Project

NORTHUMBRIA UNIVERSITY
DEVELOPING AN IN VITRO
LIPOYLATION REACTION TO
SCREEN A PANEL OF CHEMICALS
HYPOTHESISED TO TRIGGER
AUTOIMMUNE PRIMARY BILIARY
CIRRHOSIS.
A Project report submitted in partial fulfilment of the requirements for the
Degree BSc (Hons) in Biomedical Science.
By Katie Montgomery
W12032193
1/1/2015
Faculty of Health and Life Sciences
Department of Biomedical Sciences
Northumbria University
I Katie Montgomery confirm that I have read and understood the University
regulations concerning plagiarism and that the work contained within this
project report is my own work within the meaning of the regulations.

Abstract
Primary Biliary Cirrhosis is a chronic inflammatory, autoimmune disease. Characteristically
damages to biliary epithelial cells, and subsequently portal tract inflammation, are highly
associated with PBC. As a result of these damages, fibrosis, cirrhosis and ultimately liver
failure occur. There is a general consensus that PBC arises from a combination of genetic and
environmental factors. This study aims to focus on particular environmental triggers of
autoimmunity, specifically xenobiotics, hypothesised to substitute lipoic acid on the E2
subunit of pyruvate dehydrogenase. Molecular mimicry is the mechanism which these
xenobiotics use to modify the lipoyl domain of PDC-E2, thereby making it highly
recognisable as a neo-antigen to antimitochondrial antibodies (AMA). AMAs directed against
the modified domain are detected in 95% of patients, and recognise these more so than native
PDC-E2, prompting cross-reactivity of the neo-antigen and subsequent breakdown of self
tolerance, thus creating an autoimmune response. The current study aimed to optimise the
lipoylation reaction in order to substitute a panel of xenobiotics commonly found near waste-
sites, into the reaction to evaluate if they were capable of using molecular mimicry to induce a
PBC-like autoimmune response. A series of optimisation reactions were carried out, altering
amounts of lipoic acid, GTP/ATP, or the recombinant enzyme mix constituting LAE and LT.
Little success was gained from these investigations, with few conclusions to draw. Time did
not allow for substitution of the xenobiotics, thus further work is required. Conclusion: Both
LAE and LT are required for lipoylation to occur, however varying the amount does not have
a great effect on lipoylation. This prompted the question of the recombination and purification
of the enzymes, and hinted in the direction of future studies.
Word Count: 275

Introduction
Primary Biliary Cirrhosis (PBC) is a chronic inflammatory autoimmune disease of the liver,
characterised by the progressive destruction of intrahepatic bile ducts and the biliary epithelial
cells lining them. This gradual damage, without treatment, leads to chronic cholestasis, and
fatally fibrosis, cirrhosis and liver failure over a period of between 10-20 years. The name
PBC was used by Ahrens and colleagues in 1950 to describe a disease presenting with
jaundice, pruritus and hepatosplenomegaly, in middle aged women. (Ahrens et al., 1950).
Following this, anti-mitochondrial antibody (AMA) was discovered to be the disease specific
hallmark, in 1965 by Walker and colleagues, and has since then permitted earlier and assured
diagnosis for patients. (Walker et al., 1965). Together with serological techniques, increased
awareness of the condition allows for enhanced diagnosis, thereby improving the prognosis
for the patient. (Jones, 2007).
A high female predominance is typical of most autoimmune diseases, but PBC in particular
sees a staggering 90% of patients being female, with the majority diagnosed after the age of
40. (Smyk et al., 2012). The exact reason for this is unknown but speculations include the
immune system becoming less self-tolerant with age, along with an increased risk of exposure
to disease triggers such as environmental chemicals, recurrent UTI infections, and with
women in particular 2-octynoic acid (found in cosmetics). (Smyl et al., 2011).
There is now a broad consensus that PBC arises from a combination of environmental triggers
within a genetically susceptible individual. The link between environmental factors and PBC
can be demonstrated by looking at the geographical locations of high frequency of the disease.
One such ‘hot spot’ is found in the North East of England, renowned for its coal mining fame,
Prince et al noted 32.2 new cases per million, both men and women, with a case density of

7.1/km2. (Prince et al., 2001).. Similarly a study in New York found a high proportion of the
disease in areas of highly toxic federal waste. Another hot spot identified early on (1980) by
Triger, showed Sheffield had a high prevalence of PBC, 88% of these patients were supplied
with water from the same reservoir. No real evidence could be drawn implementing the water
source, but PBC clustering was certainly questioned. (Prince et al., 2001). Smyk et al cover
findings of PBC clustering in areas of high industrialisation and toxic waste extensively.
(Smyk et al., 2010). Together these findings propose a potential chemical environmental
cause of PBC, either by a direct toxic effect, or a simple trigger. (Jones, 2007). These
chemicals, namely xenobiotics are defined as a substance not normally produced but found in
an organism, for example environmental pollutants or drugs. There is a site situated in the
North East of England in close proximity to a refuse site, with a cluster of PBC case. Personal
communication with Dr. Walden states researchers are interested in chemicals released from
this site and their possible involvement in PBC cases.
As most xenobiotics are metabolised in the liver, the burden falls on this organ to bear.
Hepatic enzymes metabolise xenobiotics by activating, and conjugating a secondary
metabolite with glucaronic acid or glutathione for their excretion with bile or urine. However
some xenobiotics are resistant to degradation and so can accumulate within the organism,
worsening the condition. (Long & Gershwin, 2002).
In the case of PBC, xenobiotics have the capacity to either change or complex to self proteins
and are capable of inducing molecular mimicry through cross reactivity. Molecular mimicry is
defined as a foreign antigen sharing sequential or structural similarities with self-antigens,
producing cross reactive antibodies able to recognise self HLA. These can then activate CD4+
or CD8+ cells on presentation to MHC, and hence sustain a proinflammatory response.
(Cusick et al., 2012). Particular xenobiotics are hypothesised to substitute lipoic acid residues

on PBC-specific autoepitopes, creating cross-reactive neo-antigens, and loss of self-tolerance
thereby causing autoimmunity. (Selmi et al., 2009).
PBC is identifiable serologically by the presence of AMA (anti mitochondrial antibody) to the
lipoyl domain of the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). This is a
member of the 2-oxo-axid dehydrogenase complex family, along with the E2 subunit of the
branched chain 2-oxoacid dehydrogenase complex (BCOADC-E2), the E2 subunit of the 2-
oxogltarate dehydrogenase complex (OGDC-E2), and the dihydrolipoamide dehydrogenase
binding protein (E2BP). Together these form the target antigens of both AMA and
autoreactive T cells, and all contain a lipoate binding site in the form of immunodominant
epitopes. (Long & Gershwin, 2002). Collectively these target antigens are found on the inner
mitochondrial matrix of cells. (Poupon, 2010). In 95% of PBC patients, serum AMA is a
definitive hallmark of the disease, and presents well before the onset of symptoms.
Lipoic acid is bound to components of the PDC-E2, PDC-E3BP and 2-OADC-E2 through an
amide linkage, this is formed between its carboxylic acid group and E-amino group of a
specific lysine residue, and functions as a fundamental carrier between active sites of the 2-
OADC enzyme complexes. (Walden et al., 2008). Lipoylation is an essential mechanism for
metabolism in eukaryotes and involves the integration of lipoic acid into suitable 2-OADC
components via lipoate activating enzyme (LAE) and lipoyltransferase (LT). (Posner et al.,
2013). This lipoic acid is derived in vivo from dietary sources, and enters the exogenous
lipoylation pathway. Alternatively lipoic acid synthase is used to generate lipoic acid for the
endogenous pathway. (Morikawa et al., 2001). See Figure 1.

Figure 1. Lipoic Acid Metabolism Pathways
Figure 1. Lipoylation pathways. The synthesis pathway,also called the endogenouspathway,shows
the transfer of an octanoyl chain froman octanoyl-ACP(acyl carrier protein) to the correct LCP
(lipoyl carrier protein)catalysed by octanoyl-ACP protein transferase, in E.Coli; LipB. LipA then
catalyses the insertion of two sulphur atoms to create lipoic acid in its cofactor form, lipoamide.(Patel
& Packer,2008).The second pathway (salvage) termed the exogenous pathway, involvesthe
conversion of lipoic acid as a cofactor,into the E2 subunit of 2-OADHcomplex.Here lipoate is
conjugated to GMP by lipoate activating enzyme (LAE), specifically MACS1, a GTP dependant LAE
which generates the substrate for lipoyltransferase, and is commonly found in the liver.(Nextprot,
2011). Source: http://mmbr.asm.org/content/74/2/200.full
Xenobiotic modification becomes a possible trigger for PBC when it modifies the PDC-E2 by
molecular mimicry. Within PBC, immune reactivity is seen against the lipoic acid binding
domain of the 2-oxo-acid dehydrogenase, forming the core of the dominant autoepitope.
(Walden et al., 2008). This has a highly conserved domain structure which contains a lipoic
acid factor. Structurally related xenobiotics target the exposed lipoic acid on the binding
domain hence creating immunological cross reactivity towards native antigens. Together with
antibodies produced against the neoantigen, tolerance begins to breakdown.. (Chen et al.,
2013). Self tolerance refers to the immune system recognising a self antigen and mounting an

immune response against it, (Ghaffar & Naggarkatti, 2010), particularly a primary IgM-
specific response. Hence terming PBC an autoimmune disease as the loss of self tolerance
increases.
In this current set of experiments, the aim was to optimise the lipoylation reaction (similar to
that previously performed by Walden et al) by altering amounts of variables, in particular a
new batch of lipoylation enzymes, to enhance lipoylation for the addition of a selection of
chemicals hypothesised to take the place of lipoic acid. Variables amended included
lipoylation enzyme mix (V10), unlipoylated domain (PDC-E2-IlD U-lip), lipoic acid, and the
energy source, ATP or GTP. Of high importance are the lipoylation enzymes LAE and LT,
recombinant versions of these have been produced in large quantities using E.Coli as the
plasma vector. Both enzymes are theorized to be capable of ‘substrate promiscuity’ (Walden,
2008), allowing xenobiotics to bind to the lipoyl domain peptide of the 2-OADC family,
promoting the formation of a neo-antigen, and the development of autoimmune PBC. This
study is of great importance in demonstrating this mechanism.
Word Count: 1176

Materials and Methods
1 .Preparation of Materials
1. 1. Plasmid Construction & Expression and Purification of Recombinant Protein.
Recombinant lipoylation enzymes and PDC-E2-ILD (U-Lip) were provided and prepared as
described by Walden et al., 2008.
1.2 PAGE Gel Components and Materials
1.2 i) Buffers for Non-denaturing gels. Buffer B (Running Gel). 45.4g of Tris/HCl was added
to 250ml of distilled water, pH checked and adjusted to be 8.8. Buffer D (Stacking Gel).
6.055g of Tris/HCl was added to 100ml of distilled water, pH checked and adjusted to be 6.8.
Both buffers were sterile filtered with a 0.45µl syringe filter and stored in the fridge at 4°C.
1.2 ii) 15% Non-denaturing SDS-PAGE Gel Components. Running Gel. 5.7ml H2O were
added to 12.5ml 30% Acrylamide, 6.3ml Buffer B, 250µl 10% APS and 12.5µl TEMED.
Stacking Gel. 8.9ml H2O were added to 2.0ml 30% Acrylamide, 3.8ml Buffer D, 150 µl 10%
APS and 7.5µl TEMED. Once gels had been polymerised and set in the glass slides, they
were wrapped in damp tissue and left in the fridge and used within two weeks.
1.2 iii) 10x Tank Buffer. 144.2g of glycine (Melford Laboratories, Ipswich, UK) was added
to 30.3g Tris (Melford Laboratories, Ipswich, UK), and was made up to 1L with distilled
H2O. pH was checked to be 8.3, and the solution was diluted 1/10.
1.2 iv) Loading Buffer. 100ml was prepared by mixing 1.5g Tris, 10ml glycerol (Sigma-
Aldrich Company, Dorset, UK), 90ml distilled H2O and 1mg Bromophenol Blue (Sigma-
Aldrich Company, Dorset, UK).
1.2 v) 10x PBS. 80g of 137mM NaCl, 2g of 2.7mM KCl, 14.4g 10mM Na2HPO4 and 2.7g of
2mM KH2PO4 (all Sigma-Aldrich) were made up with 800ml distilled H2O, pH was checked

and adjusted to be 7.4, and the remaining volume was added. This was diluted 1:100 with
distilled H2O before use.
2. Lipoylation Reactions.
2.1. Lipoylation reaction components. Initial reactions were made up to the same as
published by Walden et al. and then subsequently individual components, lipoic acid, V10
and U-Lip, were altered in an attempt to optimise the response. Final concentrations
included; 40mM potassium phosphate, 0.3mg/ml BSA, 20mM Tris/HCl, 4mM GTP/ATP.
2.2. Primarily, the lipoylation reaction contained core components; 40mM potassium
phosphate, BSA (0.3mg/mL), 4mM MgCl2, 20mM Tris/HCl, 4mM ATP/GTP, with the
addition and variation of the amount of the lipoylation substrate lipoic acid, the enzyme
complex (v10) 2.8mg/ml, and the unlipoylated domain (PDC-E2-ILD ULip) at 37mg/ml. The
first assay contained the above constituents and differed between GTP or ATP as the energy
source, along with a varying enzyme (V10) volumes concentratio from 0µl to 4µl. The
reactions were left for six hours in a water bath to incubate at 37ºC.
2.3. Secondary to the above assay, further lipoylation reactions were induced using the core
components and changing three elements. Four reactions were set up, the first contained 5µl
of BSA, Tris/HCl, potassium phosphate, MgCl2, ATP, lipoic acid and v10 enzyme complex,
and 0.3µl ULip. The second included the same components but differed with no v10 enzyme,
the third comprised of the original elements but double the amount of ATP/GTP (10µl), and
the fourth contained double the volume of lipoic acid (10µl). Again these were left to run for
six hours at 37ºC.

2.4. To further optimise the lipoylation assay, altered amounts of v10 and ULip were adjusted
to try and get a higher proportion lipoylated. This assay included all the core components, the
six reactions contained varying v10 volumes; 0,4 and 8µl, while the first three used 10µg
ULip, and the last three 7µg. Assays were left for six hours at 37ºC.
2.5. Results were becoming difficult to obtain, so it was hypothesised the components were at
fault. Fresh batches of all assays were made up, including all buffers and GTP. Lipoylation
was tried again. All tubes contained the core ingredients, and differed in v10, lipoic acid and
GTP. The first three included 3µl V10 and 4mM GTP, and lipoic acid at 0mM, 1mM and
100mM. The second three had constant GTP at 4mM and lipoic acid at 100mM, but V10 at
0µl, 3µl and 6µl. The final three included constant V10 (3µl) and lipoic acid (100mM), and
varied with 0mM, 4mM and 8mM GTP.
2.6. The lipoic acid stock remained to be changed to fresh, new lipoylation assays were set up
using the new lipoic acid. All reaction contained the same core components but the first four
reactions contained 3µl of V10 and a range of lipoic acid; 0µM, 10µm, 100µm and 1000µM.
The last three contained 6µl V10, and the same range of lipoic acid, without 0µM.
3. Electrophoresis Analysis. All the assays were analysed electrophoretically using 15% non-
denaturing SDS-PAGE gels. 0.75mm gels were cast using a BioRad vertical electrophoresis
system. All components from 1.2 ii) were introduced together on a containment tray, TEMED
was added last as it is the setting agent that polymerises the gels. The running gel solution was
added in-between the glass slides to set. After approximately twenty minutes, the stacking gel
solution was pipetted on top of the running gel, and the comb was added to set the wells. This
was again left to set, before removing the comb and adding the samples to the individual

wells. Before addition, 25µl loading buffer was included in all the samples (1.2 iv), and they
were then centrifuged at 30000 x g for 10 seconds. The gels were mounted in the
electrophoresis equipment, and 10µl of each sample were aliquoted into each well. Constant
amplitude was applied to the system, 0.35A per gel. The gels were left to run for
approximately 1 hour 45 minutes, until the proteins moved through to the bottom. After
removal from the equipment and careful separation of the slides, the gels were submerged in
Coomassie Blue (Sigma-Aldrich Company, Dorset, UK) overnight, followed by PBS solution
to enhance staining.
4. Immunoblotting Analysis. All samples were subject to a Western Blot after electrophoresis
analysis. The polyacramide gels, along with filter pads and Immobilon-P nitrocellulose
membrane (Millipore, Herts, UK) (cut to size), were immersed in 10% CAPS solution
(transfer buffer) . Using a semi-dry press, the proteins from the gels were transferred
electrophoretically to the nitrocellulose membrane at 20V for 20 minutes?. This membrane
was blocked with 5% (weight/volume) skimmed milk powder in PBS for 1 hour, and then
washed thoroughly with PBS/Tween (1% vol/vol). Affinity-purified patient antibody in
dilutions of 1/10,000 to 1/5000 from a pooled serum of PBC patients) was added to the
membranes and incubated for 2 hours at room temperature. Membranes were thoroughly
washed in 1% PBS/Tween and 4µl of a 1/5000 of secondary anti-human IgG secondary
antibody-conjugated with Horse Radish Peroxidase (Sigma Aldrich) was added. Membranes
were again left to incubate for 2 hours at room temperature before being extensively washed
in 1% PBS/Tween. Peroxide reactivity and bound antibodies were to be detected using
enhanced chemiluminescence, with two reagents; Peroxide Solution and Luminol Enhancer
Solution. (Thermo Scientific, UK).
Word Count: 1149

Results
The lipoylation reaction, in particular, the incorporation of lipoic acid, requires an energy
source, in the form of GTP or ATP, it is known that GTP is the favoured source within the
mitochondria (Fujiwara et al., 2001), thus these were they first set of variables changed in the
optimisation series. From figure 2 (A) it can be seen that with an increasing enzyme
concentration, more domain becomes lipoylated, as demonstrated with darker bands. (B) only
shows very faint bands across the middle of gel, prompting a range of possible explanations.
1. Competitive use of GTP or ATP and the effect of a varying enzyme (V10) concentration.
Figure 2.
B.
ATP
4µl
GTP
unlipoylated
lipoylated
unlipoylated
A.
3µl2µl1µl0µl
0µl 1µl 2µl 4µl3µl

2. Comparison of three independent variables within In Vitro Lipoylation. To advance the
optimisation of the lipoylation reaction, three variables were altered in three separate
reactions, along with a standard control reaction. Figure 3 shows these results after
electrophoresis and subsequent staining. It can be seen lane 5, GTP 4, and lane 6, ATP 1,
show some domain become lipoylated. After collating the initial two lipoylation reactions
(above) together with this one, immunoblotting analysis was performed. A western blot was
done with all three gels, and figure 3 shows the products, with very little to describe as being
‘reliable’.
Figure 3.
Figure 3. (A) SDS-PAGE Electrophoresis gel stained with Coomassie Blue. Lane 1
contains ULip alone, lanes 2, 3, 4 & 5 contain the GTP reactions (GTP 1/2/3/4), while
lanes 6-9 contain ATP reactions (ATP 1/2/3/4). The first of both sets of reactions is the
standard control consisting of 5µl BSA, Tris/HCl, potassium phosphate, MgCl2, 0.3µl
ULip 37mg/ml, and 5µl ATP/GTP, 5µl V10 enzyme 2.8mg/ml, and 5µl lipoic acid. The
second excludes the use of V10 enzyme mix, the third increases the amount of
ATP/GTP by double, and the fourth doubles the amount of lipoic acid.
Figure 2. SDS PAGE Electrophoresis gels stained with Coomassie Blue. Both figures show
increasing concentrations from 0-4µl of V10, along with the use of constant ATP or GTP
as the energy source. (A) Here demonstrates 4mM ATP as the energy source, the first lane
contains the Unlipoylated domain (ULip) only. Subsequent lanes include 4mM ATP along
with 0µl (lane 2), 1µl (lane 3), 2µ (lane 4)l, 3µl (lane 5) and 4µl (lane 6) respectively. (B)
The same lipoylation reaction is shown but with 4mM GTP.
1 2 3 4 5 6 7 8 9
lipoylated
unlipoylated

3. Altered Amounts of V10 Enzyme Mix and Unlipoylated Domain (PDC-E2-ILD ULip). As
GTP is known to be a more efficient energy source than ATP, GTP was the choice to be used
in subsequent optimisation reactions. We went on to look how varying the amount of ULip
along with V10 had an influence on the reaction, with the hope of achieving a higher
proportion being lipoylated.
A.
B.
Figure 4.
Figure 4. Western Blot Assays of the three previously mentioned gels. There is very little to
no visible protein. Repeated attempts were made to re-apply the reagents, and re-run the
chemiluminescence, as can be seen from the above photographs. As the gels illustrated
some domain had become lipoylated, the error is most likely to lie within the transfer from
the gels to the nitrocellulose membrane.
Figure 5.
1 2 3 4 5 6 7
lipoylated
unlipoylated

As can be derived from figure 5, increasing the amount of V10 did not have a great effect on
the amount of domain lipoylated, this is seen in lanes 3 and 4. Both contain 10µg ULip but 3
has 4µl of V10 while 4 has 8µl, no clear difference can be seen between the bands of
lipoylated domain. However the difference between lane 2 and lane 3 is obvious in that some
enzyme is required for lipoylation to occur. Another point inferred from this particular assay
is the use of lower amounts of ULip. Lanes 5-7 contain 7µg of ULip, and as can be seen it is
difficult to visualise the bands. Thus in future experiments, 10µg was to be the standard
amount of ULip used.
4. Lipoylation from Fresh Reaction Series. All components of the exogenous lipoylation
reaction had been made up entirely new, excluding lipoic acid. In particular focus were new
buffers and GTP stocks. Variables changed within this new series of reactions included; V10
enzyme mix, lipoic acid concentrations ranging from 0-100mM, and GTP concentration, in
addition a lipoylated domain marker was used in place of an unlipoylated domain marker.
Figure 6 (A) presents the results of the SDS-PAGE Electrophoresis gel.
Figure 5. Altered amounts of 37mg/mg ULip and 2.8mg/ml V10 from a SDS-PAGE
Electrophoresis gel stained with Coomassie Blue. Lane 1 contains the marker
unlipoylated domain alone. Lanes 2-4 include 10µg ULip while lanes 5-7 hold 7µg.
Respectively, amounts of V10 increased from 0µl to 4µl to 8µl, in lanes 2,3,4 and again
in 5,6,7.

Figure 6 reveals all reactions which contained the lipoylation enzyme mix V10, were
lipoylated to some extent. Lane 7 did not contain any V10. From fig.6 (A) it could be induced
lane 5 shows a slightly darker band, implying this reaction has been lipoylated further.
5. Lipoylation from a New Batch of Lipoic Acid. A new stock of lipoic acid was found, and
as it was the only component to have not been upgraded or amended, a new series of reactions
were created. Again lipoylated domain was used as a marker in the place of unlipoylated
domain, as was previously used.
Figure 6.
Figure 6.SDS-PAGE Electrophoresis gel stained with Coomassie Blue.
Optimisation of the lipoylation reaction with varying V10, lipoic acid and GTP.
Lane 1 contains Lipoylated domain (L-Lip) as a marker alone. Lanes 2-4 contain
3µl V10 and 4mM GTP and different lipoic acid amounts; 2 has 100µm, 3 has
1000µm, and 4 contains 0µm. Lanes 5-7 differ in V10 amounts; 5 contains 6µl, 6
contains 3µl, and 7 contains 0µl, while all 3 lanes remain constant with 100µm
lipoic acid and 4mM GTP. Lanes 8-10 differ in GTP amounts; 8 has 4mM, 9 has
8mM and 10 contains 0mM.
lipoylated
1 2 3 4 5 6 7 8 9 10
unlipoylated

Clearly, lane 1 is the lipoylated marker and distinguishes the line at which bands become
lipoylated. Lane 2 consists of no lipoic acid and as such there is no band to imply lipoylation.
As both the enzyme concentration and the lipoic acid amount increase, so does lipoylation in
the form of darker bands appearing along the same horizontal stain produced by the marker.
Word Count: 598
Figure 7.
Figure 7. SDS-PAGE Electrophoresis gel stained with Coomassie Blue. All componentswere as
original lipoylation reaction, but altered V10 enzyme mix and lipoic acid amount were the
variables changed. Lane 1 asstandard contains the marker; L-Lip (lipoylated domain). Lanes
2-5 contain 3µl V10, and increasing lipoic acid amounts; 0µm, 10µm, 100µmand 1mM
respectively. Lanes6-8 contain 6µl of V10, and similarly 10µm, 100µmand 1mM of lipoic acid.
1 2 3 4 5 6 7 8
unlipoylated
lipoylated

Discussion
Previous studies have indicated the fidelity of the exogenous lipoylation reaction, in that it
demonstrates significant ‘substrate promiscuity’. (Carbonell & Faulon, 2010). The end aim of
the current study was to screen a panel of environmental chemicals suspected to substitute
lipoic acid within the reaction, after optimisation. Unfortunately, as can be seen from the
results, time only allowed for the multiple optimisation reactions to be carried out which were
unfortunately unsuccessful, and as a consequence, no chemicals were added to be analysed.
The first step in the optimisation series was the differentiation between GTP and ATP as the
energy source to attach lipoic acid to proteins within the reaction. As seen in figure 8, the first
phase of the exogenous lipoylation reaction is the activation of lipoic acid by LAE. This is
catalysed by GTP or ATP to form either Lipoyl-GMP or Lipoyl-AMP.
Figure 8.
Figure 8. Lipoylation Pathways in Mammalian
Mitochondria and E.Coli. Diagrammatical
representation of the lipoylation pathway, (A)
shows E.coli, while (B) showsthe pathway in
mammalian mitochondria. The relevance to the
current study is optimal due to the
demonstration of the enzymes involved,
specifically LAE and LT, which constituted the
V10 enzyme mix used in the optimisation series.
Source: Patel & Packer 2008.

The second phase of lipoylation is the transfer of the lipoyl moiety by LT, to activate an
unlipoylated apoprotein, becoming lipoylated. Both enzymes have previously been purified
from bovine liver mitochondria (Fujiwara et al., 2001), and LAE has been shown to also be a
medium-chain acyl-CoA synthetase. (Patel & Packer, 2008). LAE synthesises Lipoyl-AMP,
but does not readily release it thus GTP was used in a similar experiment to investigate. These
findings showed Lipoyl-GMP was far easily released from the enzyme (LAE) to provide a
substrate for LT, proceeding at 1000 times faster with GTP than with ATP. The results from
the current study are inconsistent with this suggestion, as figure 2 (B) demonstrates an
inconclusive assay done with GTP. No clear results are available from this set of reactions.
Fig 2 (A) reveals a clear picture to interpret: as the V10 amount increases to 3µl and 4µl there
is a faint band beneath the U-Lip marker, indicating these particular lanes have been
lipoylated. (lanes 5 & 6). Reasons for the difference between the current study’s GTP/ATP
choice and other studies include technical/human errors, or of particular focus; stock solutions
of components, specifically GTP, which was changed in Lipoylation Reactions 2.5.
With no conclusions to draw from the first set of optimisation reactions, three variables were
altered in Lipoylation Reactions 2.3. Results from the PAGE analysis (figure 3) showed lanes
5 and 6 demonstrated similar amounts of lipoylation, while all others did not. Lanes 3 and 7
contained no V10 hence no lipoylation. But 5 and 6 were different in their contents; 5
contained GTP as the energy source and double the amount of lipoic acid, and 6 contained
ATP as energy, but all components as standard. Both lanes correlate with the previously
mentioned proposal from Fujiwara et al regarding GTP acting as a faster and more efficient
energy source than ATP, as the only difference (other than GTP/ATP) between the two was
lane 5 having double lipoic acid. It is surprising that lipoylation is only seen with high levels
of GTP but only low levels of ATP, this contradicts previous enzyme knowledge and suggests

some sort of error has occurred. Some lipoylation should potentially have been seen in lanes
2, 4, and 8, but reactions were not efficient enough for any lipoylation to be detected.
After electrophoresis analysis of the lipoylation reactions, immunoblotting analysis was
undertaken. As shown by figure 4 both (A) and (B), there are no visual bands. Multiple
attempts at chemiluminescence were made, with the same ultimate results. Mistakes may have
been made either during i) addition of the secondary conjugated antibody, ii) the transfer from
the gels to the nitrocellulose membrane or iii) use of the machine/software. With regard to the
transfer, the time may have been too long or too short, Cell Signalling® states the optimum
time of 1.5hours at 70V, and the time used here was around 30 minutes. Alternatively, the
choice of membrane could be the issue. PVDF (polyvinylidene difluoride) membranes have
higher binding capacities than nitrocellulose, 150-160 µg/cm2 to 80 µg/cm2 . This ensures
higher sensitivities, which could have improved the efficiency of the transfer in the current
study. (Davies, 2013). Amendments were tried across multiple angles of error and still no
Western Blots could be produced on this occasion, however bands became visible on different
gels from figure 6.
As is known from previous studiesV10 was required in order for any lipoylation to occur. The
aim of this experiment was to increase V10 and try a lower U-Lip amount, in order to get a
higher proportion lipoylated. This experiment used GTP as the energy source based on
previous studies findings (Walden et al., 2008), due to our failed reaction series (figure 3).
Looking at figure 6, lanes 3 and 4 show the only lipoylation, with lane 7 demonstrating a very
faint band. 3 and 4 contained 10µg U-Lip and 4µl and 8µl of V10 in that order. The only
conclusion that can be drawn from this particular study is that the lipoylation reaction does
indeed require both LAE and LT (V10), and U-Lip domain to be lipoylated. Increasing the
enzyme mix did not seem to increase lipoylation, as both lanes 3 and 4 show a similar band.

However, as an observation, 7µg of U-Lip was not to be used in future experiments as it made
visualising any bands (as illustrated by figure 6 lane 7).
As there had been very little success with any of the results, new reactions were set up with all
new reagents, particularly fresh GTP stocks. Lipoylated domain was used as the marker in
place of unlipoylated, to give a clear indication of where any lipoylated bands would appear
on the gel. Figure 7 (A) shows the gel and all lanes lipoylated, apart from lane 7, as there was
no V10. If the wells are split into three groups to analyse, it becomes clear to evaluate.
Between lanes 2-4, lane 2 shows the most lipoylation: 100µm must be the optimum amount of
lipoic acid to use within the reaction, with 4mM GTP and 3µl V10. Lanes 5-7 differ in
enzyme amount, and as already demonstrated altering V10 does not affect lipoylation to a
great extent. Between lanes 8-10, 10 shows no lipoylation (clearer to see from fig 7 (B)
western blot), as it contains no GTP, thus no attachment of lipoic acid to create Lipoyl-GMP.
However all this lipoylation could be due to ‘background’ from the original purification of the
lipoylation enzymes LAE and LT, described by Fujiwara et al, 1994. Both are able to hold a
small amount of lipoic acid, which independent of the conditions, is able to be incorporated
into the reaction, and hence demonstrate this lipoylation shown.
The only variable unchanged as far was the lipoic acid. Lipoic acid is vulnerable to UV
irradiation, particularly the five membered dithiolane ring necessary for the function of acting
as an enzyme cofactor. (Wada et al., 2009). This leads to loss of lipoic acid’s physiological
activity, hence it is essential it is stored away from any potential UV sources. Figure 7 shows
a clear stained electrophoresis gel demonstrating lipoylation faintly in lanes 3 and 4, and more
visibly in lanes 5,6,7 and 8. Interestingly, it is the increase in concentration of V10, along with
the presence of lipoic acid that causes the increase in lipoylation between lanes 2-5, and 6-8.
The increase of concentration of lipoic acid does not cause any significant lipoylation
increase, demonstrating that lipoic acid is not the cause of the problems faced throughout the

study. This deduction contradicts previous findings within the study, posing more questions
than were answered.
With the end aim of the study to be to screen a panel of environmental chemicals, it was
essential to create the ideal conditions within the lipoylation reaction. From the set of results
achieved, a few assumptions can be made, but the main obvious conclusion is that more
reactions need to be completed to provide clear cut results to infer. Of particular interest are
the recombinant enzymes used. Given the extensive optimisation steps carried out, with little
success, it could indicate undetected problems during expression and purification of the
enzymes, which need to be addressed before progressing this study.
The current studies’ findings should confirm and back up previous work carried out
suggesting that certain chemicals can indeed take the place of lipoic acid in lipoylation
reactions, this could potentially create a neoantigen and in susceptible people lead to an
autoimmune response due to molecular mimicry (Walden et al., 2008). Together with already
known information regarding the environmental hotspots (Jones, 2007), these bring a strong
argument forward in support of an ‘environmental-exposure model’. Moving onwards, a
suggested list of environmental chemicals, commonly found close to land fill sites, can be
found in Appendix 1.1. These should be substituted into the optimised lipoylation reaction, to
demonstrate whether or not lipoylation occurs, and hence whether or not these individual
chemicals can cause a PBC like disease process. As phrased by Smyk et al, with regard to
environmental PBC triggers, ‘we may not yet know them, but we hope that we will find
them.’
Word Count: 1505

Further Work
As evident from the current study there are a few clear directions in which to take further
work. Due to time restrictions and problems encountered, most noticeably with stock
solutions, techniques (SDS-PAGE and Chemiluminscence) and perhaps some bad luck,
optimisation of the lipoylation reaction was never fully achieved. Therefore the first step
would be to take principles of the current study, along with some from Walden et al., and try
to achieve the optimum lipoylation reaction. Preferably, new stock solutions of all
components should be used to eradicate any errors that may arise from old or used solutions.
GTP should be the choice of energy source, as it favours lipoylation (Walden et al., 2008),
10µg U-Lip to be the standard amount due to clear visualisation, expression of the lipoylation
enzymes LAE and LT in sufficient amounts, perhaps 6µl as the findings from this study
suggested altered amounts of V10 does not severely affect lipoylation. This provides the basis
for incorporating a selection of xenobiotics commonly found in close proximity to land fill
sites, and hypothesised to induce lipoylation in the place of lipoic acid. Each individual
xenobiotic would be added to the lipoylation reaction (all components the same as above,
minus lipoic acid), and would follow the same method as stated in the current study. With
hope, lipoylation would be definitively achieved or not, giving a clear indication of which
xenobiotics cause autoimmunity.
Word Count: 233

Acknowledgements
With thanks to Dr. Hannah Walden for the guidance and advice, and Sam Jamieson (and all
other technical staff) for their continued support throughout the project duration.

References
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cirrhosis. Medicine (Baltimore) 1950;29:299-64.
Carbonell P, Faulon JL. Molecular signatures-based prediction of enzyme
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Chen. R, Naiyanetr. P, Shu. SA, Wang. J, Yang. GX, Kenny. T, Guggenheim. K, Butler. G,
Bowlus. C, Tao. M, Kurth. MJ, Ansari. AA, Kaplan. M, Coppel. RL, Lleo. A, Gershwin. ME
& Leung. SC. (2013). Antimitochondrial Antibody Heterogeneity and the Xenobiotic
Etiology of Primary Biliary Cirrhosis. Hepatology. 57 (4), 1498-1508.
CST. (2013). Western Blot Troubeshooting Guide. Available:
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Jan 2015.
Cusick. M.F, Libbery. J.E & Fujinami.R.S. (2012). Molecular Mimicry as a Mechanism for
Autoimmune Disease. Clinical Reviews in Allergy and Immunology. 42 (1), 102-111.
Davies, T. (2013). The Magic’s In the Membrane: Choose Wisely for Best Western Blot
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Membrane-Choose-Wisely-for-Best-Western-Blot-Results/. Last accessed 5th Jan 2015.
Gershwin. ME & Selmi. C.. (2009). The role of environmental factors in primary biliary
cirrhosis. Trends in Immunology. 30 (8), 415-420.
Ghaffar. A & Nagarkatti. P. (2010). Tolerance & Autoimmunity. Available:
http://pathmicro.med.sc.edu/ghaffar/tolerance2000.htm. Last accessed 4th Nov 2014.

Jones, D. (2000). Autoantigens in primary biliary cirrhosis. Journal of Clinical Pathology. 53
(11), 813-821.
Jones. DE. (2007). Pathogenesis of Primary Biliary Cirrhosis. Gut. 56 (11), 1615-1624.
Kanehisa Laboratories. (2009). Lipoic Acid Metabolism. Available:
http://www.genome.jp/kegg-
bin/show_pathway?org_name=rn&mapno=00785&mapscale=&show_description=hide. Last
accessed 2nd Dec 2014.
Long SA, Van de Water J, Gershwin ME (2002) Antimitochondrial antibodies in primary
biliary cirrhosis: the role of xenobiotics. Autoimmunology Review 1:37–42.
Morikawa T, Yasuno R, Wada H. Do mammalian cells synthesise lipoic acid: identification of
a mouse cDNA encoding a lipoic acid synthase located in mitochondria. FEBS Lett
2001;498:16-21.
nextprot. (2011). ACSM1 - Acyl coenzyme A synthetase ACSM1 mitochondrial. Available:
http://www.nextprot.org/db/entry/NX_Q08AH1. Last accessed 2nd Dec 2014.
Patel, M & Packer, L (2008). Lipoic Acid: Energy Production, Antioxidant Activity and
Health Effects. USA: CRC Press. 19-21.
Posner. MG, Upadhyay. A, Crennel. SJ, Watson. AJ, Dorus. S, Danson. MJ & Bagby. S.
(2013). Post-translational modification in the archaea: structural characterization of multi-
enzyme complex lipoylation. Biochemical Journal. 449 (2), 415-425.
Poupon. R. (2010). Primary Biliary Cirrhosis: A 2010 Update. Journal of Hepatology. 52 (1),
745-758.

Prince. M.I, Chetwynd. A, Diggle. P, Jarner. M, Metcalf. J.V & James. O.F (2001). The
geographical distribution of primary biliary cirrhosis in a well-defined cohort. Hepatology 34.
1038-1088.
Selmi. C, Cocchi. CA & Zuin. M. (2009). The Chemical Pathway to Primary Biliary
Cirrhosis. Clinical Reviews in Allergy & Immunology. 36 (1), 23-29.
Selmi. C, Gershwin. ME & Mackay. IR. (2011). The autoimmunity of primary biliary
cirrhosis and the clonal selection theory. Immunology & Cell Biology. 89 (89), 70-80.
Smyk, D.S, Rigopouloi, E.I., Pares, A,. Billins, C., Burroughs, A.K., Muratori, L., Invernizzi,
P. & Bogdanos, D.P. (2012). Sex Differences Associated with Primary Biliary Cirrhosis.
Clinical & Developmental Immunology. Pp. 610504.
Smyk. DSS, Rigopoulou. E, Lleo. A, Abeles. RD, Mavropoulos. A,Billinis. C, Invernizzi. P &
Bogdanos. D. (2011). Immunopathogenesis of primary biliary cirrhosis: an old wives' tale.
Immunity and Ageing. 8 (12),
Smyk. D, Mytillianiou. M.G, Rigopoulo. E. I & Bogdanos. D.P. (2010). PBC triggers in water
reservoirs, coal mining areas and waste disposal sites: From Newcastle to New York. Disease
Markers. 29 (1), 337-344.
Spalding, MD & Prigge, ST. (2010). Lipoic Acid Metabolism in Microbial Pathogens.
Microbiology & Molecular Biology Reviews. 74 (2), 200-228.
Wada. N, Wakami. H, Konishi. T, Matsugo. S. (2009). The Degradation and Regeneration of
α-Lipoic Acid under the Irradiation of UV Light in the Existence of Homocysteine. Journal of
Clinical Biochemistry and Nutrition. 44 (3), 218-222.
Walden. H, Kirby. J, Yeaman. SJ, Gray. J, Jones. D, Palmer. JM. (2008). Xenobiotic
Incorporation into Pyruvate Dehydrogenase Complex Can Occur Via the Exogenous
Lipoylation Pathway. Journal of Hepatology. 48 (6), 1874-1884.

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cirrhosis. Lancet. 1965;1:827-831.

Appendix
(i) Personal Ethics Consideration
(ii) Completed COSHH Long Forms
 Acetic Acid
 Acrylamide
 Ammonium Persulphate
 Methanol
 NNNN-Tetramethylethylenediamine (TEMED)
 SDS
(iii) Completeted COSHH Short Form

Ethical Considerations
Research Ethics Number: RE-19-07-121068
Project Title: In Vitro Lipoylation
Project Supervisor: Hannah Walden
Ethics must be addressed in research in order to establish important ground rules to adhere to
absolutely. The Oxford English Dictionary defines ethics as ‘the whole field of moral
science’, and to act in a ‘morally right, virtuous and honourable way’. In the context of
scientific research, all work is subject to ethical scrutiny and review. Important ethical
considerations include the use of human/animal samples and the extent of injury or harm that
would come to the participant, the degree of truth and honesty to which the researcher must
abide by, including no falsifications of data, and the overall contribution of the experiment to
the field and to the public.
The experiment aims to investigate whether during lipoylation, enzymes use inappropriate
substances to modify proteins with the potential to trigger autoimmunity. Common laboratory
chemicals will be used in these lipoylation experiments to judge whether they are
contributors. The project uses no human or animal samples so therefore only requires
University ethical clearance, and has been classified as a ‘green’ risk experiment. The
chemicals used do not require any other control measures than COSHH regulations and good
laboratory practice.

Long Form COSHH Assessment record
Faculty of Health & Life Sciences
COSHH Risk Assessment
A COSHH risk assessment is required for work with hazardous substances including source materials,
products, known intermediates and by-products. The form should be completed electronically and
approved and signed by the responsible person then forwarded to the Faculty Safety Adviser (Dave
Wealleans) for storage online.
Chemical Name(s) Acetic Acid
CAS number 64-19-7
Title ofproject or activity Primary Biliary Cirrhosis
Principal investigator /
Responsible person
Dr Hannah Walden
Department Immunology- Biomedical Science
Date ofassessment
Location ofwork
(Buildings and room numbers)
EBA603
Section 1 Project or Activity
1.1: Briefdescription ofproject or activity
Used as part of a buffer for protein purification

Section 2 Hazards
2.1: Hazardous substances used and generated
Hazardous substance Hazard Statements Precautionary
Statements
Workplace
exposure
limit (WEL
Chemicals Acetic Acid H226
H314
P280
P305/351/338
P310
Europe.
Commission
Directive
91/322/EEC
on
establishing
indicative
limit values
Carcinogens,
mutagens or
reproductive toxins
Dusts or fumes
Asphyxiants
Other substances
hazardous to health

Section 3 Risks
3.1: Human diseases,illnesses or conditions associated with hazardous substances
Material is extremely destructive to tissue of the mucous membranes and upper respiratory tract,eyes,and
skin., spasm, inflammation and edema of the larynx, spasm, inflammation and edema of the bronchi,
pneumonitis, pulmonary edema, burning sensation, Cough, wheezing, laryngitis, Shortness of breath,
Headache,Nausea,Vomiting, Ingestion or inhalation of concentrated acetic acid causes damage to
tissues of the respiratory and digestive tracts. Symptoms include: hematemesis, bloody diarrhea, edema
and/or perforation of the esophagus and pylorus, pancreatitis, hematuria, anuria, uremia, albuminuria,
hemolysis, convulsions, bronchitis, pulmonary edema,pneumonia, cardiovascular collapse, shock, and
death. Direct contact or exposure to high concentrations of vapor with skin or eyes can cause:erythema,
blisters, tissue destruction with slow healing, skin blackening, hyperkeratosis, fissures, corneal erosion,
opacification, iritis, conjunctivitis, and possible blindness., To the best of our knowledge, the chemical,
physical, and toxicological properties have not been thoroughly investigated.
.
3.2: Potential routes ofexposure
Inhalation  Ingestion  Injection  Absorption  Other  Select all that
apply
3.3: Use ofhazardous substances
Small scale  Medium scale  Large scale  Fieldwork  Animals 
Plants  Maintenance  Cleaning  Other 
Select all that
apply
3.4: Frequency ofuse
Daily  Week  Monthly  Other  Select one

3.5: Maximum amount or concentration used
Negligible  Low  Medium  High  Select one
3.6: Potential for exposure to hazardous substances
3.7: Who might be at risk (*Contact the University Occupational Health Service)
Staff  Students  Visitors  Public  Young people (<18yrs) 
*New and expectant mothers  Other 
3.8: Assessment ofrisk to human health (Prior to use of controls)
Level of risk Low  High  Very High  Select one
3.9: Assessment ofrisk to environment (Prior to use of controls)
Level of risk Low  High  Very High  Select one
Likelihood ofharm Severity of Harm Risk : the risk in using the substance =
Likelihood x Severity
Remote 1 Negligible (no injury) 1 Low 1 to 10 Good lab practice
required
Unlikely 2 Minor injury 2 High 12 to 18 Specific Identified
Control Measures
must be used

May Occur 3 Lost time injury 3 Very
High
20+ Trained personnel
only
Likely 4 Major injury 4
Very Likely 5 Single fatality 5
Certain 6 Multiple Fatalities 6

Section 4 Controls to Reduce Risks as Lowas Possible
4.1: Containment
Laboratory  Room  Controlled area  Total containment 
Glove box  Fume cupboard  Local exhaust ventilation (LEV) 
Access control Other 
Select all that
apply
Dispense in a fume hood add acid to water as exothermic reaction can take place. Dilute solutions only to be used on
the bench
4.2: Other controls
Avoid contact with skin and eyes. Avoid formation of dust and aerosols.
Provide appropriate exhaust ventilation at places where dust is formed.Keep away from sources of ignition
- No smoking.Take measures to prevent the build up of electrostatic charge.
4.3: Storage ofhazardous substances
Store in cool place. Keep container tightly closed in a dry and well-ventilated place
4.4: Transport of hazardous substances
4.5: Personal protective equipment (PPE)
Lab coat  Overalls  Chemical suit  Disposable clothing 
Apron  Spectacles  Goggles  Face shield 
Gloves  Special headwear  Special footwear  Other 
Select all that
apply
[ENTER DETAILS HERE]
4.6: Respiratory protective equipment (RPE)
Disposable mask  Filter mask  Half face respirator 
Full face respirator  Breathing apparatus  Powered respirator 
Other 
Select all that
apply

4.7: Waste management and disposal
Liquid  Solid  Gas  Inorganic  Organic  Aqueous  Mixed 
Other 
In dilute form can be disposed of with water waste with additional water flush
4.8: Monitoring exposure (If you need advice contact the University Occupational Health Service)
N/A
4.9: Health surveillance (If you need advice contact the University Occupational Health Service)
N/A
4.10: Instruction, training and supervision
Special instructions are required to safely carry out the work (If yes enter details below) Yes
Instructed on mixing with water
Special training is required to safely carry out the work (If yes enter details below) Yes 
A: Work may not be carried out without direct personal supervision (If yes enter details below) Yes 
B: Work may not be started without the advice and approval of supervisor (If yes enter details
below)
Yes 
C: Work can be carried out without direct supervision Yes 

Section 5 Emergency Procedures
5.1: Emergency procedures
Use personal protective equipment. Avoid dust formation. Avoid breathing vapours, mist or gas. Ensure
adequate ventilation. Evacuate personnel to safe areas. Avoid breathing dust.
5.2: Minor spillage or release
Specify procedure
Pick up and arrange disposal without creating dust. Sweep up and shovel. Keep in suitable,
closed containers for disposal.
Other actions Evacuate and secure laboratory / area Yes 
Inform competent person (eg principal investigator / school safety
officer etc)
Yes 
5.3: Major spillage or release
Specify procedure
Other actions Evacuate building by fire alarm Yes 
Call security and fire brigade (3200 on campus) Yes 
officer etc)
Yes 
5.4: Fire Precautions
Carbon dioxide  Water  Powder  Foam  Blanket  Automatic fire suppression 
Other 
5.5: First aid
Wash with copious amounts of water and apply polyethylene glycol (PEG) 300 for phenol 
Wash with copious amounts of water and apply calcium gluconate gel for hydrofluoric acid 

Remove affected clothing and wash with copious amounts of water for skin contact 
Oxygen for cyanide 
Eye wash station 
Emergency shower 
Other 
Consult a physician
If breathed in, move person into fresh air. If not breathing, give artificial respiration.
If in contact with skin, wash well with soap and water
If in eyes,flush with water
5.6: Emergency contacts
Name Position Supervisor Telephone 4307
Hannah Walden Responsible person

Section 6 Approval
6.1: Assessor
Name Signature Date
6.2: Responsible person
Name Hannah Walden Signature Hannah Walden Date 3/10/14

Chemical Name(s) Acrylamide
CAS number 79-06-1
Responsible person
Dr Hannah Walden
Date ofassessment
Location ofwork
(Buildings and room
numbers)
EBA603
Use in PAGE gels for electrophoresis

Section 2 Hazards
Hazardous
substance
Hazard Statements Precautionary
Statements
Workplace
exposure
limit
(WEL)
Chemicals Acrylamide
H301, H312, H315,
H317, H319, H332,
H340, H350, H361f,
H372
P201, P280,
P301/310,
P305/351/338,
P308/313
N/A
Carcinogens,
mutagens or
reproductive
toxins
Acrylamide
H301, H312, H315,
H317, H319, H332,
H340, H350, H361f,
H372
P201, P280,
P301/310,
P305/351/338,
P308/313
N/A
Dusts or fumes Acrylamide
H301, H312, H315,
H317, H319, H332,
H340, H350, H361f,
H372
P201, P280,
P301/310,
P305/351/338,
P308/313
N/A
Asphyxiants
Other substances
hazardous to
health

Section 3 Risks
Carcinogen and neurotoxin.
Toxic if swallowed. Harmful to skin. Causes skin irritation. May cause allergic skin reaction. Causes
serious eye irritation. Harmful if inhaled. May cause genetic defects. May cause cancer. Suspected of
damaging fertility. Causes damage to organs through prolonged or repeated exposure.
Inhalation  Ingestion  Injection  Absorption 
Other 
Select all that
apply
Small scale  Medium scale  Large scale  Fieldwork 
Animals  Plants  Maintenance  Cleaning 
Other 
Select all that
apply
Negligible  Low  Medium  High  Select one

Remote 1 Negligible (no
injury)
1 Low 1 to 10 Good lab practice
required
Control Measures
must be used
High
only

4.1: Containment
Laboratory  Room  Controlled area  Total
containment 
Glove box  Fume cupboard  Local exhaust ventilation (LEV)
 Access control Other 
Select all that
apply
Must only be used in liquid form and only used on a drip tray
If gloves are contaminated must be changed immediately using good technique to ensure no exposure
of skin. If clothing is contaminated must be removed immediately and underling skin washed
thoroughly. Seek medical advice if concerned
4.2: Other controls
Wear protective gloves, remove immediately if contaminated without contaminated surface contacting
skin. Only use on drip tray to prevent contamination of bench
Store at 4O
C. Keep container tightly closed in a dry and well-ventilated place.
Lab coat  Overalls  Chemical suit  Disposable
clothing 
Select all that
apply
Safety glasses with side shields
Gloves – Nitrile rubber, 0.11mm thickness, 480 minute break through time

Other 
Select all that
apply
Liquid  Solid  Gas  Inorganic  Organic  Aqueous 
Mixed  Other 
Should not be disposed of in liquid form. Solid waste should be disposed of in specialised laboratory
gel waste
4.8: Monitoring exposure (If you need advice contact the University Occupational Health
Service)
N/A
N/A
Special instructions are required to safely carry out the work (If yes enter details
below)
Yes 
Training from supervisor required before use
Special training is required to safely carry out the work (If yes enter details below) Yes 

A: Work may not be carried out without direct personal supervision (If yes enter
details below)
Yes 
B: Work may not be started without the advice and approval of supervisor (If yes
enter details below)
Yes 
C: Work can be carried out without direct supervision Yes
Supervisor(s) Dr Hannah
Walden

Use personal protective equipment. Avoid dust formation. Avoid breathing vapours, mist or gas.
Ensure
Specify procedure Wearing PPE make solid by the addition of APS and TEMED before
disposal in laboratory gel waste
Inform competent person (eg principal
investigator / school safety officer etc)
Yes 
Specify procedure Spillages should be treated with APS and TEMED and removed once set
Inform competent person (eg principal
investigator / school safety officer etc)
Yes 
Carbon dioxide  Water  Powder  Foam  Blanket  Automatic fire
suppression  Other 
Special hazards arising from the substance or mixture
5.5: First aid

Eye wash station 
Other 
Consult a physician

Chemical Name(s) Ammonium Persulfate (APS)
CAS number 7727-54-0
Responsible person
Dr Hannah Walden
Date ofassessment
Location ofwork
(Buildings and room
numbers)
EBA603
Use in making PAGE gels

Section 2 Hazards
Hazardous
substance
Statements
Workplace
exposure
limit
(WEL)
Chemicals Ammonium
persulfate
H272
H315
H317
H302
H319
H334
H335
P220
P261
P280
P305/351/338
P342/311
/
Carcinogens,
mutagens or
reproductive
toxins
Dusts or fumes
Asphyxiants
Other substances
hazardous to
health

Section 3 Risks
Inhalation May be harmful if inhaled. Material is extremely destructive to the tissue of
the mucous membranes and upper respiratory tract. Causes respiratory
tract irritation.
Ingestion Harmful if swallowed. Causes burns.
Skin Harmful if absorbed through skin. Causes skin burns.
Eyes Causes eye burns.
Inhalation  Ingestion  Injection  Absorption 
Other 
Select all that
apply
Other 
Select all that
apply

Remote 1 Negligible (no
injury)
1 Low 1 to 10 Good lab practice
required
Control Measures
must be used
High
only

4.1: Containment
containment 
Select all that
apply
4.2: Other controls
Provide appropriate exhaust ventilation at places where dust is formed.Keep away from sources of
ignition
clothing 
Select all that
apply
Disposable mask  Filter mask  Half face respirator  Select all that

Other 
apply
Liquid  Solid  Gas  Inorganic  Organic  Aqueous 
Mixed  Other 
Disposed of in gel waste as part of gel
Service)
N/A
N/A
below)
Yes 
Advised on safe use of substance
details below)
Yes 
Yes 

Supervisor(s) Hannah Walden

Use personal protective equipment. Avoid dust formation. Avoid breathing vapours, mist or
gas. Ensure
Specify
procedure
Pick up and arrange disposal without creating dust. Sweep up and shovel. Keep in
suitable, closed containers for disposal.
Inform competent person (eg principal investigator / school
safety officer etc)
Yes 
Specify procedure
Inform competent person (eg principal investigator / school
safety officer etc)
Yes 
Carbon dioxide  Water  Powder  Foam  Blanket  Automatic fire
suppression  Other 
5.5: First aid

Eye wash station 
Other 
Consult a physician

Chemical Name(s) Methanol
CAS number 67-56-1
Responsible person
Dr Hannah Walden
Date ofassessment
Location ofwork
(Buildings and room
numbers)
EBA603
Use in buffers for PAGE gel transfer

Section 2 Hazards
Hazardous
substance
Statements
Workplace
exposure
limit
(WEL)
Chemicals Methanol H25
H301/311/331
H370
P210
P260
P280
P301/310
P311
UK.EH40
250 ppm
333 mg/m3
Carcinogens,
mutagens or
reproductive
toxins
Dusts or fumes
Asphyxiants
Other substances
hazardous to
health

Section 3 Risks
: Lungs, Thorax, or Respiration:Dyspnea. Ingestion may cause gastrointestinal irritation, nausea,
vomiting and diarrhoea
Methyl alcohol may be fatal or cause blindness if swallowed.
Effects due to ingestion may include:, Headache,Dizziness, Drowsiness, metabolic acidosis, Coma,
Seizures.
Symptoms may be delayed., Damage of the:, Liver, Kidney .
Inhalation  Ingestion  Injection  Absorption 
Other 
Select all that
apply
Other 
Select all that
apply
Daily  Week Monthly  Other  Select one

4.1: Containment
containment 
Select all that
apply
4.2: Other controls
Provide appropriate exhaust ventilation at places where dust is formed.Keep away from sources of
ignition
clothing 
Select all that
apply
Skin must be covered to prevent absorption
Disposable mask  Filter mask  Half face respirator  Select all that

Other 
apply
Liquid  Solid  Gas  Inorganic  Organic  Aqueous 
Mixed  Other 
Dilute volumes down sink with water waste and copious amounts of water
Service)
N/A
N/A
below)
Yes 
Told of exposure risks and instructed on proper PPE use
details below)
Yes 
Yes 

Chemical Name(s) N,N,N′,N′-Tetramethylethylenediamine (TEMED)
CAS number 110-18-9
Responsible person
Dr Hannah Walden
Date ofassessment
Location ofwork
EBA603
Use in gels for electrophoresis

Section 2 Hazards
Statements
Workplace
exposure
limit (WEL
Chemicals N,N,N′,N′-
Tetramethylethylenedi
amine (TEMED)
H225, H302, H314,
H332
P210, P280,
P305/351/338, P310
N/A
Carcinogens,
mutagens or
reproductive toxins
Dusts or fumes
N,N,N′,N′-
Tetramethylethylenedi
amine (TEMED)
H225, H302, H314,
H332
P210, P280,
P305/351/338, P310
N/A
Asphyxiants
Other substances
hazardous to health

Section 3 Risks
Highly flammable liquid and vapour. Harmful if swallowed. Causes severe skin burns and eye damage. Harmful i
inhaled.
apply
Select all that
apply

required
Control Measures
must be used
High
only

4.1: Containment
Select all that
apply
4.2: Other controls
Wear protective gloves, use on drip tray. Keep away from sparks/heat/flame. Dispense from high concentrations in
fume hood.
Store in cool place. Keep container tightly closed in a dry and well-ventilated place. Containers which are opened m
be carefully resealed and kept upright to prevent leakage. Handle and store under inert gas. Air and moisture sensitiv
Select all that
apply
Safety glasses with side shields
Gloves – Nitrile rubber, 0.11mm thickness, 480 minute break through time
Other 
Select all that
apply

Liquid  Solid  Gas  Inorganic  Organic  Aqueous  Mixed 
Other 
Solid waste as part of gel to be disposed of in gel waste
N/A
N/A
Special instructions are required to safely carry out the work (If yes enter details below) Yes 
Special training is required to safely carry out the work (If yes enter details below) Yes 
below)
Yes 

C: Work can be carried out without direct supervision Yes
Supervisor(s) Dr Hannah Walden

In case of eye contact
Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing
Immediately call a POISON CENTER or doctor/ physician.
Specify procedure Pick up and arrange disposal without creating dust. Sweep up and shovel. Keep in suitable,
closed containers for disposal.
officer etc)
Yes 
Specify procedure
officer etc)
Yes 
Carbon dioxide  Water  Powder  Foam  Blanket  Automatic fire suppression 
Other 
Special hazards arising from the substance or mixture
5.5: First aid

Eye wash station 
Other 
Consult a physician

Chemical Name(s) Sodium Dodecyl Sulfate (SDS)
CAS number 151-21-3
Responsible person
Dr Hannah Walden
Date ofassessment
Location ofwork
EBA603
For use in SDS-PAGE gels and protein denaturation buffers

Section 2 Hazards
Statements
Workplace
exposure
limit (WEL
Chemicals Sodium Dodecyl
Sulfate (SDS)
H228
H302
H311
H315
H318
H335
P219
P261
P280
P305/351/338
P312
Carcinogens,
mutagens or
reproductive toxins
Dusts or fumes
Asphyxiants
Other substances
hazardous to health

Section 3 Risks
Prolonged or repeated exposure may cause allergic reactions in certain sensitive individuals.
sneezing, The sodium salt of dodecyl sulfate has been reported to cause pulmonary sensitization resulting
in hyperactive airway dysfunction and pulmonary allergy accompanied by fatigue, malaise, and aching.
Significant symptoms of exposure can persist for more than two years and can be activated by a variety of
nonspecific environmental stimuli such as automobile exhaust, perfumes, and passive smoking.
Liver - Irregularities - Based on Human Evidence
apply
Select all that
apply

required
Control Measures
must be used
High
only

4.1: Containment
Select all that
apply
4.2: Other controls
Dispense only in dust hood
Provide appropriate exhaust ventilation at places where dust is formed.Keep away from sources of ignition
Select all that
apply
Select all that
apply

Other 
Liquid  Solid  Gas  Inorganic  Organic  Aqueous  Mixed 
Other 
As part of gel waste
N/A
N/A
Special instructions are required to safely carry out the work (If yes enter details below) Yes 
Must be used in dust hood unless in soultion
Special training is required to safely carry out the work (If yes enter details below) Yes
Will demonstrate handeling of substance
below)
Yes 

Experimental Record - short COSHH record form
COSHH Assessments for: Experiment Title: In Vitro Lipoylation
Name of assessor __________________________ Signed __________________________Date_________
Substance
H
Statement1
Hazard2
Key hazard(s)
associated with
the substance
Signal
Word?3
Likelihood4
Severity5
Risk6
(before
additional
control
measures)
Specific Risk Control
Measures7
Controlled
Risk8

Acrylamide
H301,
H312,
H315,
H317,
H319,
H332,
H340,
H350,
H361f,
H372
Toxic if
swallowed H301
Harmful to skin
H312
Causes skin
irritation H315
May cause
allergic skin
reaction H317
Causes serious
eye irritation
H319
Harmful if
inhaled
H332
May cause
genetic defects
H340
May cause
cancer H350
Suspected of
damaging
fertility H361f
Causes damage
to organs
through
prolonged or
repeated
exposure H372
Danger 3 4 12
Wear protective gloves,
remove immediately if
contaminated without
contaminated surface
contacting skin
Only use on drip tray to
prevent contamination of
bench
8

N,N,N′,N′-
Tetramethylethylenediamine
(TEMED)
H225,
H302,
H314,
H332,
Highly
flammable
liquid and
vapour H225
Harmful if
swallowed H302
Causes severe
skin burns and
eye damage
H314
Harmful if
inhaled H332
Danger 3 3 9
Wear protective gloves,
use on drip tray.
Keep away from
sparks/heat/flame
6
Sodium Dodecyl Sulfate
(SDS)
H228,
H302,
H311,
H315,
H318,
H335
Flammable solid
H228
Harmful if
swallowed H302
Toxic in contact
with skin H311
Causes skin
irritation H315
Causes serious
eye damage
H318
May cause
respiratory
irritation H335
Danger 3 3 9
Keep away from
heat/sparks/open flame
Avoid breathing dust,
only weigh out powder in
a hood with positive
pressure,or use face
mask. Use on open bench
only in weak solution.
Wear protective gloves
and eye protection
3

Ammonium Persulfate
(APS)
H302,
H315,
H317,
H319,
H272,
H334,
H335
May intensify
fire (oxidant)
H272
Causes skin
irritation H315
May cause
allergic skin
reaction H317
Harmful if
swallowed H302
Causes serious
eye irritation
H319
May cause
allergy or
asthma
symptoms or
breathing
difficulties
H334
May cause
respiratory
irritation H335
Danger 3 2 6
Keep and store away from
clothing and combustible
materials
Avoid breathing
dust/fume
Wear protective gloves
2
2-mercaptoethanol
Danger 2 3 6
Use in well ventilated
area,do not inhale fumes,
open container for
minimum time only
3

Acetic acid
Danger 3 3 9
Use only in weak
concentrations on the
bench, make up from
concentrated stock in a
fume hood, always add
acid to water to reduce
exothermic reaction
3
Methanol
Danger 2 3 6
Use only in weak
concentrations on the
bench, PPE/GLP should
prevent any skin contact,
If contact occurs wash
immediately with copious
quantities of water
3
Lipoic Acid
H302
Harmful if
swallowed H302
Warning 2 2 4
Handle in accordance
with good industrial
hygiene, PPE/GLP should
prevent any skin contact.
Wash hands before breaks
and at the end of
workday. Wear protective
gloves. Avoid breathing
dust/fumes. Use a dust
mask.
2

Menadione
H302,
H315,
H319,
H335
Harmful if
swallowed H302
Causes skin
irritation H315
Causes serious
eye irritation
H319
May cause
respiratory
irritation H335
Warning 2 3 6
hygiene and safety
practice. , PPE/GLP
should prevent any skin
contact. Wash hands
before breaks and at the
end of workday. Wear
protective gloves. Avoid
breathing dust/fumes.
3
Methapyrilene
H301
Toxic if
swallowed H301
Danger 3 3 9
Avoid contact with skin,
eyes and clothing. ,
PPE/GLP should prevent
any skin contact. Wash
hands before breaks and
immediately after
handling the product.
Wear protective gloves.
Use a face mask.
3

Tartrazine
H317,
H334
May cause
allergic skin
reaction H317
May cause
allergy or
asthma
symptoms or
breathing
difficulties if
inhaled H334.
Danger 2 3 6
hygiene and safety
practice. , PPE/GLP
contact. Wash hands
breathing dust/fumes.
3
Sulphanilic Acid
H315,
H317,
H319
Causes skin
irritation H315
May cause an
allergic skin
reaction H317
Causes serious
eye irritation
H319
Warning 2 3 6
hygiene and safety
practice. , PPE/GLP
contact. Wash hands
breathing dust/fumes. Use
a face mask.
3

Nonylophenol
H302,
H314,
H361fd,
H410
Harmful if
swallowed H302
Causes severe
skin burns and
eye damage
H314
Suspected of
damaging
fertility and the
unborn child
H361fd
Facilities storing
or utilizing this
material should
be equipped
with an eyewash
facility and a
safety shower.
Use
adequate
ventilation to
keep airborne
concentrations
low.ery toxic to
aquatic life with
long lasting
effects H410
Danger 3 4 12
Do not breathe dust,
vapor, mist, or gas. Do
not get in eyes, on skin, or
on clothing. , PPE/GLP
contact. Use only in a
chemical fume hood.
Useadequate ventilation
to keep airborne
concentrations low. Wear
protective gloves.
4

Troglitazone
N/A N/A N/A 1 1 1
Contains no substances
with occupational
exposure limit values.
General industrial hygiene
practice. Wear protective
gloves. , PPE/GLP should
1
Paracetamol
H302,
H319,
H335
Harmful if
swallowed H302
Causes skin
irritation H319
Causes serious
eye irritation
H335
Warning 2 2 4
Avoid breathing dust.
IF IN EYES: Rinse
cautiously with water for
severalminutes. Remove
contact lenses, if present
and easy to do. Continue
rinsing. Wear protective
gloves. , PPE/GLP should
2
Aminopyrazolone
H315,
H319,
H335
Causes skin
irritation H315
Causes serious
eye irritation
H319
May cause
respiratory
irritation H335
Warning 2 3 6
Avoid breathing dust/
fume/ gas/ mist/ vapours/
spray. IF IN EYES: Rinse
cautiously with water for
severalminutes. Remove
contact lenses, if present
and easy to do. Continue
rinsing. , PPE/GLP should
Wear suitable gloves and
eye/face protection. Wear
impervious clothing.
3

α-naphthylisothiocyanate
H301,
H312 +
H332,
H315,
H319,
H334,
H335
Toxic if
swallowed.H301
Harmful in
contact with
skin or if
inhaled H312 +
H332
Causes skin
irritation.H315
Causes serious
eye irritation.
H319
May cause
allergy or
asthma
symptoms or
breathing
difficulties if
inhaled H334
May cause
respiratory
irritation H335
Danger 2 3 6
Avoid contact with skin,
eyes and clothing. Wash
hands before breaks and
immediately after
handling
the product.
Avoid breathing dust. Use
powdered form only in
fume hood. Only very
dilute forms to be used on
bench and then only on
containment trays.
,PPE/GLP should prevent
any skin contact. Wear
protective gloves.
Complete suit protecting
against chemicals
3
Substance P Statement9
Storage10
Emergency Procedures (in event ofspillage, fire etc)11
Detail
Disposal12

Acrylamide
P201
P280
P301/310
P305/351/338
P308/313
Store at 4O
C. Keep
container tightly
closed in a dry and
well-ventilated place.
Spillages should be treated with APS and TEMED and
removed once set
Fire - Use water spray,alcohol-resistant foam, dry
chemical Spillage - Pick up and arrange disposal without
creating dust. Sweep up and shovel. Keep in suitable,
closed
containers for disposal.
Should be disposed
of only in solid state
in specialised gel
waste route in the
laboratory

N,N,N′,N′-
Tetramethylethylenediamine
(TEMED)
P210
P280
P305/351/338
P310
Store in cool place.
Keep container tightly
closed in a dry and
Containers which are
opened must be
carefully resealed and
kept upright to prevent
leakage.
Handle and store
under inert gas. Air
and moisture
sensitive.
In case ofeye contact
Rinse cautiously with water for severalminutes. Remove
contact lenses, if present and easy to do. Continue rinsing.
Immediately call a POISON CENTER or doctor/ physician.
chemical Spillage - Contain spillage, and then collect with
an electrically protected vacuum cleaner or by wet-
brushing and
place in container for disposal according to local
regulations
Should be disposed
of in specialised gel
waste as part of gels
route in the
laboratory
Disposal of large
quantities is unlikely
but should burn in a
chemical incinerator
equipped with an
afterburner and
scrubber but exert
extra care in igniting
as this material is
highly flammable.
Offer surplus and
non-recyclable
solutions to a
licensed disposal
company.

Sodium Dodecyl Sulfate
(SDS)
P219
P261
P280
P305/351/338
P312
closed in a dry and
well-ventilated place. In case ofeye contact
Rinse cautiously with water for severalminutes. Remove
contact lenses, if present and easy to do. Continue rinsing.
Call a POISON CENTER or doctor/ physician if you feel
unwell.
chemical Spillage - Sweep up and shovel. Contain spillage,
and then collect with an electrically protected vacuum
cleaner or
by wet-brushing and place in container for disposal
according to local regulations. Keepin suitable, closed
containers for disposal. Contain spillage, pick up with an
electrically protected vacuum
cleaner or by wet-brushing and transfer to a container for
disposal according to local regulations
Should be disposed
route in the
laboratory
Disposal of large
but burn in a
equipped with an
afterburner and
scrubber but exert
as this material is
highly flammable.
Offer surplus and
non-recyclable
solutions to a
licensed disposal
company.

Ammonium Persulfate (APS)
P220
P261
P280
P305/351/338
P342/311
closed in a dry and
Moisture sensitive.
Store away from
combustible materials
chemical May intensify fire; oxidiser.Use water spray to
cool unopened containers.
Spillage - Sweep up and shovel. Contain spillage, and then
collect with an electrically protected vacuum cleaner or
by wet-brushing and place in container for disposal
according to local regulations Keep in suitable, closed
containers for disposal.
Should be disposed
route in the
laboratory
Disposal of large
but burn in a
equipped with an
afterburner and
scrubber but exert
as this material is
highly flammable.
Offer surplus and
non-recyclable
solutions to a
licensed disposal
company.

2-mercaptoethanol Store in cool place.
closed in a double
container in toxic
cabinet in 603
Moisture sensitive.
Store away from
combustible materials
Should be disposed
route in the
laboratory
Disposal of large
but burn in a
equipped with an
afterburner and
scrubber but exert
as this material is
highly flammable.
Offer surplus and
non-recyclable
solutions to a
licensed disposal
company.
Acetic acid Store in specialised
acid storage in
laboratories
Dispose of dilute
solutions in sink as
water waste with
plenty of water.
High concentrations
will not be disposed
of

Methanol Store in specialised
alcohol storage in
laboratories. Weak
dilutions can be stored
in cool dark cupboards
Dispose only of
extremely dilute
solutions in sink as
water waste with
plenty of water.
Lipoic Acid
N/A
closed in a dry and
Recommended storage
temperature: 2 - 8 °C Fire - Use water spray,alcohol-resistant foam, dry
chemical
Pick up and arrange disposal without creating dust. Sweep
up and shovel. Keep in suitable, closed containers for
disposal.
Do not let product
enter drains.
Disposal of large
quantities is
unlikely. Offer
surplus and non-
recyclable solutions
to a licensed
disposal company.
Dissolve or mix the
material with a
combustible solvent
and burn in a
equipped with an
afterburner and
scrubber.

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