1. Maximizing the extraction efficiency of arsenic in solid matrices followed by
graphite furnace atomic absorption spectrometric analysis.
Mikki Wakeman and Cielito DeRamos King,
Bridgewater State University, Bridgewater, MA 02325
Abstract
Arsenic is an element of concern due to its toxicity even at low
levels and it is a known human carcinogen. Research studies were done to
determine the arsenic content of rice, chicken and shrimp. Previous studies
in our lab involving rice and standard reference rice material showed that
the extraction efficiency of arsenic is very low (40 % or less) using sample
digestion on a hot block. Thus, we need to modify our sample digestion
method in order to increase the extraction efficiency of arsenic in rice,
chicken and shrimp using hot block digestion since we do not have a more
efficient microwave digester. SRM 1568b rice flour and a variety of rice
samples were digested in 0.28 M HNO3 in a closed vessel at 95˚C
following FDA EAM Section 4.1, then analyzed for arsenic using graphite
furnace atomic absorption spectrometry. Results showed that % recovery
of As in rice is still very low, so we shifted our studies to chicken meat and
shrimp to see if we can get better results. Ground chicken meat and shrimp
samples were digested in concentrated HNO3/30 % H2O2 on a hotblock at
95 0C (HNO3) and 65 0C (H2O2) using two variations in sample
preparation: with and without drying the meat. SRM1566b oyster tissue
was used as reference sample. Our data indicates still very low arsenic
recoveries in chicken and shrimp. We plan on refining our techniques and
method to see if we can increase the extraction efficiency of hot block
digestion for both chicken meat and seafood to improve our % recoveries.
Introduction
Arsenic can be found in the environment from both natural
(volcanoes, rocks and minerals, microbial action on soil) and
anthropogenic sources, such as burning of fossil fuels.1 It is also used to
make glass, wood preservatives called chromated copper arsenate (CCA),
and in semiconductor as gallium arsenide.2 People can be exposed to
arsenic from contaminated food, water and air. Because it is a known
carcinogen, arsenic contamination of water and soil has raised global
concern in the past decade. Arsenic exposure is regarded as a major
worldwide environmental health concern and has been referred to as the
“worst chemical in the world”.3 While drinking arsenic-contaminated
water is the main source of human’s exposure to arsenic, chronic exposure
from secondary sources, which has been linked to developmental effects,
cancer, gastrointestinal and respiratory diseases, and neurotoxic
symptoms4,5 must not be overlooked.
The inspiration for this research came from a 2006 article
published by The Institute for Agriculture and Trade Policy titled Playing
Chicken: Avoiding Arsenic in your Meat.6 According to this article,
inorganic arsenic can be found in chicken meat from feed additives such
as Roxarsone.6,7 Their samples of chicken (raw and cooked, bought from
various supermarkets and fast food chains) were cut into pieces, digested
in HNO3/H2O2 on a hot block, then analyzed for arsenic using ICPMS.6
Their results showed varying levels of arsenic in chicken, from below
detection to as high as 21 ppb in raw meat, and 2-46 ppb in cooked meat,6
with all the results showing arsenic levels below the current limit of 500-
2000 ppm As in animal meat treated with veterinary drugs,8 but some are
above the drinking water limit of 10 ppb As.9
The goal of this project was to determine the best extraction
method for the analysis of arsenic in chicken meat and shrimp purchased
from local supermarkets near Bridgewater, MA, using graphite furnace
atomic absorption spectroscopy (GFAAS). The results of this study can be
used to further test other types of food that is commonly consumed that
may contain arsenic. It is important to study arsenic exposure in food to
reduce the chances of cumulative inorganic arsenic exposure from both
food and water, which can lead arsenic poisoning, DNA damage, and
worst, cancer.
Methodology
Sample Preparation
Raw chicken meat and shrimp were purchased from local supermarkets and were kept
frozen in the laboratory until needed.
After thawing, chicken meat were placed in a meat grinder to create a composite sample
while shrimp samples were cut into tiny pieces with a kitchen knife.
Method 1: Raw meat
Around 2 grams of ground chicken or cut-up shrimp and 0.5 grams of NIST oyster tissue
were weighed onto separate 50-mL metals-free plastic centrifuge tubes. Duplicate chicken
and shrimp samples were spiked with 10.00 ppm As to achieve a spike level of 40 ppb
when diluted to a fixed mass following digestion. Five mL of concentrated, trace-metal
grade HNO3 was added to each tube and, after 15-20 minutes, was digested at 95˚C for an
1.5 hours on a ModBlock digester.
Instrumental Analysis
Total arsenic in the digested samples were
measured on a graphite furnace atomic absorption
spectrometer (GFAA) following EPA method
7060A.10 The method detection limit of 2.3 ppb As
was determined earlier using spiked DI water.
Treatment of Data
The % recovery of As in spiked samples
and in the NIST 1566b oyster meat samples were
calculated to determine the accuracy of extraction
method. Precision was measured by obtaining
triplicate readings of each sample on the GFAA.
All five brands of raw and cooked chicken, both red and white meat
purchased from local supermarkets and fast food restaurants and processed through
Method 1 (no drying) showed non-detectable levels of arsenic. Similarly, the three types
of shrimp (one local and 2 imported) processed through Method 1 had < LOD levels of
arsenic. For Method 2 (dried samples), not enough samples were left except for Halal
chicken, local (Key West) and imported (Vietnam) shrimp, which again did not show
detectable levels of arsenic.
The recovery of As in all spiked chicken and shrimp samples was 21.85 % or
less using either Method 1 or Method 2. The low recoveries indicate that some of the
added arsenic in the spiked samples are lost during the digestion process. Although the
loss from effervescence and “boiling over” of raw samples during the initial addition of
concentrated HNO3 can be accounted for the low % recoveries from Method 1,
our % recoveries from Method 2 where the raw samples were first dried only showed
improvement for the NIST 1566b oyster meat sample. Thus, we are still unsure of the
reason for the loss of arsenic during the sample digestion procedure. We can presume
that some of the organic forms of arsenic is loss from volatilization, but we think that our
digestion procedure need to be refined further and done with much more care in order to
improve our % recoveries. We propose to do the initial addition of concentrated HNO3 in
2 to 3 increments, capping the digestion vessel after each incremental addition, and
leaving the mixture in the acid longer prior to heating at 95 0C.
Results and Discussion
Table 1: Percent recoveries of arsenic in chicken, shrimp and standard reference material, NIST 1566b.
Extraction Method 1 (raw
meat)
Extraction Method 2 (oven-
dried meat)
Sample ID
% recovery of spike or As
in NIST
% recovery of spike or As in
NIST
Key West Pink shrimp 21.80 21.85
Mexico jumbo shrimp 16.75 N/A
Vietnam shrimp <LOQ <LOD
Halal chicken, drumstick <LOQ <LOD
NIST 1566b oyster tissue 42.73+ 71.64
+ Low recovery is partly due to loss of sample to effervescence
during initial HNO3 addition
Accuracy using QC sample = 86.7 (method 1) and 84.88 (method 2)
Method LOD= 2.3 ppb As; LOQ= 7.0 ppb As
References
1. Water Treatment Solutions. (2014, January 1). Retrieved March 9, 2015, from
http://www.lenntech.com/periodic/elements/as.html
2. Arsenic Element Facts. (n.d.). Retrieved March 10, 2015, from
http://www.chemicool.com/element/arsenic.html
3. Deborah Blum. Is Arsenic the Worst Chemical in the World?, Wired Science, June 1, 2012,
http://www.wired.com/wiredscience/2012/06/is-arsenic-the-worst-chemical-in-the-
world/
4. Yoshida, T., Yamamuchi, H. and Fan Sun, G. “Chronic health effects in people exposed to
arsenic via the drinking water: dose-response relationships in review.” Toxicol. Appl.
Pharmacol. 2004. 198, 243–252
5. Kozul-Horvath, C., Zandbergenm F., Jackson, B., Enelow, R., and Hamilton, J. “Effects of
Low-Dose Drinking Water Arsenic on Mouse Fetal and Postnatal Growth and
Development.” PLoS ONE. 2012. 7, (5), 1, www.plosone.org.
6. Wallinga, D., M.D. (2006). Playing Chicken: Avoiding Arsenic in Your Meat. Institute for
Agriculture and Trade Policy, 1-33.
7. U.S. Food and Drug Administration. (2014, July 3). Retrieved March 10, 2015, from
http://www.fda.gov/AnimalVeterinary/SafetyHealth/ProductSafetyInformation/ucm2583
13.htm
8. Agency for Toxic Substances and Disease Registry (ATSDR). “Arsenic Toxicity.” Available at
http://www.atsdr.cdc.gov/csem/csem.asp?csem=1&po=8 (Accessed March 2015)
9. USEPA. “Arsenic in Drinking Water.” Available at
http://water.epa.gov/lawsregs/rulesregs/sdwa/arsenic/index.cfm (Accessed March 2015)
10.US Environmental Protection Agency SW-846 Method 7060A. 1994. “Arsenic (Atomic
Absorption Furnace Technique),” Test Methods for Evaluating Solid Waste,
Physical/Chemical Methods, Third Edition, Update II. http://www.caslab.com/EPA-
Methods/PDF/EPA-Method-7060A.pdf
Acknowledgements
Without support from the following groups, this research would not be possible.
• Bridgewater State University through the Adrian Tinsley Program for Undergraduate
Research for funding
• Department of Chemical Sciences for funding
• Dr. Cielito King, my mentor, for helping me take on this research
Method 2: Dried meat
The same procedure as Method 1 above was followed
except that the ground (chicken) or cut-up (shrimp)
samples were weighed onto acid-washed evaporating
dish and dried in a 40 0C oven for 2 days prior to
analysis. Another modification was volume fractions
were used instead of mass fractions during the final
process of dilution prior to GFAA analysis.
After cooling for 30 min, 2 mL of 30 % H2O2 was
added to each tube to solubilize any remaining
organic matter, followed by heating at 65˚C for 20
min. The previous step was repeated once,
followed by final heating to 90˚C for 30 minutes.
After cooling, the final mass was brought to 20.0 g
with deionized water, transferred to a clean, 50-mL
metals-free plastic centrifuge tube, and centrifuged
for 15 minutes at 2800 rpm prior to GFAA analysis
for total arsenic.
Figure 1. Dried shrimp meat prior
to digestion
Figure 2. Shrimp and chicken
samples after digestion
Figure 3. Perkin-Elmer PinAAcle 900T
GFAA spectrometer with AS-80
autosampler

2. Galactosemia type III UDP-galactose-4-epimerase
Mikki Wakeman
A Biochemistry II Course Project, Instructor: Dr. Emily Garcia Sega
Department of Chemistry, Bartlett College of Science and Mathematics, Bridgewater State
University
Abstract
Introduction
References
• Timson, D. J. (2006). The Structural and Molecilar Biology of type 3 Galactosemia.
IUBMB Life, 83-
• Dobrowolski, S. F., Banas, R. A., Suzaw, J. G., Berkley, M., & Naylor, E. W. (2003,
February). Analysis of Common Mutations in the Galactose-1-Phosphate Uridyl
Transferase Gene. Journal of Molecular Diagnostics, 5(1), 42-47.
• Fridovich-Kell, J., Bean, L., He, M., & Schroer, R. (2013, October 24). Epimerase
Deficiency Galactosemia. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK51671/
• Lai, K., Willis, A. C., & Elsas, L. J. (1999). The Biochemical Role of Glutamine 188 in
Human Galactose-1-phosphate Uridultransferase. Journal of Biological Chemistry,
274(10), 6559-6566.
Acknowledgements
Without support from the following groups, this research would not be possible.
• Bridgewater State University through the Adrian Tinsley Program for
Undergraduate Research for funding
Galactosemia is a disease that prevents the making of
glucose from galactose. The mutation that leads to this disease is a
recessive trait that can be inherited. When galactose builds up in the
body to toxic levels which could lead to vomiting, low blood sugar,
brain damage and worst case would be death. There are three types
of galactosemia where three different enzymes are to blame for the
disorder. For type 3 galactosemia uridyl diphosphogalactose is the
enzyme responsible for the inability to turn galactose into glucose.
Galactosemia type 3 is the rarest form with a wide range
of severity. A person with the lesser form of the disease will hardly
have any symptoms and can enjoy a normal diet. Those that have the
more advanced form of the disease can experience vomiting,
diarrhea, fatigue and low blood sugar like experienced in type 1
galactosemia. Those with the most severe form have neurological
disorders such as seizures.
Figure 1
• Two forms of (GALE) one that
affects the blood cells and has
no symptom
• The peripheral form of GALE
activity is reduced in the blood
but normal in other tissues. The
symptoms have altered levels of
blood galactose and galactose-
1-phosphate.
• The severe case (rare) can lead
to brain damage, cataracts,
enlarged liver, vomiting and
sometimes death.
• Treatable with some side affects
• Mutation occur in GALE gene
altering the ability to convert
UDP-galactose to UDP-glucose
• Galactosemia affects newborns from all ethnic groups but is most
commonly detected in Ireland.
• 1 in 24,000 newborns are born with this disorder.
• 1 in every 30,000- 60,000 newborns in the US are born with
galactosemia.
• Enables the body use galactose and make glucose
• Galactose is a part of lactose
• Tested positive for GALT and is given a lactose restricted diet to see if the
affects are changed
• The loss of activity of GALE can lead to accumulations of glucose or
galactose can lead to cataracts, intellectual disability, and damage to the
liver, kidneys, and brain and some cases premature death.
• Galactose eliminated from diet  build up of toxic intermediate
galactose-1-phosphate
Figure 3. structure of UDP-galactose 4- epimerase
• Dimer with binding sites in each subunit
• Contains NAD+ tightly bound, required for catalytic activity
• Sugar held less tightly which allows it to move around the active site
• Mutations occur in enzyme UDP-galactose-4-epimerase
• Substrate binding in each sub unit of dimer
• Converts UDP-galactose to UDP-glucose in presence of NAD+
• 22 known mutations in UDP-galactose-4-epimerase
Biological significance
Figure 1. Galactose Pathway
Figure 2. The Leloir Pathway.
Treatment
• Restrict the dietary intake of galactose
• Small amounts of galactose needed for type 3 for the biosynthesis of UDP-
galactose
• Gene therapy to enable partial restoration of GALE activity
• Replacement of GALE gene
• re-engineering other pathways of galactose pathway (improve quality and
quantity of life)
• Problems with gene therapy in small number of patients suggest there is no solution
in the near future for galactosemia
• Inhibition of GALK 1 therapeutic strategy
Summary
Galactosemia is a rare metabolic disorder that affects the production of
glucose from galactose. It is an inherited recessive disorder that affects 1 in 30,000-
60,000 newborns in the US and 1 in 24,000 in newborns in Ireland. The GALE
activity is affected in the Leloir pathway of glucose metabolism. The Leloir pathway is
needed to convert galactose to the more stable and metabolically useful glucose-1-
phosphate. Some symptoms include reduced blood activity, it can lead to early onset
cataracts, intellectual disability, and damage to the liver, kidneys, and neurological
disorders. There are some treatments depending on the severity of the disorder, the
most common is to restrict the intake of galactose, gene therapy to enable the
restoration of GALE activity. The inhibition of the GALK 1 is a possible therapeutic
strategy. Re-engineering the pathways of GALE activity can increase the quantity or
quality of life. Another possibly treatment depending on the severity is a
reconfiguration of the galactose pathway. In the patients that don’t respond to gene
therapy and any other treatment there is no solution to the disorder in the near future.
This is only in a small number of patients that can be categorized to no future solution.

3. Investigation into the lesion bypass activity of human DNA polymerase
kappa following a mutation of the residue D109N in the active site.
Mikki Wakeman; Kayla Ruggiero; Dr. Samer Lone
Bridgewater State University, Bridgewater, MA 02324
Acknowledgements
I would like to thank Dr. Samer Lone helping me do this research
Abstract
Polymerase kappa is a member of the Y family polymerases. To better understand
the structure and function of pol kappa, a mutation was made by changing a single
amino acid. Residue D109N located in the active site of the palm domain was mutated
from the negatively charged aspartic acid to the hydrophilic asparagine. This region is in
particular interest because it is in close proximity to D107, D198 and E199, all of which
catalyze the nucleotidyl transfer reaction in the active site. The residues differ in that
aspartic acid has a negatively charged OH group and asparagine has a NH2 group
making asparagine neutral and hydrophilic. This mutation may inhibit or eliminate the
interactions necessary to catalyze the transfer reactions. To determine if these changes
occur, site directed mutagenesis, protein purification and extraction, and DNA
polymerase assays will be carried out. A mutation in the active site residue 109 should
eliminate all nucleotide transfer reactions.
Introduction
Polymerase kappa is a member of the Y-family DNA polymerases. Pol kappa is
thought to function in translesion synthesis and act as an extender through DNA lesions.
The full length of pol k is 1-870 amino acids long but only the residues from 19-526
shows catalytic activity. The other remaining amino acids may be involved in
interactions with other proteins. Pol k consists of four domains and the N-clasp where
each domain has a different function. The palm domain contains the active site and is
involved in the transfer of nucleotides. Finger domain is located between the active site
and the new nucleotides. The thumb is located near the minor groove surface of the
DNA. The PAD domain provides stability by holding onto the DNA and is larger than
some of the other polymerases. The N-clasp stabilizes the other domains around the
DNA.
Methods
Site-directed mutagenesis
Site directed mutagenesis is accomplished by obtaining the QuickChange II kit. This
kit works by altering a plasmid that contains the gene sequence for pol k. In order to
change the template DNA, primers were designed with a specific mutation to create
the desired change. The primers were ordered from Integrated DNA Technologies
and used in PCR for DNA amplification. The plasmid DNA created contains the
desired change in amino acids. The plasmid is then amplified using the restriction
enzyme DpnI then transformed into XL-1 blue supercompitent cells and was
amplified in LB/amp media overnight.
Aspartic Acid Asparagine
Expression and Purification of pol k
Human DNA pol k is synthesized. The plasmid that contains the gene for pol k is
incorporated into E.coli bacterial cells. Following site- directed mutagenesis the DNA
was purified and transformed into BL-21 gold supercompetent cells that were then
grown up in LB media, then were grown with the presence of IPTG to initiate
transcription. Pol k was purified by extracting from bacterial cells by sonicating then
centrifuging to separate from the remaining cell debris. Final purification of pol k was
completed by using GST-chromatography where the GST tag was used
to further purify the protein from the other components where only kappa was eluded.
To determine if the protein was purified a SDS-PAGE was carried out and a Bradford
assay to determine how much was made.
DNA Polymerase Assay
DNA polymerase assay was carried out to determine the functionality of the mutated pol
k. There were four assays carried out with both normal and mismatched DNA with both
wild type polymerase and mutated D109N polymerase. The template used for the wild
type polymerase was GATTC and for the mutated D109N polymerase was CGTTCT.
Results
Figure 1. SDS-PAGE of protein purification. The first half of the gel is D109N.
Lane 1 and 12 are the mw ladder lanes 2-6 are flow through, high salt, low salt, protein
and glutathione.
y = 0.0371x + 0.0246
R² = 0.9859
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 2 4 6 8 10 12
Absorbance
µl BSA
Figure 2. Results of the Bradford Assay. Standard curve used to calculate the protein concentration of
D109N
ALL A T G C ALL A T G C
ALL A T G C ALL A T G C
WT
D109N
NORMAL
NORMAL
MISSMATCH
MISSMATCH
Figure 3. DNA Polymerase gel. Gel of both normal and mismatched DNA interacting with wild
type polymerase kappa.
Figure 4. DNA polymerase gel. The gel of both normal and mismatched DNA interacting with
D109N mutated pol kappa.
References
• Lone, S., Townson, S., Uljon, S., Johnson, R., Brahma, A., Nair, D., Aggarwal, A.
(n.d.). Human DNA Polymerase κ Encircles DNA: Implications for Mismatch
Extension and Lesion Bypass. Molecular Cell, 601-614.
Discussion
Polymerase kappa is a member of the Y family polymerases that was
mutated in the active site that is located in the palm domain. Residue D109N was
mutated from aspartic acid to asparagine. This mutation being close to the residues
that catalyze the nucleotidyl transfer reactions is supposed to either inhibit or
eliminate all activity of the polymerase on mismatched DNA. Running the SDS-
PAGE determined that the protein was purified from all other cell debris (figure 1).
Using the standard curve (figure 2) from the Bradford assay it was determined that
there is a concentration of 3.213mg/ml of the protein that was collected from the ion
exchange and size exclusion chromatography. After obtaining the concentration of
the protein it was used in the DNA polymerase assay.
The DNA polymerase assay was run to test the lesion bypass activity of
polymerase kappa after the mutation was carried out and the ability to replicate over
mismatched DNA. The wild type polymerase kappa used DNA template that read
GATTC. For normal DNA the polymerase had was able to insert adenine across
from adenine on the DNA template. The correct base of thymine was also inserted
across from adenine, the same with guanine (figure 3). The mismatched DNA, the
wild type pol kappa was able to insert all bases across from the DNA template. The
mutated D109N polymerase kappa used DNA template CGTTCT (figure 4). The
mutation in polymerase kappa seemed to eliminate all replication activity on both
the normal and mismatched DNA. The lack of base insertions in the mutated
polymerase proves the initial hypothesis that mutations done in the active site will
eliminate all replication activity. There was some pipetting issues during the DNA
polymerase assay leading to the bands being hard to visualize in both the wild type
and D109N sequencing gels. There are a few more steps that can be done before
sequencing the polymerase to determine the folding and functionality of pol kappa.
The mutation of residue D109N in the active site is supposed to inhibit or eliminate
the nucleotidyl transfer activity of the polymerase. Based on the gels being partially
visible the inhibition of all activity was completed.
Palm
Thumb
N-Clasp
Fingers
PAD