Sublethal effects of low pH in two fish
species (Gasterosteus aculeatus and
Gadus morhua)
Fredrik Gunnarsson
Degree project for Master of Science in
Environmental Science
Department of Plant and Environmental Sciences
University of Gothenburg
March 2010
List of contents
List of contents ........................................................................................................................................ 1
Summary ................................................................................................................................................. 2
Sammanfattning ...................................................................................................................................... 2
1.
Introduction..................................................................................................................................... 3
1.1.
The other CO2-problem............................................................................................................ 3
1.2.
Art of problem ......................................................................................................................... 3
1.3.
The buffering capacity of the oceans ...................................................................................... 3
1.4.
Biomarkers............................................................................................................................... 7
1.5.
Proteomics............................................................................................................................... 9
1.
Aim................................................................................................................................................. 10
2.
Method.......................................................................................................................................... 10
2.1.
Exposure ................................................................................................................................ 10
2.2.
Samples ................................................................................................................................. 11
2.4.
2D gel electrophoresis........................................................................................................... 12
2.4.1.
1st dimension ..................................................................................................................... 12
2.4.2.
2nd dimension ..................................................................................................................... 12
2.4.3.
Spot identification (performed by the Proteomic facility) ................................................. 13
3.
Results ........................................................................................................................................... 13
4.
Discussion ...................................................................................................................................... 17
4.1.
Protein functions ................................................................................................................... 17
4.2.
Discussion of protein regulation ........................................................................................... 23
5.
Conclusions.................................................................................................................................... 24
6.
Acknowledgements ....................................................................................................................... 24
7.
References..................................................................................................................................... 25
8.
Attachments .................................................................................................................................. 31
8.1.
Annex A – Routes of H+ in the organism............................................................................... 31
8.2.
Annex B – Buffers .................................................................................................................. 32
1
Summary
Gunnarsson, F. 2010. The honour thesis in ecotoxicology - Sublethal effects of low pH in two
fish species (Gasterosteus aculeatus and Gadus morhua). Göteborg university. Department of
zoological institution. 30 pp.
Ocean acidification is a consequence from the burning of fossile fuels and long term effects
on marine habitats and organisms are not fully investigated. The physiological effects on adult
fish species are even less known. Proteomic 2D-gel electrophoresis (2D-GE) is a good
approach for comparative analyses giving a specific 2D pattern of expressed proteins. 2D-GE
was used to identify differences of pH acclimatization in two marine fish species; cod (Gadus
marhua) and stickleback (Gasterosteus aculeatus). These were chosen to reveal differences in
their inherent capacity to resist low pH exposure. The 2D protein expression pattern gave
significantly regulated spots (three in cod and two in stickleback) in low pH exposure groups
and four of the protein spots were significantly matched with mass spectrometry
identification. The spots detected were: Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and hemoglobin beta chain in cod and annexin max3 and proteasome subunit N3 in
stickleback. These results indicate that 2D-gel electrophoresis is a good hypothesis generating
approach and allows for easy screening in organism protein expressions in the search for
potential biomarkers.
Sammanfattning
Gunnarsson, F. 2010. Examensarbetet i ekotoxikologi – Subletala effekter av lågt pH i två
marina fiskar (Gasterosteus aculeatus and Gadus morhua). Göteborgs universitet. Zoologiska
institutionen. 30 sidor.
Försurningen av havet är en konsekvens av förbränningen av fossila bränslen och kroniska
effekter på marina habitat och dess organismer är dåligt utrett. De fysiologiska effekterna på
vuxna fiskar är till stor del förbisett. Proteomisk 2D-Gel elektrofores (2D-GE) är en bra metod
för jämförande studier. Metoden användes för att identifiera skillnader i pH acklimatisering i
två marina fiskar; torsk (Gadus morhua) och storspigg (Gasterosteus aculeatus). Dessa valdes
för att identifiera skillnader i deras medfödda förmåga att motstå lågt pH. 2D- proteinuttrycket
gav signifikant uppreglerade prickar (tre i torsk och två i spigg) för de grupper exponerade för
lågt pH. Fyra av dessa prickar kunde matchas signifikant med hjälp av masspektrometri. De
proteiner som identifierades var: Glyceraldehyd-3-fosfat dehydrogenas (GAPDH) och
hemoglobin beta unit i torsk, annexin max3 och proteosome subunit N3 i storspigg.
Resultaten visar att 2D-gel elektrofores är en bra hypotesgenererande metod och ger bra
möjlighet till att i organismers proteinuttryck söka efter potentiella biomarkörer.
2
1. Introduction
1.1.
The other CO2-problem
Climate changes are not the only consequence of increasing CO2 concentration in the
atmosphere. The increase in atmospheric CO2 concentration increases the partial pressure of
CO2 (pCO2) in surface waters, reducing the pH of all open waters leading to ocean
acidification and hypercapnia, a consequence called “the other CO2 problem” (Doney et al.,
2009). The CO2 concentration in the atmosphere is increasing in a steady rate (approximately
0,5% each year (Forster et al., 2007)) due to increasing use of anthropogenic CO2- releasing
processes, e.g. burning of fossil fuels and the heating of calcium carbonate in cement
production (The Cement Sustainability Initiative: Progress report, 2002, pp. 20) and
deforestation. Natural increased partial pressure of CO2 (a process known as hypercapnia)
have been observed in many natural marine habitats (Diaz, Rosenburg, 1995). The average
CO2- concentration in surface waters has been stable for about 25 million years and there is
no evidence that the pCO2 in surface waters has increased by the rate observed to date
(Pörtner et al., 2004).
1.2.
Art of problem
Short term exposure scenarios might not raise the same physiological effects as the ones for
long term exposure which means that many experiments performed today does not reflect the
physiological effects in hundred years. Pörtner (2008) use a strong suggestion that:
“…previous investigations are invalid because they have used high CO2 levels that are
beyond expected scenarios of ocean acidification” and argues that when new results on longterm experiments have been made, the view of physiological adaption in marine animals
might be totally different. Organisms living in a constant environment adapt to their
environment; they become specialists and more sensitive to habitat changes. In 2000,
Hofmann et al., made a study showing that the capacity of the organism for producing heat
shock proteins have been lost in antartarctic teleost fish after being in a constant temperature
for around 14-25 million years. According to Widdicombe and Spicer (2008), adaption to
hypercapnia is more easily achieved by species with small body-size than larger species
across generations during a long timeframe. Key-species tend to have a larger body than other
species and might be more sensitive. Not much research has been performed on comparing
the sensitivity for marine species with different body sizes and different roles in the
ecosystem. Cod (Gadus morhua) is an important top-predator and function as a keystone
species through that it affects the community more than would be expected by its abundance.
Stickleback (Gasterosteus aculeatus) is a small fish living close to the shoreline and not very
deep down in the water column. This makes the two species very interesting in comparing
when investigating physiological responses in low pH exposure scenarios.
1.3.
The buffering capacity of the oceans
Oceans provide a natural sink for inorganic carbon because of its buffering capacity and
photosynthetic carbon fixation by plants and algae. To date, oceans have absorbed
3
approximately half of all anthropogenic CO2 releases (Sabine et al., 2004). They stand for
about half the net primary production on earth, resulting in approximately 45 gigatons of fixed
carbon each year which is almost half of all earth primary production (Borges et al., 2006).
Increased pCO2 in surface waters have been shown to increase carbon fixation by
photosynthetic species (Doney et al., 2009). This means that the oceans are a crucial CO2 sink
and an important component for all living food chains and element cycles on earth
(Falkowski, 1998). Any factor affecting the natural systems and processes such as nutrient
flow and substance cycling in the oceans might have an immense direct effect on intrinsic
species and a possible indirect effect on other populations depending on the biological
activities of the oceans (Widdicombe, Spicer, 2007; Fox, 2008; May, 1994).
In the 18th century, when the industrial revolution started, the CO2- concentration was 280
ppm, today it has increased to 380 ppm (Widdicombe, Needham, 2007; Feely et al., 2004). As
for the world’s oceans, an increased CO2- concentration increases the partial pressure of the
gas in open waters, shifting the equilibrium of the following reaction to the right:
H2O + CO2 ↔ H2CO3 ↔ H+ + HCO3This favors the production of carbonic acid (H2CO3) and thus the production of hydroxonium
ions (H3O+). This increase in hydroxonium ion concentration ([H3O+]) in ocean surface waters
(referring to depth of about hundred meters) in turn decreases the pH (Claiborne et al., 2002).
This rapid change in CO2- concentration might affect oceanic element cycles and ecosystems.
Apart from the threat of high CO2- concentrations in the waters, there are very few
investigations about other environmentally changing factors such as mixture toxicity effects in
a low pH or high CO2 partial pressure environment, increasing temperature and salinity
variations. One study indicates low synergistic effects between a low pH and reduced salinity
(Egilsdottir et al., 2009). Additionally, pH is lowered in urbanized regions because of the
release of nitrogen and sulfur compounds, released in the burning of biomass and fossil fuel.
This produces sulfur dioxide (which combines with water to give H2SO4 which is a strong
acid) and nitrogen dioxide (which gives HNO3 which also is a strong acid, completely
dissociating in water) (Holland, 1998; Doney 2007). Increased temperature conditions have
been shown to reduce pH in body fluids of frogs, toads and turtles in a study made by Howell
and coworkers (1970).
The pH values today (around 8,1) indicates a decrease in pH of 0,1 units (equal to a 30%
increase in H+ concentration) (Caldeira, Wicket, 2003) in surface waters since the middle of
the 18th century: the beginning of the industrial revolution (Widdicombe, Needham, 2007).
According to a future scenario where CO2 releasing processes are increasing as in present
pace, the pH in surface waters will decrease to 7,9 in less than hundred years (Thornton,
2009) or, according to a scenario calculated by Caldeira and Wickett (2003) the pH level
could decrease to 7,7 in year 2100 and to 7,4 in year 2250.
An inherent buffering capacity is stabilizing the gradual pH changes in the oceans as a result
of bicarbonate ions that adds to the H+ ions and thereby withstands a change in pH. Dissolved
inorganic carbon (DIC) in marine waters exists primarily in three forms. These are
bicarbonate (HCO3-) 91%, carbonate (CO32- ) 8%, carbon dioxide (CO2 ) and carbonic acid
(H2CO3 ) 1% combined (Pörtner, 2008). HCO3- is important for the buffering capacity of the
oceans, H+ adds to the compound creating H2CO3 yielding H2O and CO2. During time and
4
increased CO2 concentration, the HCO3- will be used up and the oceans will slowly lose their
buffering capacity rendering the oceans less resistant to pH changes (Salomon et al., 2003).
Furthermore, climate change and increasing temperature on earth reduces the mixing of deep
sea water through thermo cline formation which also leads to decreased buffering capacity of
the world’s oceans (Raven et al., 2005).
The seemingly most sensitive group of marine species to oceanic hypercapnia is suggested
to be the ones that use calcium combined with carbonate (Ca2+ + CO32- ↔ CaCO3) for
incorporation into their exoskeleton or other hard surfaces. This group includes for example
corals, foraminifera, mollusks and crustaceans. A decreased pH in surface waters shifts the
equilibrium of the following reaction to the right:
CO2 + CO32- + H2O ↔ 2HCO3This lowers the concentration of CO32- and thereby the availability of this compound, this in
turn reduces the capacity for biogenic CaCO3 incorporation in these species (Gazeau et al.,
2006). The corrosive effect of these shells is mainly dependent on the saturation state (Ω) of
CaCO3 in the water which is calculated by the product of the concentration of Ca2+ and CO32divided by the solubility coefficient of either aragonite or calcite (Ω= [Ca2+][ CO32-]/Ksp
cal/arag). If this Ω for calcite or aragonite is <1, a corrosive process begins upon the shell (Fabry
et al., 2008) if the shell is not protected by an organic coating (Doney et al., 2009).
Echinoderms (juveniles and adults) use a chrystal form of CaCO3: Aragonite, in building
shells. Aragonite is particularly sensitive to ocean acidification (Politi et al., 2004).
Additionally, Echinoderms are sensitive because they do not contain any respiratory pigment
in their vascular fluids which is important for HCO3- storage; they also have a very poor
capacity for regulating interior acid base balance (Miles et al., 2007). Miles and coworkers
showed that an echinoderm named Psammechinus miliaris could only regulate very small
differences in pH (up to 0,5 units) and when exposed to a pH of 6,16, P. miliaris died within 7
days of exposure. Gao et al. (1993) showed an enhanced calcification in Hydrolithon sp.
through the elevation of pH. Semesi et al. (2009) found that the biogenic calcification process
seem to be rather directly correlated to the surrounding pH.
Photosynthetic pigments on corals give an increased pH close to the organism.
Photosynthetic CO2- fixation of these pigments on coral species increase pH in the vicinity by
reducing the partial pressure of CO2, this alters the CO32- equilibrium in favor of CO32production which enhances the species ability of CaCO3 incorporation (Gao et al., 1993). A
high CO2- concentration have been showed leading to a bleaching process of photosynthetic
pigments of three different photosynthetic algae where an increased temperature enhances the
process (Anthony et al., 2008). This reduction in photosynthetic activity leads to a decreased
pH in the coral environment.
Species residing in the most constant temperature and chemical conditions is another
sensitive group of organisms (Childress, 1995). For example, in the north Atlantic, where CO2
is taken up by the highest degree (Sabine et al., 2004), deep sea fishes live in stable conditions
and are therefore another particularly sensitive group according to Childress (1995). In
addition, Goffredi et al., (2001) suggested that deep sea organisms tend to have lower activity
in ion regulation than organisms living in shallow habitats. Long term stress symptoms are
not clearly understood and the investigations have not been very thorough. For long term
5
symptoms of low pH conditions, Jung and Jagoe (1995) showed that growth is inhibited in
fish and green tree frog (Hyla cinerea) tadpoles. Keystone species not tolerant to low pH
conditions might disrupt a whole ecosystem if vanished (e.g. the mysid shrimp (Mysis relicta)
that died out in a Canadian lake when pH was lowered to 6,0) (Schindler, 1996). Rosa and
Seibel made a study in 2008 about a top-predator: the jumbo squid (Disidicus gigas) and
found that metabolic rate and activity is significantly reduced in shallow acidified water. In
investigating energy turnover in hepatocytes of two Antarctic fish species: Grey notothen
(Lepidonotothen kempi) and the Antartic eelpout (Pachycara brachycephalum), Langenbuch
and Pörtner (2003) observed a reduction in oxygen demand (up to 34 %) and thereby a
reduced metabolic activity in hepatocytes at a pH of 6,5. An overall change in the oceanic pH
might cause more harm to specialist species than generalists, which would lead to a changed
oceanic ecosystem and a reduction in diversity.
The physiological effects of marine fishes exposed to low pH have not been fully
investigated, studies indicate that sensory abilities in different developmental stages of fishes
might be impaired (Munday et al., 2009). Dixson and coworkers found in a study 2010, that
settlement-stage larva of the marine fish Amphiprion percula could not distinguish between
predator and non-predator olfactory cues while controls could. Other studies have shown that
fishes have the ability to totally compensate for ocean acidification pH levels, sustain a
normal oxygen demand, normal growth and metabolism (Larsen et al., 1997; Fivelstad et al.,
1999).
Many different factors act on how the exposure will affect the individual e.g. surrounding
chemistry and toxic mixture of the water, ionic activity, elimination possibilities, buffering
capacity, bioavailability, exposure history and general fitness of the organism. Water
chemistry affects bioavailability of toxic compounds in the way that charged molecules does
not pass the cellular membrane as easy as uncharged species does. H+ containing species
excreted from the gills might alter the water chemistry very close to the gills in a way that
changes the toxic potential of species in the proximity (Newman, Jagoe, 1994). According to
the previous statement, Hayashi et al. (2004a) suggested that fishes are not as sensitive to a
high H+ concentration as they are to a high pCO2 because CO2 traverse easier through
membranes than charged H+ ions rendering CO2 the active molecule in the process of
hypercapnia. Hayashi et al. (2004) investigated if the toxicity arises from the low pH or CO2.
They exposed Japanese flounder (Paralichthys olivaceus) to ocean water acidified to a pH of
6,18 by CO2 in one group and by H2SO4 in another group. In the group where the water was
acidified with CO2, the fishes died within 48 hours. Not one fish died in the group where the
water was acidified by H2SO4. These results clearly show the harm of hypercapnia compared
to low pH exposure. CO2 seems not only to cause acidosis in the extracellular fluids (ECFs)
but also might cause more severe damage to cardiac cells leading to a reduction in cardiac
output and stroke volume which would be the primary reason for lethal toxicity of CO2 for all
developmental stages for marine fishes. In addition, a high CO2 concentration reduces the
affinity of O2 in the gas exchange structure of the lamellas in the gills (Lee et al., 2003;
Ishimatsu et al., 2004). Melzner et al. (2009) exposed atlantic cod (Gadus morhua) to elevated
CO2 levels (0,3 and 0,6 kPa PCO2) of seawater. They were testing swimming ability and
Na+/K+-ATPase regulation. They found that swimming ability was not altered during 12
months of exposure but Na+/K+-ATPase regulation was increased 2-fold in protein expression
and 2-fold in Na+/K+-ATPase activity. This indicates Na+/K+-ATPase to be important in ion
regulation during exposure of hypercapnia.
6
Normal pH in body fluids is about 7,4 where arterial blood is slightly more basic and
venous blood more acidic because of the dissolved CO2 in venous blood from metabolic
activity. Any increase or decrease in pH of the (extra cellular fluid) ECF leads to alkalosis or
acidosis respectively, an impairment of protein function and nerve cell synapses are the most
evident symptoms in acidosis of marine vertebrates (Widdicombe, Spicer, 2008). The
capacity for an organism to maintain a constant pH in the ECFs differs among different
species. First of all, pH dependent substances traverse between the fish and the surrounding
media as a major factor for correcting pH imbalances, this way involve several epithelial ion
exchange routes (Claiborne et al., 2002). In addition, vertebrates have four different ways to
buffer the variations in [H+]:
1. CO2 – HCO3- buffering system,
2. Peptide/Protein buffering system,
3. Hemoglobin (Hb) buffering system,
4. Phosphate buffering system.
The buffering system is a direct mechanism for maintaining proper concentrations in the
intracellular fluid (ICF), ECF and plasma in the organism. The CO2 – HCO3- buffering system
does not buffer fluctuations in H2CO3 (a buffering system cannot buffer itself). A
compensatory increase in HCO3- might occur in acidosis leading to a shift in the equilibrium
of the reaction: H2O + CO2 ↔ H2CO3 ↔ H+ + HCO3- to the left leading to a higher pCO2
which results in a higher excretion of CO2. The peptide/protein buffering system involves all
H+ transporters in the cell membrane, albumins, chaperons and other proteins which have
reducing and oxidizing domains. This buffering system is important because of the great
abundance of intracellular proteins. Hemoglobin (Hb) not bound to oxygen has greater
affinity to H+ than the oxygenated state in the tissues whereas the reaction is reversed at the
respiratory organ where H+ can be released by Hb. The phosphate buffering system acts
through the absorption of H+ by Na2HPO4 which leads to the production of free sodium ions
through the chemical equation: Na2HPO4 + H+ ↔ NaH2PO4 + Na+ (Sheerwood et al., 2005).
The second step for pH regulation is acclimatization through different excretion routes. For
marine fishes, renal H+ excretion is relatively abundant compared to the low amount of urine
produced (Claiborne et al., 1994). Most substances such as heavy metals and H+ are excreted
by the gills. Gills consist of many filaments which have thousands of small respiratory
surfaces (lamellas) facilitating gas exchange processes. Except the fine capillaries in the
lamellae, the tissue is made up of membranes and ion transporter proteins. Cells active in
balancing acid-base chemistry is chloride cells (CC) and pavement cells (PVC). Active
transporters in the gills handling H+ stress include H+ V-ATPases, Na+H+ antiport and
exchange transporters (See attachment 1). One known exchanger able to remove bases is the
Cl- - HCO3- exchanger (Hayashi et al., 2004). The CO2- molecule is distributed among the
ECF, ICF and tissues through combining with water giving H+ and HCO3-. CO2 and these two
substances are then transported by active and passive control to maintain homeostasis.
Maintenance of homeostasis in acid-base balance takes much energy in stressful conditions
(Pörtner et al., 2000).
1.4.
Biomarkers
Since ocean acidification is a relatively new area of research, not many biomarkers have
been revealed. Rimoldi et al., (2009) describes in their article that two genes (Na+ /H+
exchanger (NHE)-1 and c-Fos) regulating gill proteins are activated during hypercapnia
7
exposure, suggesting that these genes could be used as biomarkers for hypercapnia exposure.
O’Donnell et al. (2008) describes a way to use genetic expression of a gene encoding the
stress hormone heat shock protein 70 (hsp70) to study the stress compatibility of ocean
acidification in sea urchin. More research is needed to be done to evaluate possible
biomarkers for ocean acidification and physiological hypercapnia.
It is difficult to understand which impacts an elevated hypercapnia in the oceans might have
on marine populations. One similar scenario in the history of earth where oceanic surface
temperature and CO2 concentration was increased in a relatively fast pace is the “Paleocene
Eocene Thermal Maximum” (PETM). PETM was an era that occurred about 55,8 million
years ago which sustained during a time of 100.000 to 200.000 years. During this time,
geological evidences suggest that the CO2 concentration in the air was increased to values
higher than ever before and the pH of the oceans was drastically decreased in a similar way as
calculated by future combustion scenarios. Though this period was not as drastically started as
future scenarios predict in about some decades from our time, it is the most valuable source of
geological proof in what possible actions a decreased pH in surface waters may cause (e.g.
Fossils of the era show that many benthic species went extinct as a result of the low pH)
(Zachos et al., 2005). Another proposed in situ study is to observe effects around
hydrothermal vents in the Pacific Ocean near Hawaii. Brainard, a marine scientist studying
this possibility, have measured the pH close to a hydrothermal vent where the pH is about 6,1,
a few meters away, in an unstable pH environment, corals are thriving (Pala, 2009) suggesting
a possibility for environmental adaption of the species in a pH fluxing habitat.
Low pH effects have been observed on benthic oceanic species: Echinoderms act as
bioturbators and biomineralizizers in the ocean sediment, oxygenating this habitat and
substantially increasing the biological diversity. They affect the oxygen and nutrient flux
through increasing the aerobic nature of the sediment and increasing the surface area of the
sediment for chemical processes. These species seems to be very sensitive to a lowered pH in
the ocean because they use CaCO3 in their exoskeleton which depends on pH and CO32- saturation. Echinoderms are not only important because of their burrowing activity; they are
also important species in the marine habitat because they act as keystone predator species and
as a food source for certain commercial fish species (Dupont et al., 2008). Batten and Bamber
(1996) noticed that at a pH of 7,5, the burrow making polychaete: Nereis virens, significantly
reduced its burrowing activity. Results show that many echinoderm species affected are
particularly sensitive in different life stages, especially the larval stage (Politi et al., 2004).
Several ways for decreasing the atmospheric CO2 concentration (sequestration) have been
suggested such as CO2 incorporation into rocks and dumping it into the deep sea where the
high pressure and low temperatures transform the CO2 into liquid. Storage of CO2 into rock
formations is successfully being used (Korbol, Kaddour, 1995; Torp, Gale, 2004). Whereas
the dumping of CO2 into deep sea might cause great harm to fishes in vicinity because of the
mixing of the deep sea water (Ishimatsu et al., 2004).
8
1.5.
Proteomics
Proteomics is the study of proteomes. The word “proteome” originate from the two words
“protein” and “genome” and was coined by Wilkins and coworkers in 1996, the proteome
includes all the proteins expressed by the cell’s genome (Carlsohn, 2005). Different
techniques (e.g. immunofluorescence, gene therapy, shotgun proteomics, gene knockouts, the
development of antibody recognition, 2-D gel electrophoresis, differential in-gel
electrophoresis (DIGE), together with surface-enhanced laser desorption and ionization
(SELDI), matrix assisted laser desorption and ionization – time of flight mass spectroscopy
(MALDI-TOF MS)) of proteomics will help in quantification of proteins, determine
functional structures and modifications and the complex interplay between genomes and
proteins (Carlsohn 2005) (James, 1997). In measuring sublethal effects in fishes, Albertsson et
al., (2007) suggests that: “the advantages of proteomics, compared with transcriptomics, are
that proteins are more likely to reflect functional disturbances in the organism”, suggesting
that proteins are the final product of many redundant gene expression processes making
protein level results very specific for the tested variable and treatment. Protein expression is
not altered by epigenetic factors such as feedback-loops or post transcriptional activities; they
are genomic endpoints and are often the organism’s final response to physiological
disturbances to maintain homeostasis. With proteomics, it is possible to “decipher the
mechanisms of gene expression control” (Anderson and Anderson, 1998). An advantage with
proteomics compared to genomics is that proteins are usually more stable than nucleotides in
vito (Anderson and Anderson, 1998).
Proteins are amphoteric molecules and can be acidic, neutral or basic, allowing them to
migrate according their individual isoelectric point (pI) in a pH gradient powered by an
electric field. In 2D-gel electrophoresis (2D-GE), proteins are separated according to their pIs
in the first dimension and molecular weights (MWs) in the second dimension by isoelectric
focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
respectively. The results are comparable 2D-patterns of the expressed proteins in the analyzed
tissue leading to an effective visualization and screening for sensitive biomarkers (Kling and
Förlin, 2009). Problems lay in handling expertise, gel-to-gel variation, contamination risks,
protein solubilization difficulties etc. (Martyniuk and Denslow, 2008). Much effort has been
laid to advance the technique into a standardized methodology. The development of
immobilized pH gels (IPGs) has made the method more reproducible, reliable and increased
its resolution. The production of standardized equipment results in even more reproducible
2D-patterns. The Dodeca criterion cell (Biorad), for example, master 12 gels at the same time
and thereby reduce the variation between runs. In a study made by KiBeom Lee et al. (2008),
spot resolution in the gel depends much on the lengths of the IPG strip in the first dimension
while length of the gel in the second dimension does not affect resolution much. The method
is often followed by mass spectroscopy (MS) to identify interesting regulated or modified
spots.
The uses of 2D-GE are quite abundant. 2D-GE has been used to look at “in action” changes
of the genom (e.g. Biron et al., 2005) used 2D-GE to actively search for genomic changes in
9
host insects infected by the Nematomorph hairworm (Spinochordodes tellinii), a parasite
making the insects land in water resulting in an appropriate reproduction site for the adult
parasite. Biron and coworkers results show that certain proteins involved in the central
nervous system were regulated during parasite infection leading to an impairment of the
host’s neurotransmitter molecules suggesting a possible cause of the manipulated behavior of
the host insect. According to searches on published articles (www), 2D-GE has not been used
in comparing pH sensitivity of marine fishes.
1. Aim
The aim of the study was to analyze the protein expression in the gills of two marine fish
species; Gasterosteus aculeatus and Gadus morhua exposed to low pH. This was done by
exposing the fishes to control (pH 8,1) and low pH (pH 7,7) sea water for three weeks and
analyzing the gill tissue with 2D-gel electrophoresis to reveal regulated proteins between the
two species. This study is part of a larger project (POAP) that investigates the effects of ocean
acidification on marine organisms.
2. Method
2.1.
Exposure
The experiment started the 9th of September when twenty cods (Gadus morhua) were placed
in two tanks, each containing approximately 400 liters. 10 cods of varying size and sex were
distributed randomly to each of the two tanks. pH was 7,7 (with an initial variation of 0,04
units) in one. This was done by bubbling CO2 to the water from a pressure tank and pH was
controlled by AquaMedic pH computer. The pH in the other tank was controlled solely by the
flow from the sea and kept at 8,1 pH units. The flow was 6000 mL/min and was taken directly
from the sea outside the marine research facility of Kristineberg. The temperature was about
11 Co. Oxygen level was measured three times and the average value was 5,7 mg/l in low pH
tank and 5,6 mg/l in the control tank, the values had a variation of 0,3 mg/l. The surface water
of the sea outside the facility had a value of 10,8 mg/l. 25 sticklebacks (Gasterosteus
aculeatus) where placed three days before the experiment in each of the 400 liter tanks.
Sticklebacks were placed in cages inside the tanks to protect them from predation and stress.
Fig 1. pH in fish tanks: control and low pH (∆pH 0-0.04 units)
10
2.2.
Samples
Sampling was performed the 29th of September. The fishes were killed by a blow to their
heads after which samples of blood and gills were taken, length and weight measured and sex
determined. Plasma was stored in -80Co and gill samples were stored in liquid nitrogen for
future analyses.
Only female samples were used to reduce biological variation. Ten fishes were analyzed of
each species. In cod, six samples from control pH and 4 from low pH exposure were
analyzed. In stickleback five samples from control pH and five from low pH exposure were
analyzed.
Low pH (7,1)
Control pH (8,1)
Cod
4
6
Stickleback
5
5
Table 1. Samples taken for analysis.
2.3.
Protein extraction and measurement
All buffers were prepared from milliQ-water (annex B). Proteins were extracted by sonication
of the sample in 250 µl lysisbuffer (annex B). They where later centrifuged at 100000 x g in 1
h to clean the cytosolic fraction. A 25 µl sample was then used for protein quantification
while the remaining was stored in freezer for later analysis.
Protein quantification was performed by using the RcDc Protein assay (reducing agent and
detergent compatible), BioRad catalog number: 500-0120. Microfuge Tube assay protocols
were used. 5 µl of DC reagent S were added to each 250 µl of DC reagent A needed. Solution
referred to A’. A dilution series of Bovine serum albumin (BSA) with 4 steps was prepared as
standard curve. 25 µl of standards and samples were pipetted into 1,5 ml centrifuge tubes. 125
µl or RC reagent I were added each tube, vortexed and incubated in room temperature for 1
min. 125 µl of RC reagent II were added each tube, vortexed and later centrifuged at 15000 x
g for 4 min. Supernatants were discarded and 127 µl of reagent A’ were added to each tube
and the tubes were incubated for 5 min to dissolve the pellet. Tubes were then vortexed. 1 ml
of DC reagent B were added each tube and instantly vortexed. Tubes were then incubated in
room temperature for 15 min. Absorbance was read at 750 nm.
11
2.4.
2D gel electrophoresis
2.4.1. 1st dimension
Buffers for 2D-gel electrophoresis were prepared according to protocol (See attachment 2)
one day before and put in fridge until use, biolyte and DTT were added instantly before use
because of loss of function when freezed.
Isoelectric focusing (IEF) was done by diluting samples from the protein extraction with
lysisbuffer. 0,5 µg protein/µl in the diluted sample giving 100 µg of protein per sample. 200
µl diluted sample was distributed evenly along the lengths of the channel in the IEF focusing
tray. Wicks were placed over the electrodes to prevent ionic movements during focusing. The
plastic covers were gently removed from IPG strips and the IPG strips were placed with the
gel side down and the plus in the right direction. Air bubbles were carefully avoided. Mineral
oil was added to cover the strips. The lid for the IEF focusing tray was put on the tray to
ensure firm contact of the gel by the electrodes. Then the trays were placed in the Protean IEF
cell, rehydrated and focused according to a preprogrammed schedule. The rehydration was
active and performed at 12 h at 50 V and 20oC. The isoelectric focusing program exhibited
was: 250 V for 1 hour, 8000 V until approximately 35,000 V h was reached (total time was
approximately 22 hours). The strips were then added to the gels for 2nd dimension.
2.4.2. 2nd dimension
Criterion Dodeca cell (BioRad nr: 165-4130) was used for the 2nd dimension of the
electrophoresis. Agarose was heated in microwave oven and then kept at 60oC in water bath.
Runningbuffer (annex B) was diluted 5 times and 2% DDT was added to the
equilibrationbuffer (annex B). The strips were washed in runningbuffer and put in a
rehydration tray. 3 ml equilibrationbuffer was added to each channel in the rehydration tray
and the tray was gently shaked for 25 min. The gels were prepared by rinsing the well with
runningbuffer. Agarose was added to the channel in the gel. The strips were washed with
runningbuffer and later put onto the gel. 3 µl of a prestained proteinstandard was pipetted onto
a wick and put into the gel. The gels were put into the electrophoresis beaker and
runningbuffer was filled to the mark. The gels were run in 50 volts for 10 min and 200 volts
for 50 min. The gels were washed in milliQ water 3 times and then incubated with Bio Safe
Comassie for 60 min. The gels were then destained with milliQ water for 30 min.
After staining, the gels were scanned and saved as 400 dpi and 16 bit grayscale. The spots
were detected and matched manually using landmarks in PDQuest (Biorad). Statistical tests
(Mann-Whitney U) were performed in the program to reveal significant differences between
12
groups. When observing significant spots, the gels were brought to the Proteomic facility for
spot identification.
2.4.3. Spot identification (performed by the Proteomic facility)
Protein spot samples were sent to the proteomic facility and protein digestion was
performed according to Carlsohn et al. (2005). Samples were then identified using nanoflow
Liquid- chromatography masspectrometry Fourir transform ion cyclotron resonance
(Nanoflow LC-MS/MS FT/ICR). Significance of matched proteins followed the 95%
confidence interval in at least two peptide sequences.
3. Results
Significans was based on 95% confidence intervals and spot quantity should be detected in
at least 80% of the total number of exposure groups. Quantity is measured in pixel intensity
after scanning. The results gave significantly regulated spots in the 2D protein patterns: three
spots (out of 269) in cod and two (out of 169) in stickleback. All significant spots where
upregulated in the low pH exposure groups.
The Nanoflow LC-MS/MS FT/ICR analysis significantly matched four proteins with a score
higher than 25 (a lower than 0,05 expected value) and at least two peptide matches which are
the criteria for significans. The analysed sequences were run through NCBI database having
approximately 7000 sequenced cod proteins and 500 sequenced stickleback proteins. None of
the matched peptides was found in these two species but found as analogs in other species.
The matched proteins were: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
hemoglobin beta chain in cod. Annexin max3 and proteasome subunit N3 in stickleback.
13
Protein identity
Mr (Da)
pI
Expect
Function
Times
regulated
Glyceraldehyde-3-phosphate
dehydrogenase
21214
7,00
0,003
Creating
NADH
2,37
Hemoglobin beta chain
16449
n.d.
0,0096
Oxygen
transport
2,48
Unmatched protein
n.d.
n.d.
n.d.
n.d.
2,48
Annexin max3
21523
8,70
0,013
Calcium,
actin and
phospholipid
binding
1,95
Proteosome subunit N3
28460
6,00
0,015
Protein
digestion
2,07
Table 2. Protein identity with mass (Mr), isoelectric point (pI), Expected value (significant
when Expect<0,05), function and how many times it was regulated. (n.d. = no data.)
14
Fig 1. Marked significant spots after 2D-GE and gel scanning. Protein indentification gave the
following matches to known peptide sequences: Cod: 1. Glyceraldehyde-3-phosphate
dehydrogenase. 2. Hemoglobin beta chain. Stickleback: 1. Annexin max3. 2. Proteasome
subunit N3.
15
Cod
Glyceraldehyde-3-phosphate dehydrogenase
Matched sequence: 14%
Protein sequence from: Gillichthys mirabilis
1 TXXDGRQXRS CTTNCLAPLA KVIXDNFGIV GGXXEHXXRX HXHSERPWDG
51 PSGKXWRDGR GAXQNIIPAS TGAAKAVGKV IPELNGKLTG MAFRVPTPNV
101 SVVDLTVRLE KPAKYEDIKK VVKEAANGPL KGYLGYTEDQ VVSTDFNGDT
151 HSSIFDAGAG IALNEHFVKL VSWYDNEFGY SHRVCDLMAH MASKE
Hemoglobin beta chain (highest score)
Protein sequence from: Gadus morhua
The sequence also matched:
chemokine CCL-C25a (from Danio rerio)
Pi-class glutathione S-trasferase (from Anguilla Anguilla)
Unmatched protein
Stickleback
Annexin max3
Matched sequence: 8%
Protein sequence from: Pagrus major
1
51
101
151
ARGNQYKKDL EDDIKSDTSG DFRSALFELC KAGRTEGVCE QLIDSDARAL
YEAGEGRKGK DCSVFIEILT TRSALHLRKV FERYSKYSKV DVAKAIDLEM
KGDIESLLTA VVKCAGSRPA YFAERLYLSM KGKSPRKNVL TRIMVARSEI
DMKRIRDEYK KTYGKTLHQE ILDDTKGDYE KILLALCGEN
Proteosome subunit N3
Matched sequence: 12%
Protein sequence from: Oncorhynchus mykiss
1
51
101
151
201
251
MDSSGLKLNF WENGPKPGQF YSFPGSSLTP GCGPIKHTLN PMVTGTSVLG
VKFTGGVIIA ADMLGSYGSL ARFRNISRLM KVNDSTILGA SGDYADYQYM
KQIIEQMVID EELLGDGHSY SPKAIHSWLT RVMYNRRSKM NPLWNTVVIG
GFYNDESFLG YVDKLGVAYE APTVATGFGA YLAQPLMREV VENKVEITKD
EARALIERCL KVLYYRDARS YNRHEIAIVT KEGVEIVGPM SCETNWEIAH
MVSGFE
Table 2. Peptide sequence matches from nanoflow LC/MS/FT data, matching percent and the
species from which the protein is sequenced from. Matched peptides are shown in Bold.
Significant results needs to have a matching of at least 80% residue match in at least two
peptides. For the 2nd protein in cod, hemoglobin matched 3 proteins but with the highest score
for hemoglobin beta chain.
16
4. Discussion
Proteomic 2D-GE was used to generate hypotheses about biological defensive mechanisms in
cod and stickleback exposed to low pH. The results indicate that cod and stickleback can be
affected by a low pH exposure. Proteomic analyses revealed 5 significantly regulated spots in
the two fish species indicating a low frequency of responses. The Nanoflow LC-MS/MS
FT/ICR analyses matched four of the five proteins to sequenced proteins: GAPDH and
hemoglobin beta chain in cod and annexin max3 and proteasome subunit N3 in stickleback.
The third spot in cod protein expression did not give a matching result according to the
Nanoflow LC-MS/MS FT/ICR identification indicating a non sequenced protein.
4.1. Protein functions
4.1.1. Glyceraldehyde-3-phosphate dehydrogenas
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme catalyzing the sixth step
of the glycolysis; turning glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate, releasing
one molecule of NADH. NADH functions as an electron donor in energy turnover reactions.
The formation of 1,3-bisphosphoglycerate from glyceraldehyde-3-phosphate takes place in
two reactions: The first is thermodynamically favorable and the second is not favorable. In
order to excise energy from the reaction, a thioester intermediate is formed by the active site
in the enzyme. The thioester intermediate is higher in free energy than the carboxylated
product otherwise formed, rendering the reaction more favorable and thereby much faster.
The intermediate is formed by the cystein and histidine residues in the active site of the
enzyme. The enzyme has been shown to be an important enzyme in the control of the
glycolysis in lactic bacteria. In a study made by Mercade and coworkers 2005, the enzyme
was increased 4,8 fold when pH was decreased from 6,6 to 4,4 pH units while other enzymes
were not affected (Mercade et al., 2005). The glycolysis has been shown to be directly
dependent by the regulation of GAPDH in the shift between homolactic and mixed acid
metabolism in a number of bacteria strains and that the activity of the enzyme depends of the
availability of the substrate NAD+. The concentration of the enzyme increases with lowered
pH in lactic bacteria (Garrigues et al., 1997).
17
Fig 2. GAPDH activity in Lactococcus lactis ssp. cremoris MG 1363 is dependent of the
NADH/NAD+ ratio and decreases with low pH. This is compensated by an increase in the
concentration of the enzyme (Mercade et al., 2005).
GAPDH has been shown to be involved in several non-glycolytic reactions. It is involved
with the binding of human telomeres with high affinity protecting them from degradation
(Demarse et al., 2009). The enzyme has also been shown to directly associate with
microphage scavenger receptors (MSRs) together with hsp90 and hsp70. MSRs are membrane
proteins with a cytosolic domain that binds lipoproteins and is associated with the
development of artherosclerosis (thickening of arterial walls). The proposed effect of GAPDH
is that GAPDH binds to MSRs in signal transduction (Nakamura et al., 2002). It has also been
proposed to be a binding partner with a certain cell adhesion molecule L1. L1 is a protein
mediating the outgrowth of neurites. In binding GAPDH to L1 costs ATP and neurite
outgrowth was shown to be inhibited (Makhina et al., 2009). In a study made by Zhong et al.
(1999), they showed that the genetic expression of GAPDH varies in blood and tissues in the
search of housekeeping genes from newborn infants. The article concludes that GAPDH
should be avoided as a housekeeping gene because the expression varies in the relatively low
pH of the blood in newborn infants.
18
Fig 3. Ribbon diagram of Glyceraldehyde-3-phospate dehydrogenase
(Wikipedia).
The regulation of GAPDH suggests that this step of the glycolysis is a limiting step and
increasing the concentration of this enzyme would increase the entire flux through the
glycolysis. The regulation could indicate an increase in the glycolytic flux and NADH
production. The hypothesis is that the low pH exposure would be energy demanding for the
organism and processes producing energy would be regulated to maintain homeostasis and
ionic balances (Barton, Schreck, 1987). Energy could be used to produce cellular defense
mechanisms such as ion transporters and stress proteins. Other functions might be involved in
the upregulation of the enzyme to prevent the cell from the stressor.
4.1.2. Hemoglobin beta chain
In red blood cells (erythrocytes) of vertebrates, hemoglobin (hb) exists in great abundance. Hb
increases the binding capacity of oxygen in the blood by approximately 70 times (Costanzo
2007). Hb is a metalloprotein and its main function is to carry oxygen through the interior of
the organism. In fish, hb transports oxygen from the gills to the desired tissues throughout the
organism. Hb consists of four subunits (two alpha and two beta subunits) of approximately 17
kDa, each carrying a heme molecule binding an iron atom that has a high affinity for oxygen
through ion-induced dipole forces. When oxygen is bound to the protein it is called
oxyhemoglobin (hb-O2) and will be in a so called “R-state”. The conformational change that
occurs when hb binds oxygen is called “the Root effect”, creating a higher affinity for oxygen
for each oxygen atom that is bond. When the protein is fully oxygenated, it will be stabilized
until it reaches anoxic environments. In metabolically active tissues, H+-ions and CO2
molecules binds allosterically to hb causing a reduced affinity to oxygen because of reduced
cooperativity of the subunits. This causes the oxygen to be released where it is most needed (a
phenomenon called “the Bohr effect”). Hemoglobin not bound to oxygen (deoxy or T-state)
has a higher affinity for H+ ions and CO2 molecules, causing acids to be buffered and bound
to hb for later excretion through expiration (Animal physiology, pp 601). CO2 binds to a
particular site on the α-subunit of hemoglobin. In the state of ocean hypercapnia, CO2
competes with oxygen and according to Terwilliger et al. (1998), environmental hypoxia is:
19
“a key stimulus to increase the production of an oxygen-transport protein and thereby to
increase the oxygen-carrying capacity of the blood.” When in hypoxic environment, Hb uses
ATP energy and binds 2,3-bisphosphoglycerate (2,3-BPG) to stabilize the T-state which could
lead to a reduction in glycolytic substrates and an impaired energy production (Kinoshita et
al., 2007).
Fig 4. Ribbon diagram of Hemoglobin (red subunits are α- and blue
are β-chains) (Wikipedia).
Regulating the hemoglobin beta chain indicates an increase in the concentration of the entire
tetramer of hemoglobin. This is necessary for the organism to cope with hypoxia or a reduced
affinity of hemoglobin to oxygen. Another theory is that the fish needs higher levels of
hemoglobin because of more energy demanding metabolism. When the level of oxygen is
low, or pH is low and CO2 high, 2,3-BPG attaches to hemoglobin to stabilize the T-state. This
reduces the availability to glycolytic substrates giving another possible explanation for the
regulation of GAPDH and energy production.
20
4.1.3. Annexin max3
Annexins has various functions and can bind membranes through regulation of Ca2+-ions, link
Ca2+ to membrane mechanisms, e.g. they have been shown to be involved in the endocytosis
of membrane receptor proteins. Annexin V has been revealed to be a marker for apoptosis on
cell membranes (BD biosciences). Annexins are a highly conserved family of proteins with
close resemblance in many phyla of organisms. The proteins consist of repeated alpha helix
sequences that make up a homologous core that binds Ca2+-ions and phospholipids. The Nterminal of the proteins has been shown to be the most flexible region with a probable
function of being a second site for membrane binding, which would explain membrane
aggregation in association with the proteins (Shesham et al., 2008). Annexin max3 is a protein
that binds calcium, phospholipids and actin threads. The protein has been proposed by
Spenneberg et al. (1998) to function as a vesicular guide in high concentration calcium
environments.
Fig 5. Ribbon diagram of Annexin III, core unit shown in red helixes
and loops shown in green. (Wikipedia)
Regulating annexin max3 might suggest disturbed ion balance in the fish and that vesicles are
needed to transport ions in a controlled direction to maintain membrane potential and ion
balances. The ability of some annexins to promote apotosis agrees with the hypothesis that
exposure to low pH would be a stressor for the organism. Cells that are excessive or
disordered are programmed for apoptosis. Apoptosis removes cells that e.g. have been
damaged or becoming cancerous. This is a natural response to toxicants or other harmful
substances that impairs with cellular function.
21
4.1.4. Proteasome subunit N3
The proteasome subunit N3 is part of the 26S1 proteasome; a proteolytic enzyme
recognizing ubiquitin labeled proteins. There are several thousands of proteasomes per cell
and works with ATP to degrade proteins marked with ubiquitin. The proteasome consists of
two 19S caps and one 20S catalytic core subunit. The 19S caps recognize ubiquitin with the
expense of ATP. The cap then denatures the protein and leads it through the 20S core subunit
where degradation starts. The core subunit consists of four heptameric rings (two alpha and
two beta rings) with their proteolytic sites facing inwards. The rings in the middle of the core
subunit are the beta rings consisting each of seven subunits, one of these are the subunit N3.
The proteasome has a high processivity meaning that the protein being degraded is not
released until the process is complete. The protein digestion is essential for several reasons,
e.g. for reuse of amino acids that are needed for new protein synthesis, to remove enzymes
and transcription factors that are no longer needed and for producing energy. Protein
degradation has been observed in liver of salmonoid fishes in a study focusing on stress
responses by Wiseman et al. (2007). They found an upregulation of genes involved in e.g.
metabolism, immune function and protein degradation.
Fig 6. The proteasome (A) showing the four heptameric rings and
proteolytic sites in red. (B) The proteasome 19S subunit in blue and the
20S proteolytic core subunit in yellow (Baumeister et al., 1998).
Proteasome subunit N3 is needed if a larger amount of proteasome is needed. This could be
because the cell needs to prioritize between the proteins expressed in the cell if different
enzymes, ion transporters or transcription factors are needed. The degradation of proteins
might also indicate that the cell needs energy.
22
4.2. Discussion of protein regulation
Stress responses of marine fishes can, according to Iwama et al., 1999, be induced by e.g.
“extreme conditions or changes in water quality such as dissolved oxygen, ammonia,
hardness, pH, gas content, partial pressures, and temperature”. The physiological response
involves production of certain stress hormones e.g. catecholamines and cortisol, which
promotes increased oxygen carrying capacity and decrease tissue pH for a more effective
tradeoff of oxygen. The stress response in fish also involves metabolic changes such as an
elevated level of glucose in the blood through the breakdown of stored glycogen for fast
energy turnover. Energy is thereby prioritized to be used for “fight and flight” responses of
the organism. In addition, different stressors make the epithelia of the organism more
permeable to small molecules and ions (Bonga, 1997). Long term exposure to stressors may
lead to reduced reproductive and swimming ability and inhibited growth. The physiological
response of the two fish species in this study coincide well with the literature findings of
stress symptoms.
The results give a small indication of possible physiological mechanisms leading to
hypotheses for future studies. 2D-GE is a good approach for detecting specific responses in
fields of little knowledge but continued studies needs to be done to verify the different stress
responses in the 2D-GE results. For example, Surface- enhanced laser desorption/ionization
(SELDI) works in TOF-MS manner, using a laser pulse to ionize the sample to give the
individual quantity response for each protein in the sample. This is a relevant technique in the
continued studies of cod and stickleback pH exposure analysis.
Many cycles of crucial compounds have been shown to be altered as a result of ocean
acidification and many mechanisms of actions are yet to be revealed. Researchers should
focus on investigating long term effects on different marine organisms and pay attention to
the most sensitive group, effect and stressor. To investigate the protein involved in regulating
the acid-base balance, focus must attend toward localizing the specific proteins with
antibodies for a better understanding of transport routes (Freire et al., 2007) in order to
prevent changed natural habitats and reduced biodiversity.
23
5. Conclusions
In this pilot study, the protein expressions of the two fish species cod (Gadus morhua) and
stickleback (Gasterosteus aculeatus) were altered following a 22 days exposure, indicating a
physiological response from low pH or hypercapnia. 2D-GE gave that all significantly
regulated spots observed in the gels were expressed approximately 2 times higher in the low
pH exposure groups.
6. Acknowledgements
Special thanks to the Proteomics Core Facility at Sahlgrenska Academy, Gothenburg’s
University, which was funded by a grant from the Knut and Alice Wallenberg Foundation.
Thanks also to Joachim Sturve, Lars Förlin and the rest of the Ål-group at the department of
Zoological institution. Thanks to Peter Kling for all the help in 2D-GE analysis and to Sam
Dupont for controlling the pH at the marine research facility of Kristineberg.
24
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8. Attachments
8.1. Annex A – Routes of H+ in the organism
CO2 and H+ distribution in marine organism (Pörtner, 2004).
31
8.2. Annex B – Buffers
Tris (1 M)
Substance
Amount
Tris (Tris(hydroxymethyl)-aminomethan)
1,2114 g
Milli Q water
Up to 10 ml
TrisHCl (1,5 M, pH 8,8)
Substance
Amount
Tris (Tris(hydroxymethyl)-aminomethan)
9,09 g
Milli Q water and HCl are added to a pH of 8,8 and a volume of 50 ml
Lysisbuffer
(used in rehydration step in IEF)
Substance
Amount/Volume
Final conc
Urea (60,06 g/mol)
4,2 g
7M
Thiourea (
1,5 g
2M
Chaps (614,9 g/mol)
0,4 g
4%
Ampholyte (Biolyte 20% 3-10)
100 µl
0,2 %
DTT (Dithiotreitol) (154,3 g/mol)
0,154 g
100 mM
Tris ( 1 M)
0,4 ml
40 mM
BPB (Bromophenol blue)
200
µl
of
stocksolution
Milli Q water
Up to 10 ml
32
0,1
% 0,002 %
5 x SDS running buffer
Substance
Amount/Volume Final conc
Tris (Tris(hydroxymethyl)-aminomethan, 121,1 g/mol)
37,8 g
25 mM
Glycine (75,07 g/mol)
180,3 g
192 mM
SDS (288,38 g/mol)
12,5 g
0,1 %
Milli Q water
Up to 2,5 l
OBS! 5 x SDS running buffer are diluted to 1 x SDS running buffer before use.
SDS equilibration buffer
Substance
Amount/Volume
Final conc
TrisHCl (1,5 M, pH 8,8)
6,7 ml
50 mM
Urea (60,06 g/mol)
72,07 g
6M
Glycerol (100 %, 92,10 g/mol)
60 ml
30%
SDS (288,38 g/mol)
4g
2%
BPB (Bromophenol blue)
4 ml (0,1% stocksolution)
0,002%
Milli Q water
Up to 200 ml
OBS! 4 ml DTT is added right before use.
Rehydration buffer
Substance
Amount/Volume
Final conc
Urea (60,06 g/mol)
9,6 g
8M
Thiourea
3g
2M
Chaps (614,9 g/mol)
0,8 g
4%
Triton X-100
100 µl
0,5 %
Ampholyte (Biolyte 20% 3-10)
500 µl
0,5 %
BPB (Bromophenol blue)
A few grains
Milli Q water
Up to 20 ml
Buffers where stored in fridge until use.
33