1. Purification and Quantification of Cytochrome C from Bovine Liver Tissue
Amol Agarwal, Michael DeBrota, Logan Fenimore
Introduction
Results
Conclusion
References
Acknowledgements
Rose-Hulman Institute of Technology, CHEM291
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.
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. 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
• Create buffer by mixing 40 mL of 0.1 M solution of Tris, 4
mL of 1 mM solution of EGTA, and 80 mL of 1 M solution
of sucrose. Lower pH to 7.4 by adding 1.2759 g of HEPES.
• Rinse, mass, cut and blend 99.8 g of bovine liver.
• Homogenize blended liver using Dounce tissue grinder.
• Centrifuge at 600g for 10 min at 4 °C.
• Discard supernatant, resuspend pellet in buffer and centrifuge
at 10000g for 10 min at 4 °C.
• Discard the supernatant and resuspend the pellet again and
add 0.1380 g of succinic acid, 0.535 mL of 6 M NaOH
solution and 2 mL of water.
• Centrifuge suspension at 2500g for 10 min at 4 °C.
• Collect supernatant and centrifuge again at 25000g for 10
min at 4 °C.
• Collect the supernatant for analysis.
• Create 0.8 mL dilutions of 200 ug/mL BSA of concentrations
7.5, 6, 4.5 and 3 ug/mL using a micropipette and water and
add 0.2 mL of blue dye
• Collect absorption spectra of standards using Vernier
SpectroVis Plus and create calibration curve of concentration
of BSA versus absorption at 595 nm.
• Collect absorption spectrum of pure supernatant and 800-fold
dilution of supernatant.
After the liver was blended, it was observed that there were suspended chunks of liver in
the mixture, and to homogenize it, a dounce tissue grinder was used. When the homogenate
was centrifuged for the first time at 600g, it was found that this speed was not high enough
to centrifuge out all the mitochondria, and only the big chunks were centrifuged. The
supernatant at this point looked red and opaque, and there was a possibility that the
supernatant contained some amount of mitochondria. After the final centrifugation
(purification step), a red and translucent solution was obtained which was optimal for
making samples for the spectroscopic analysis and hence the assumption was made that
almost all of the mitochondria was centrifuged out of the suspension during the first
centrifugation step.
.
Absorption spectrum of 800-fold dilution Absorption spectrum of pure supernatant
Calibration curve of BSA at 595 nm
Cytochrome C
The main objective was to extract and quantify
Cytochrome C from beef liver using UV-vis
absorption and Beer’s Law. We were successful in
both extracting and quantifying Cytochrome C from
the liver tissues. Approximately 1.46 mg of
Cytochrome C was extracted form 99.8 g of beef
liver with a degree of purification of 0.385%.
Cytochrome C Cuvette with BSA and blue dye
[1] 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
[2] 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.
[3] 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.
[4] Bushnell, G., Louie, G., & Brayer, G. (1990). High-
resolution three-dimensional structure of horse heart
cytochrome c. Journal of Molecular Biology, 214(2), 585-
595. doi:10.1016/0022-2836(90)90200-6
We would like to thank Dr. Weatherman, Cyndi
Erwin, and the Department of Chemistry at Rose-
Hulman Institute of Technology.