This document summarizes an experiment analyzing the diatom Thalassiosira pseudonana and the protein Tp34211. Tp34211 is a putative monoheme cytochrome c that may act as a catalyst in the reaction between carbon dioxide and Rubisco, an important carbon fixation enzyme. The experiment transformed Tp34211 into E. coli cells and purified the protein. Testing showed that Tp34211 increased the rate of a peroxidase reaction as hydrogen peroxide concentration increased, indicating it may act as a catalyst, although not a strong one. Further analysis of Tp34211's role in carbon dioxide and Rubisco interconversion in T. pseudonana is needed.
SIMONA CAVALU_Rotational Correlation Times of proteinsSimona Cavalu
Noncovalent spin labeled proteins (ovalbumin, bovine serum albumin, hemoglobin, and cytochrome c) were
investigated in order to follow the different type of interactions between the nitroxide radical of 3-carbamoyl-
2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy spin label and functional groups of heme and nonheme proteins as
well as the pH influence on molecular motion of the label with respect to these proteins. EPR spectra were
recorded at room temperature and the computer simulation analysis of spectra was made in order to obtain
the magnetic parameters. Noncovalent labeling of proteins can give valuable information on the magnetic
interaction between the label molecule and the paramagnetic center of the proteins. The relevance of this
interaction can be obtained from line shape analysis: computer simulations for nonheme proteins assume
a Gaussian line shape, whereas for heme proteins, a weighted sum of Lorentzian and Gaussian components
is assumed. In the framework of the “moderate jump diffusion” model for rotational diffusion, the rotational
correlation time is strongly influenced by pH, because of the electrostatic interactions and hydrogen bonding.
SIMONA CAVALU_Rotational Correlation Times of proteinsSimona Cavalu
Noncovalent spin labeled proteins (ovalbumin, bovine serum albumin, hemoglobin, and cytochrome c) were
investigated in order to follow the different type of interactions between the nitroxide radical of 3-carbamoyl-
2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy spin label and functional groups of heme and nonheme proteins as
well as the pH influence on molecular motion of the label with respect to these proteins. EPR spectra were
recorded at room temperature and the computer simulation analysis of spectra was made in order to obtain
the magnetic parameters. Noncovalent labeling of proteins can give valuable information on the magnetic
interaction between the label molecule and the paramagnetic center of the proteins. The relevance of this
interaction can be obtained from line shape analysis: computer simulations for nonheme proteins assume
a Gaussian line shape, whereas for heme proteins, a weighted sum of Lorentzian and Gaussian components
is assumed. In the framework of the “moderate jump diffusion” model for rotational diffusion, the rotational
correlation time is strongly influenced by pH, because of the electrostatic interactions and hydrogen bonding.
Thermally Stimulated Discharge Current study of PMMA:PVP blendsinventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Computational and Experimental Studies of MTO Catalyzed Olefin HydrogenationKaram Idrees
The poster that I presented at the 253rd American Chemical Society National Meeting and Exposition in San Francisco,
CA. It highlights some of my REU research at North Carolina State University under the mentorship of Dr. Elon Ison.
Mechanistic Aspects of Oxidation of P-Bromoacetophen one by Hexacyanoferrate ...IJERA Editor
The kinetics of oxidation of p-bromoacetophenone by hexacyanoferrate (III) has been studied in alkaline
medium. The order of reaction with respect of both acetophenone and hexacynoferrate (III) has been found to be
unity. The rate of reaction increases with increase in the concentration of sodium hydroxide.On addition of
neutral KCl, reaction rate increases. The effects of solvent and temperature have been also studied. The product
p-bromophenyl glyoxal have been characterized by IR studies.
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
Determination of DNA Methylation Using Electrochemiluminescenc.docxkhenry4
Determination of DNA Methylation Using Electrochemiluminescence
with Surface Accumulable Coreactant
Ryoji Kurita,*,† Kumi Arai,† Kohei Nakamoto,†,‡ Dai Kato,† and Osamu Niwa†,‡
†National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki,
Japan 305-8566
‡Graduate School of Pure and Applied Science, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, Japan 305-8573
ABSTRACT: Cytosine methylation in DNA was determined by
an enzyme linked immunosorbent assay (ELISA) with electro-
chemiluminescence (ECL) detection and employed for the DNA
methylation assay of a long and real genomic sample for the first
time. The developed method employed an antimethyl cytosine
antibody labeled with acetylcholinesterase, which was added to
recognize single methylated cytosine in a DNA oligomer. The
acetylcholinesterase converted acetylthiocholine (substrate) to
thiocholine (product), which was accumulated on a gold electrode
surface via gold−thiol binding. This surface accumulated
preconcentration made it possible to observe bright and distinctive
ECL by applying a potential to the gold electrode in the presence
of a tris(2,2-bipyridyl)ruthenium complex luminophore when the
analyte DNA contained a methylation region. Methyl-cytosine was measured quantitatively in the 1−100 pmol range, which
exhibits sufficiently high sensitivity to achieve real DNA measurements without amplification by a polymerase chain reaction
(PCR). The proposed ECL method also exhibited high selectivity for methyl-cytosine against nonmethylated cytosine, guanine,
thymine, and adenine nucleotides. Finally, original and methylated DNA samples were clearly distinguished with our method
using a real DNA bacteriophage sample (48 502 base pairs).
DNA methylation is a well-known epigenetic modificationmechanism that regulates gene expression and plays
crucial roles in embryonic development.1 Cytosine methylation
in CpG islands has received particular attention because it is
thought to be involved in controlling genetic expression,
including that in cancer,2 genomic imprinting,3 cellular
differentiation, and Alzheimer’s disease.4 5-Methyl-cytosine is
now recognized as the fifth DNA base containing heritable
information. Therefore, highly sensitive, accurate, and quanti-
tative information concerning cytosine methylation in DNA
would be valuable with respect to genetic disease diagnosis.
Two major cytosine methylation assay methods have been
reported. One is hydrolysis and sequencing with a bisulfite
salt,5,6 and the other is a cleavage assay with methyl-cytosine
sensitive (or insensitive) restriction enzymes.7 A bisulfite based
determination method is very widely used to distinguish
between cytosine and methyl-cytosine. Treatment with bisulfite
converts cytosine to uracil, while methyl-cytosine remains
unaffected. Therefore, information about methyl-cytosine in
DNA can be obtained by combining bisulfite treatment and a
polymer.
Thermally Stimulated Discharge Current study of PMMA:PVP blendsinventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Computational and Experimental Studies of MTO Catalyzed Olefin HydrogenationKaram Idrees
The poster that I presented at the 253rd American Chemical Society National Meeting and Exposition in San Francisco,
CA. It highlights some of my REU research at North Carolina State University under the mentorship of Dr. Elon Ison.
Mechanistic Aspects of Oxidation of P-Bromoacetophen one by Hexacyanoferrate ...IJERA Editor
The kinetics of oxidation of p-bromoacetophenone by hexacyanoferrate (III) has been studied in alkaline
medium. The order of reaction with respect of both acetophenone and hexacynoferrate (III) has been found to be
unity. The rate of reaction increases with increase in the concentration of sodium hydroxide.On addition of
neutral KCl, reaction rate increases. The effects of solvent and temperature have been also studied. The product
p-bromophenyl glyoxal have been characterized by IR studies.
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
Determination of DNA Methylation Using Electrochemiluminescenc.docxkhenry4
Determination of DNA Methylation Using Electrochemiluminescence
with Surface Accumulable Coreactant
Ryoji Kurita,*,† Kumi Arai,† Kohei Nakamoto,†,‡ Dai Kato,† and Osamu Niwa†,‡
†National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki,
Japan 305-8566
‡Graduate School of Pure and Applied Science, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, Japan 305-8573
ABSTRACT: Cytosine methylation in DNA was determined by
an enzyme linked immunosorbent assay (ELISA) with electro-
chemiluminescence (ECL) detection and employed for the DNA
methylation assay of a long and real genomic sample for the first
time. The developed method employed an antimethyl cytosine
antibody labeled with acetylcholinesterase, which was added to
recognize single methylated cytosine in a DNA oligomer. The
acetylcholinesterase converted acetylthiocholine (substrate) to
thiocholine (product), which was accumulated on a gold electrode
surface via gold−thiol binding. This surface accumulated
preconcentration made it possible to observe bright and distinctive
ECL by applying a potential to the gold electrode in the presence
of a tris(2,2-bipyridyl)ruthenium complex luminophore when the
analyte DNA contained a methylation region. Methyl-cytosine was measured quantitatively in the 1−100 pmol range, which
exhibits sufficiently high sensitivity to achieve real DNA measurements without amplification by a polymerase chain reaction
(PCR). The proposed ECL method also exhibited high selectivity for methyl-cytosine against nonmethylated cytosine, guanine,
thymine, and adenine nucleotides. Finally, original and methylated DNA samples were clearly distinguished with our method
using a real DNA bacteriophage sample (48 502 base pairs).
DNA methylation is a well-known epigenetic modificationmechanism that regulates gene expression and plays
crucial roles in embryonic development.1 Cytosine methylation
in CpG islands has received particular attention because it is
thought to be involved in controlling genetic expression,
including that in cancer,2 genomic imprinting,3 cellular
differentiation, and Alzheimer’s disease.4 5-Methyl-cytosine is
now recognized as the fifth DNA base containing heritable
information. Therefore, highly sensitive, accurate, and quanti-
tative information concerning cytosine methylation in DNA
would be valuable with respect to genetic disease diagnosis.
Two major cytosine methylation assay methods have been
reported. One is hydrolysis and sequencing with a bisulfite
salt,5,6 and the other is a cleavage assay with methyl-cytosine
sensitive (or insensitive) restriction enzymes.7 A bisulfite based
determination method is very widely used to distinguish
between cytosine and methyl-cytosine. Treatment with bisulfite
converts cytosine to uracil, while methyl-cytosine remains
unaffected. Therefore, information about methyl-cytosine in
DNA can be obtained by combining bisulfite treatment and a
polymer.
SIMONA CAVALU_Raman and surface-enhanced Raman spectroscopy of tempyo spin la...Simona Cavalu
Tempyo labelled ovalbumin at different pH values was prepared and investigated using Raman and SERS spectroscopy.
Raman spectra of tempyo labelled ovalbumin in the pH range from 6.7 to 11 were compared to those of the corresponding free
ovalbumin. In the basic pH range from 6.7 to 11 the molecular conformation was found to be unaffected by the tempyo
presence. Adsorption versatility to the colloidal Ag particles of pure- and tempyo labelled ovalbumin was also found to be
unchanged in this basic pH range. As the SERS binding site of protein the a-helix conformation is favourable.
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
1Armburst, E.V. The life of Diatom in The World’s Ocean.
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.