1. Matt Bosch, Tony Rivera, Dr. Stephen U. Dunham
Department of Chemistry, Moravian College, 1200 Main Street, Bethlehem, PA 18018!
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Ref. 2011, Wang !
• E.coli broth : N,N-dimethylformamide (DMF).
• Op:cal Density of broth 0.2-0.8 at Abs595
• 5, 10, 15, 20, 30 uM cipla:n in DMF. Final
experimental concetra:on was 30 uM.
• 2D Assay: 1st assay, separa:on of proteins by
pH. 2nd assay, separa:on by molecular
weight
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Platinum based metal research has been around since the 1970’s. Since it’s discovery by Barnett Rosenberg 1, which he found that E.coli cells experienced retarded growth
due to platinum contaminated media ( 1965, Rosenburg). Scientist have continued experiments on the effects of platinum in biochemistry to: understand its negative role in
replication, the chemical mechanisms, synthesize new platinum analogs, and design effective cancer therapy treatments. Current literature has researched, the effects of
monofunctional platinum agents on bacterial cell growth, and found that platinum agents acted as a stressor to reduce cellular growth (2013, Johnstone). !
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In an attempt in trying to understand further into the stress effects of cisplatin on cell replication, this study experimented on E.coli broth exposed to 30 uM cisplatin, and we
ran a 2D gel assay to analyze the proteins. 2D gels allows us to analyze the pI and molecular weight of proteins found in E.coli cells after interaction with a chemical stressor.
This type of information gives us insight into what, or how, much proteins are expressed by the cell, which can be used to sequence back to the genome. Furthermore,
knowing the proteins affected by cisplatin exposure, future experiments can look into these proteins for gene-to-protein information and try to puzzle the pieces to build a more
clearer picture that describes the mechanisms or pathways involved, which are not fully understood.!
Introduc*on
Materials and Methods
Results
Figure 1. 2D Gel Assays of Escherichia coli. [A] Reference gel page (Expasy). [B] Experimental gel page ( Control: E.coli, no stress). [C]
Experimental gel page ( Stress: E.coli, cispla:n).
Discussion References
Protein Accession No. Name pI M w
(kDa)
Function
P69441 Adenylate Kinase 5.6 22509 Phosphotransferase ADP
P37016 Yadk 5.5 28425 Adhesion
P0A858 Triosphosphase 5.5 26972 Glycolysis
P22939 Farnesyl diphosphate
synthase
5.3 22939 Catalyst of Steriod
intermidiates
P0AGD3 Superoxide dismutase 5.5 22150 Catalyst of superoxide
radicals
P05055 PNPase 5.1 83954 RNA degrading protein
Table 1. List and characteriza:on of iden:fied proteins made by E.coli
From observing the results, Gel C containing the cisplatin sample showed fewer protein spots than the control Gel B. Using the differences in the
gels we can conclude that cisplatin had an effect on E. coli in the production of the missing proteins all found at a pI of about 5.0, and ranging from
25 to 75 kDa. The large bands found towards the bottom of the gels are presumably a build up of proteins that were two small to get caught in the
gel. This could have been caused by degradation of the proteins during preparation of cell lysis.!
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Studies show that “the regulation of two proteins, aconitate hydratase 2 and 60 kDa chaperonin 1, could be linked to a platinated amino acid in a
protein sequence” (Stefanopoulou, 2011). Other proteins that can be affected by cisplatin are: GyrB (DNA gyrase), hydrogenase-4 component G
(hyfG), malate dehydrogenase (MdH), and target enzyme enolase (Eno). Enolase is an important metabolite used in glycolysis which plays a role as
a catalyst in the pathway that converts two phosphoglycerate into phosphoenolpyruvate. Chaperone protein DnaK (DnaK), that enables the refolding
of proteins, are affected by platination of its peptide side chains, which induces a stress response.!
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The two gels in our experiment show that cisplatin had an effect on the protein profile of E.coli cells. During our preparation, we identified that at 30
uM of cisplatin, the platination of the E.coli broth acted as a stress by lowering the absorbance. Therefore, cisplatin is a chemical stressor that
impedes the success of cellular replication.!
1. Rosenberg B. In: Cisplatin: Chemistry and Biochemistry of a Leading
Anticancer Drug. Lippert B, editor. Verlag Helvetica Chimica Acta;
Zürich: 1999. p. 1.
2. Johnstone, Timothy C., Sarah M. Alexander, Wei Lin, and Stephen J.
Lippard. "Effects of Monofunctional Platinum Agents on Bacterial Growth:
A Retrospective Study." J. Am. Chem. Soc. Journal of the American
Chemical Society 136.1 (2014): 116-18. Web.
3. Stefanopoulou, Maria, Malte Kokoschka, William S. Sheldrick, and Dirk
A. Wolters. "Cell Response of Escherichia Coli to Cisplatin-induced
Stress." Proteomics PROTEOMICS 11.21 (2011): 4174-188. Web.
4. Park, G. Y., J. J. Wilson, Y. Song, and S. J. Lippard. "Phenanthriplatin, a
Monofunctional DNA-binding Platinum Anticancer Drug Candidate with
Unusual Potency and Cellular Activity Profile." Proceedings of the
National Academy of Sciences 109.30 (2012): 11987-1992. Web.