymethyl)propane-1,3-diol), 4 mL of 1 mM EGTA (Ethylene glycol-bis(2-
aminoethylether)-N,N,N’,N’-tetraacetic acid), and 80 mL of 11 M sucrose. The pH of the
resulting solution was then lowered to 7.4 by adding 1.2759 ± 0.0002 g HEPES (2-[4-(2-
hydroxyethyl)piperazin-1-yl]ethanesulfonic acid). The prepared buffer was placed on ice to
chill.
Preparation and homogenization of liver tissue.
Bovine liver was cut into small pieces using scissors and massed on an analytical balance.
The tissue was rinsed using the prepared pH 7.4 cold buffer solution. The rinsed liver was
then combined with approximately 100 mL of new, cold buffer in a beaker. This mixture
was then transferred to a blender and blended for 2 minutes. The blended liver was further
homogenized using a Dounce tissue grinder in several aliquots, stroked 3 times each with a
glass pestle. The homogenate was transferred to 3 mass-balanced centrifuge tubes and
centrifuged at 600g for 10 minutes at 4°C. The supernatant was discarded, and the pellet
was resuspended and washed using the pH 7.4 buffer. This suspension was then
centrifuged at 10000g for 10 minutes at 4°C. The supernatant of this centrifugation was
immediately discarded, and the pellet was resuspended using the pH 7.4 buffer. This
suspension was stored in a refrigerator for a week [2].
After a week of refrigeration, the suspension was removed from the refrigerator. The
mitochondria were energized to release cytochrome c by adding 0.1380 g of succinic acid,
0.535 mL of 6 M NaOH, and approximately 2 mL water. This created a solution of 116 mL
total volume with an approximate concentration of 10 mM succinate. This suspension was
centrifuged at 2500g for 10 minutes at 4°C. The supernatant containing the released
cytochrome c was then decanted from the pellet and stored in a refrigerator for a week [3].

3. After another week of refrigeration, the pellet that had formed during storage was
resuspended by adding a small volume of pH 7.4 buffer, and the suspension was centrifuged
for a final time at 25000g for 10 minutes at 4°C. After this centrifugation, the supernatant
was decanted into a beaker, covered with Parafilm, and stored.
Analysis and quantification of cytochrome c.
The overall recoveries and concentrations of protein and cytochrome c in the liver were
determined through the construction of a calibration curve. The absorption of ultraviolet-
visible light of bovine serum albumin (BSA) at 595 nm was plotted versus BSA
concentration. Dilutions of the BSA of 7.5, 6.0, 4.5, and 3.0 µg/mL were prepared using a
micropipette to volumes of 0.8 mL, with water as the solvent. 0.2 mL of blue dye were
added to each dilution to reach a final volume of 1 mL. Ultraviolet-visible light absorption
spectra for each dilution were collected using a Vernier SpectroVis® Plus
spectrophotometer. The absorptions for each dilution were recorded at 595 nm, and the
calibration curve mentioned above was created. The supernatant containing cytochrome c
was then also diluted multiple times using a micropipette with water as the solvent to a
volume of 0.8 mL, and 0.2 mL of blue dye were added. The absorption spectrum of each
dilution was collected until the absorptions at 541 nm and 595 nm were within range of the
standard solution absorptions.
The dilution of the sample that absorbed within the bounds of the calibration curve was a
1:800 dilution. To calculate the concentration of protein in the liver, the absorptions of the
sample were recorded at 541 and 595 nm, and the concentration of the protein in the
diluted sample was calculated using the calibration curve. The concentration of the protein
in the liver supernatant and the recovery of the protein in the original liver was determined
by accounting for the sample dilution factor and the volume of the supernatant. An
approximate ratio of the grams of protein per gram of cow liver was also utilized in these
calculations. The concentration of the cytochrome c and the degree of purification compared
to the total protein concentration were determined using both the absorption of the 1:800
dilution sample at 541 nm and the same calibration curve. The dilution factor was also
taken into account to calculate this concentration.
Results.
Observations made during tissue extraction and purification.
After the liver was blended, it was observed that there were large suspended pieces of liver
tissue still present in the mixture. To remedy this, a Dounce tissue grinder was used to
homogenize the mixture. The homogenate was then centrifuged at 600g, but this speed
proved not to be high enough to separate all mitochondria. After this first centrifugation,
the supernatant appeared red and opaque, indicating the possibility that the supernatant
contained some amount of mitochondria. Following the final centrifugation (purification
step), a red and translucent supernatant containing cytochrome c was obtained. Due to the

4. transparency of this solution, the assumption was made that almost all of the mitochondria
had been separated out of the mixture during the first centrifugation step. This solution
was then used to prepare standard samples for spectroscopic analysis.
Overall protein recovery and percent yield from initial tissue.
The fitted-line equation in Figure 1 models Beer’s Law. The y variable corresponds to the
absorption of BSA at 595 nm and the x variable corresponds to the concentration of BSA in
the standards.
y = 0.0449x + 0.6771
1:800 Dilution Sample Absorption at 595 nm: 0.878
y = 0.878
Solving for x:
x = 4.474387528 ug/mL
Concentration of protein in the supernatant = 4.474387528
𝑢𝑔
𝑚𝐿
× 1x10-6
𝑔
𝑢𝑔
× (Dilution
Factor)
Concentration of protein in the supernatant = 4.474387528
𝑢𝑔
𝑚𝐿
× 1x10-6 𝑔
𝑢𝑔
× 800 =
0.003579510022
𝑔
𝑚𝐿
Overall protein recovery = Concentration of protein in the supernatant × Volume of
supernatant
Overall protein recovery = 0.003579510022
𝑔
𝑚𝐿
× 106 mL = 0.3794280623 g = 0.379 g
Overall protein yield =
𝑂𝑣𝑒𝑟𝑎𝑙𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦
𝑀𝑎𝑠𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑡𝑖𝑠𝑠𝑢𝑒
× 100
Mass of initial tissue = 99.8 g
Figure 1: Calibration curve of BSA at 595 nm.

5. Overall protein yield =
0.3794280623 𝑔
99.8 𝑔
× 100= 0.3801884392% = 0.380%
Overall cytochrome c recovery and degree of purification compared to total protein
concentrations.
Overall cytochrome c recovery =
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑎𝑡 541 𝑛𝑚
𝐸𝑥𝑡𝑖𝑛𝑐𝑡𝑖𝑜𝑛 𝐶𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡
× Molar mass of cytochrome c × Volume
of supernatant =
0.459 𝑐𝑚−𝑚𝑜𝑙
29500 ∗ 1 𝑐𝑚−𝐿
× 12327
𝑔
𝑚𝑜𝑙
[4] × 0.106 𝐿 = 0.020330774847 g = 0.0203 g
Degree of purification =
𝑂𝑣𝑒𝑟𝑎𝑙𝑙 𝑐𝑦𝑡𝑜𝑐ℎ𝑟𝑜𝑚𝑒 𝑐 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦
𝑂𝑣𝑒𝑟𝑎𝑙𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦
× 100
Degree of purification =
0.020330774847 g
0.3794280623 𝑔
× 100 = 5.35826863312% = 5.36%
Discussion.
The main objective of the experiment was to extract and quantify cytochrome c, at any yield
and any degree of purity, from beef liver tissue. Through analysis and in consideration of
this objective, we were successful in both extracting and quantifying cytochrome c from the
liver tissues. From the calibration curve, a protein yield of 0.380% was obtained.
Approximately 20.3 mg of cytochrome c were extracted from 99.8 g of beef liver, resulting in
a 5.36% degree of purification. Because a nonzero yield and a nonzero degree of purification
were found for the protein and the cytochrome c, the main objective and goals of the
experiment were fulfilled.
References.
[1] Hough, M. A., Silkstone, G., Worrall, J., & Wilson, M. T. (2014). Chapter Eight - NO
Binding to the Proapoptotic Cytochrome c–Cardiolipin Complex. Vitamins and Hormones,
96, 193-209.
[2] Frezza, C., Cipolat, S., & Scorrano, L. (2007). Organelle isolation: functional
mitochondria from mouse liver, muscle and cultured fibroblasts. Nature Protocols, 2(2), 287-
295. doi:10.1038/nprot.2006.478
[3] Ghafourifar, P., Klein, S. D., Schucht, O., Schenk, U., Pruschy, M., Rocha, S., & Richter,
C. (1999). Ceramide induces cytochrome c release from isolated mitochondria. The Journal
of Biological Chemistry, 274(10), 6080-6084.
[4] Malmgren, L., Olsson, Y., Olsson, T., & Kristensson, K. (1978). Uptake and retrograde
axonal transport of various exogenous macromolecules in normal and crushed hypoglossal
nerves. Brain Research, 153(3), 477-493. doi:10.1016/0006-8993(78)90333-5