1. Tp34211 As a Catalyst
Emily and Jonathan
Department of Chemistry, Seattle University, Seattle, WA 98122
Alignment
Summary and Ongoing Work
Results / Methods
Representative cytochrome structure
Introduction
Figure 2: Phaeodactylum tricornutum, a monoheme cythochrome c. P.
tricornutum also has a Fe heme. The Lysine in the structure might have the
covalent interaction with the heme.
Section 3 – Size Exclusion Chromatography (G-75)
Section 1 – DNA Sequence Transformation
Section 2 - DEAE Anion Exchange Chromatography
References
After harvested, the protein was purified using DEAE Anion Exchange Chromatography with using equilibration buffer
(20mM Tris Base and 20mM NaCl, pH=7.5), wash buffer (1M NaCl, pH=7.5), and elution buffer (0-1M NaCl and 20mM
NaCl, pH=7.5). Twelve fractions were collected run through SDS-PAGE and analyzed with UV/VIS.
Figure 1: From this sequence alignment, the pink color determine the possibility of ion-ion
bond and hydrogen bond. Through this, it could be assumed that Tp34211 is actually more
stabile than the other protein. Moreover, the the sequence similarity are not reflected on
the later part of the sequence.
Diatoms are a unicellular, eukaryotic phytoplankton that could be found throughout the
world’s oceans and freshwater systems1. They are a major contribution to our global
climate because of their ability to photosynthetic and responsible for our global carbon
fixation,2. At this experiment, the specific diatom that is being studied about is
Thalassiosira pseudonana. As diatoms, T. pseudonana is included as an important part of
diatom carbon fixation, but it is still not known how the Carbon Dioxide could be delivered
to the active site of Rubisco, carbon fixation enzyme. Moreover, it is also identified that
there is a catalyze interconversion between Carbon Dioxide and Rubisco2. However, the
particular T. pseudonana that is analyzed is Tp34211. Tp34211 is a putative monoheme
cythochrome c. Here, the pET86C-Tp34211 is transformed into E.coli. Therefore, in this
experiment the physiological function of Tp34211 will be analyzed. This will prove
whether Tp34211 is acting as a catalyst in this reaction.
In this experiment, it is concluded that Tp34211 could work as a
reaction catalyst. However, it is not a strong catalyst. After
analyzation, it is resulted that Tp34211 is indeed a catalyst.
However, in order to develop this experiment, the next thing that
should be done is to further analyze Tp34211. We could also
relate by determining whether Tp34211 could be one of the
catalyst that affect the interconversion between Carbon Dioxide
and Rubisco in the T. pseudonana.
At first, the DNA sequence (pET22b-Tp34211) was transformed into BL21 (DE3) pEC8b (E. coli). The transformed E. coli was placed into
Ampicillin and Chloramphenicol gel. Then, the expression of Tp34211 was induced from pET22 by inserting the gene into the plasmid vector, and
then it was isolated, purified, cultured (5 min, 200rpm) and harvested by centrifugation.
In the next step, the final result of the purification were combined and purified for the second time using Size Exclusion Chromatography (G-75) with
10mM Tris Base and 25mM NaCl as the equilibration buffer (pH=7.5). Ten fractions were collected and run through SDS-PAGE. The fractions were
quantified using UV/VIS with the equilibration buffer as the baseline. Peroxidase assay is used as the analytical method with buffer (50mM HEPES,
100mM NaCl, at pH=7.5), 30mM ABTS, and H2O2 (0.1mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM). The rate of the final product will be analyzed with
UV/VIS (wavelength 405nm, extinction coefficient of ABTS=36.8M-1cm-1).
The result of the experiment is that there is an increasing rate of
reaction as the concentration of Hydrogen Peroxide is increasing.
Therefore, there is a possibility that the protein could act as a
catalyst. However, as the increasing rate of the reactions are not
very high, it could be assumed that Tp34211 is not a strong catalyst
for the reaction.
Result
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0 1 2 3 4 5 6 7 8 9
Vo
[Hydrogen Peroxide]
Enzyme Catalyzed Reactions
Enzyme Catalyzed Reactions
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Nature 459.185-192
2Armbrust, E. V. The Genome Of the Diatom Thalassiosira
Pseudonana: Ecology, Evolution, and Metabolism.
Science. 2004, 79–86.
3Arslan, E.; Schulz, H.; Zufferey, R.; Künzler, P.; Thöny-
Meyer, L. Overproduction Of TheBradyrhizobium
Japonicum c-Type Cytochrome Subunits of
thecbb3Oxidase InEscherichia Coli. Biochemical
and Biophysical Research Communications. 744–747.
4Wolfe-Simon, F.; Starovoytov, V.; Reinfelder, J. R.;
Schofield, O.; Falkowski, P. G. Localization And
Role of Manganese Superoxide Dismutase in a Marine
Diatom. Plant Physiology. 2006, 1701–1709.