Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Abstract
Snakehead fish, also known as Haruan, is recognized in Asia Pacific countries as a remedy for healing of wounds. The fish
enhances dermal wound healing and reduces post-operative pain and discomfort. The efficacy of wild type snakehead fish has
made it a common food served to women after childbirth or those who had undergone surgery. Due to high demands of snakehead
fish, farming of the fish is now carried out commercially. However, the flesh of cultured snakehead fish has been said to produce
different texture from the wild type fish. In this study, analysis of the protein composition of the flesh of snakehead fish was
carried out. Wild type snakehead fish of different sizes and caught in different months of the year were compared. The data
showed that fish of smaller sizes yielded higher protein content as compared to the bigger fish. However, protein profiles of the
fish were similar for all the different months of catching. The major group of protein in snakehead fish was enzymes, followed by
structural proteins. The protein profile displayed may be used as a reference for farming and culturing of snakehead fish.
of powdered tissue was dissolved in 1000 l of buffer removed and the gel pieces were dried in a vacuum
(for the Tris buffer extract, 10 l of extract was diluted in centrifuge. The gel pieces were swollen in digestion buffer
1000 l of buffer), it was then centrifuged at 12,000 x g containing 50 mM NH4HCO3, 5 M CaCl2, and 12.5 ng/l
for 30 min and the supernatant was recovered. Five L of trypsin in an ice-cold bath. After 45 minutes, the
of the supernatant was then added to the wells. 250 L of supernatant was removed and replaced with 10 l of the
the Bradford Reagent was added to each plate well that same buffer but without trypsin to keep the gel pieces
contained standards and samples. The plate was then wet during enzymatic cleavage at 37 C overnight.
shaken for approximately 30 seconds and incubated at Peptides were extracted from the gel matrix by adding 15
room temperature for 15 minutes. The absorbance was l of 20 mM NH4HCO3, vortexed and incubated at room
measured at 595 nm. A standard curve was plotted using temperature for 10 minutes. The supernatant was
net absorbance versus protein concentration of each recovered after a brief spin. This was followed by adding
standard. The protein concentration of unknown samples (1 to 2 times the volume of gel pieces) 5% (v/v) formic
was determined by comparing the average A595 values acid in acetonitrile:water mixture (70:30), vortexed and
against the standard curve. incubated for 20 minutes at room temperature. It was
then spun down and the supernatant was recovered. These
Sodium Dodecyl Sulphate-Polyacrlyamide Gel steps were repeated 3 times. Pooled extracts were dried
Electrophoresis (SDS-PAGE) down in a vacuum centrifuge and stored at 20C.
SDS-PAGE was performed as described by Laemmli
[7]. Ten per cent polyacrlamide gel in a vertical slab gel HPLC-MS Analysis
apparatus (Hoefer) was used. Protein samples (Tris buffer Mass spectrometric analysis was carried out using an
extraction) were then loaded into the wells of polymerized ion trap mass spectrometer (Agilent, VL). The peptides
gel. Electrophoresis was performed at a constant voltage were ionized using the electrospray soft ionization
of 200 volts when samples were in the stacking gel. technique (ESI). The mass spectrometer was operated in
When the dye front reached the resolving gel, voltage a two-mode program consisting of full MS Scan and full
was increased to 245 volts. The run was stopped when MS/MS Scan, whereby, the most intense ion in the full
the dye front was 2 to 3 mm away from the bottom edge MS Scan was isolated and subjected to full MS/MS Scan.
of the gel. At completion of electrophoresis, the glass
sandwich was disassembled. The stacking gel was The peptides resulting from the in-gel digestion was
discarded and the resolving gel was stained using reconstituted in 50 L of ddH20. The peptides were
Coomassie Blue. Molecular weights of the proteins were separated on a reversed-phase column (1mm x 250 mm,
determined by comparing relative mobility of protein 5 m, 300 A). The HPLC separation condition was at
bands to the standard protein markers. linear 5% B to 95 % B in 65 minutes at 20 l/min flow
rate. The eluent of HPLC was directed to a mass
The Coomassie Blue stained gel images were acquired spectrometer, which was interfaced with the HPLC. The
and digitized using Versadoc Imaging Scanner. Protein parameters used for acquiring the MS data were: heated
bands intensity analysis was carried out using the Camag capillary temperature of 300 C, dry gas flow rate of 8.0
TLC Scanner 3 and the densitometry analysis was L/min and nebulizer gas pressure of 30.0 psi. The
performed using the CATS software. parameters set for the MS/MS Scan were collision energy
(voltage) = 1.15 V, charge state = 2, minimum threshold
In-Gel Digestion = 5000 counts, and the isolation width = 2 m/z. The MS/
MS spectra were recorded in the automated MS to MS/
The polyacrylamide gel was washed thoroughly with
MS switching mode with an m/z dependent set.
100 mM NH4HCO3. The protein bands were then excised
from the gel. In-gel digestion using trypsin was performed
according to Shevchenko, et al. [8] with slight Sequence Database Search
modification. The gel pieces were first excised and shrunk The MS/MS data were subjected to Mascot protein
by dehydration in acetonitrile. The solvent was then database search engine (www.matrixscience.com). The
discarded and the gel pieces were dried in a vacuum search engine contains the calculated spectra for all
centrifuge. A volume of 10 mM dithiotreitol (DTT) in peptides in the National Centre for Biotechnology
100 mM NH4HCO3 sufficient to cover the gel pieces was Information (NCBI) non-redundant sequences database
added and the protein was reduced for 1 hour at 56C. [9]. The taxonomy and enzyme selected was
After cooling to room temperature, the DTT solution was Actinopterygii (Ray-Finned Fish) (29309 sequences) and
replaced with a same volume of 55 mM iodoacetic acid Trypsin, respectively, whilst Fixed Modification was
in 100 mM NH4HCO3. After 45 minutes incubation at Carboxymethyl (C). The Peptide Mass Tolerance was set
ambient temperature in the dark with occasional vortexing, at 2 Da whereas 0.8 Da was set for the Fragment
the gel pieces were washed with 50-100 l of 100 mM Mass (MS/MS) Tolerance. The data format was selected
NH4HCO3 for 10 minutes, dehydrated with acetonitrile, as Mascot Generic and only one missed cleavage was
rehydrated in 100 mM NH4HCO3 and dehydrated in the allowed. Instrument type set was ESI-TRAP i.e.
same volume of acetonitrile. The liquid phase was Electrospray Ionization and Ion Trap Mass Spectrometer
Proteomic analysis of snakehead fish muscle tissue 27
(1100 Series, Agilent, Germany). Proteins functions and The phenomenon of cannibalism in snakehead fish
characteristic information were obtained from both the may be one of the reasons why muscle protein synthesis
PubMed (www.ncbi.nlm.nih.gov/entrez) and Swiss Prot activity in smaller snakehead fish is more active and
(www.expasy.ch/sprot/sprot-top.html). rapid as compared to the larger fish. The secretion of
myofibrillar protein and collagen were greater in the
Results and Discussion smaller fish for their movement to survive in the
present of larger predator. In contrast, the consistent
Snakehead fish has long been consumed as a source protein content amongst the larger fish shows the fishes
of dietary protein. It is also well known traditionally for of 25 to 38 cm lengths have achieved their full protein
its medicinal property for healing of wounds. In this capacity.
study, the proteins extracted from the fishs muscle tissue
were analyzed. Different sizes of wild type snakehead The problem of cannibalism is believed to be the
fish caught at different seasons were used in the study. cause of the low survival of smaller fishes in culturing of
The data obtained provide useful information on the snakehead fish [10]. The alternative approach would be
nutritional and medicinal properties of snakehead fish. to provide adequate food [11] or partially control
Culturing of snakehead fish has been carried out in cannibalism by grading fishes into approximately similar
Malaysia due to the high demand of the fish. As a large size group.
proportion of fish muscle tissue is made up of proteins,
cultured fish muscle tissue texture can be monitored by Figure 2 illustrates the protein contents of the three
comparing their protein profile with those of the wild different batches of snakehead fish. It was found that
type fish. snakehead fish from B1 and B2 did not vary significantly
in their protein concentration (P>0.05). Fishes from both
In order to study the protein profile of snakehead fish, of these batches contained on average 0.3640.001 mg
fishes of different sizes that were caught at different protein/mg tissue and 0.3710.001mg protein/mg tissue,
seasons were analyzed. Three batches of snakehead fish respectively. However, snakehead fish from B3 showed
caught in November (B1), January (B2) and March (B3), significantly higher protein content than B1 and B2
respectively were used in this study. From each batch, (P<0.05). Snakehead fish from B3 contained on average
eight different lengths of snakehead fish were used, which 0.4490.001 mg protein/mg tissue.
were at 16 cm, 23 cm, 24 cm, 25 cm, 28 cm, 29 cm, 30
cm and 38 cm lengths. Different in protein concentration in the three batches
of snakehead fish (Figure 2) can be explained by the
In B1, it was found that smaller fish (16 cm and 23 seasons when the fish were caught. In Malaysia, the
cm) contained significantly higher protein content weather condition for November (B1), January (B2) and
(P<0.05) than the larger fish (Figure 1). The same March (B3) are dry, extremely dry and rainy, respectively.
observation also occurred in B2 and B3. However, only Our data have shown that the fish that were caught during
the 16 cm fish showed significantly higher protein content rainy season yielded significantly higher protein compared
(P<0.05) than the larger fish in these two batches of to those that were caught during the dry seasons. During
fishes. Generally, the protein content for the fish length rainy season food availability is much more abundant for
from 25 to 38 cm did not differ significantly with respect carnivore fish type, such as the snakehead fish.
to their sizes in all the three batches of the fish analyzed.
Protein Concentration from Haruan with Various Length Protein Content from haruan B1, B2 and B3
30.00 600.0
b
Concentration (mg / mL)
ab ab
25.00 ab
ab 500.0 b
b ab a
Content (mg / g)
a
b b ab
20.00 a ab
a ab ab a a ab
a a 400.0 a a
ab
a B1
15.00 B2 300.0
B3
10.00 200.0
5.00 100.0
0.00 0.0
16 23 24 25 28 29 30 38 B1 B2 B3
Length (cm)
Batch Number
Figure 1: Comparison of the protein concentration (50 mg
of dry tissue) of Snakehead fish muscles tissue Figure 2: Protein content of Snakehead fish from B1, B2 &
from three batches of fish. Protein concentration B3. The average protein content of all the fish
was analyzed using Bradford method. Different according to the fish length. Different alphabet
alphabet annotation represents significant annotation represents significance different
difference (p<0.05) between the different batches (p<0.05) between the different batches of fish.
of fish. Statistical analysis was carried out using Statistical analysis was carried out using analysis
analysis of variance (ANOVA). of variance (ANOVA).
Proteomic analysis of snakehead fish muscle tissue 28
Table 1: List of water soluble proteins detected in Haruans muscle tissue. Band number are refer to indication mentioned
in Figure 3.
SWISS-PROT
Protein Name Mw pI Function Band number
Accession number
KIBOA/ P00570 Adenylate kinase (EC 2.7.4.3) 21761 8.94 Enzyme 14
Q804Y1 Aldolase (Fragment) 17427 8.73 Enzyme 10
Q8JH72 Aldolase A. 40223 8.27 Enzyme 2,8,9,10
Q7ZW73 Aldolase b, fructose-bisphosphate 39700 8.75 Enzyme 10
Q6PUS4 Brain glycogen phosphorylase Pygb 97916 6.11 Enzyme 2,7
Q9YGE7 Complement factor Bf-1 85034 5.90 Enzyme 1,6
Q7ZU04 Creatine kinase, brain 43178 5.49 Enzyme 7,9
Q804Z1 Creatine kinase 43231 6.29 Enzyme 9
Q804Z2 Creatine kinase 43032 6.32 Enzyme 9
Q9I8I6 Creatine kinase (EC 2.7.3.2) 46859 8.73 Enzyme 9
S13164 Creatine kinase (EC 2.7.3.2) 43267 6.20 Enzyme 7,9,10
Q98SS7 Creatine kinase (Fragment) 29125 8.89 Enzyme 7,9
Q9DFM2 Creatine kinase (Fragment) 21139 5.79 Enzyme 9
Q7T1J1 Creatine kinase brain isoform (Fragment) 42608 5.89 Enzyme 9
Q7T306 Creatine kinase CKM3 43115 6.29 Enzyme 9
Q9YI16 Creatine kinase M1-CK 42983 6.21 Enzyme 2,9
Q9YI15 Creatine kinase M2-CK 43133 6.22 Enzyme 9
Q9YI14 Creatine kinase M3-CK 43185 6.25 Enzyme 9
Q7T1J0 Creatine kinase mitochondrial isoform precursor 47108 8.50 Enzyme 9
Q7T1J3 Creatine kinase muscle isoform 1 42713 6.32 Enzyme 9,10
Q7T1J2 Creatine kinase muscle isoform 2 42888 6.44 Enzyme 9
Q7ZZM5 Enolase (Fragment) 28757 8.15 Enzyme 6
Q6TH14 (AAQ97775) Enolase 1 (AY398342 NID) 47848 - Enzyme 6
Q6GQM9 Enolase 2 47160 4.77 Enzyme 6
O57518 Fructose-1, 6-bisphosphate aldolase 39957 6.21 Enzyme 10
Q76BF6 Phosphoglycerate kinase (Fragment) 41657 6.04 Enzyme 9
Q8AY84 Phosphoglycerate kinase (Fragment) 11317 4.67 Enzyme 9
Q6NXD1 PKM 2 protein 58598 6.36 Enzyme 5
Q803D2 Platelet-activating factor acetylhydrolase, isoform 47080 6.97 Enzyme 7
Ib, alpha subunit b.
Q76IM5 Pol-like protein 149432 9.28 Enzyme 11,12
Q7SXV3 Pygb protein (Fragment) 60118 7.30 Enzyme 7
Q8JJC2 Pyruvate kinase 58767 6.35 Enzyme 5
Q8QGU8 Pyruvate kinase 58582 7.96 Enzyme 5
Q7M558 Replicase/ helicase/ endonuclease 350347 8.68 Enzyme 9
BAD04856 (Q76B34) Reverse transcriptase 132804 - Enzyme 14
Q7T040 Solble guanylyl cylase alpha2 subunit 90214 7.54 Enzyme 10
Q8UW40 ST7 protein 58059 7.03 Enzyme 14
Q6DR47 Topoisomerase 2 (Top 2A protein) 178147 8.93 Enzyme 2,5,8,10,11,
12, 13, 14, 15
Q76BE1 Triose phosphate isomerase (Fragment) 25178 6.00 Enzyme 11
Q7T315 Triosephospahte isomerase 1b 27100 6.90 Enzyme 11,12
Q90XF8 Triosephosphate isomerase B 26476 7.60 Enzyme 12
Q9PWD1 TYK2 tyrosine kinase 129986 8.38 Enzyme 7,9
Q7ZU23 Actin, alpha 1, skeletal muscle 42304 5.23 Structural 2
Q6TNW2 Actinin, alpha 2. 104086 5.23 Structural 7
Q6DHS1 Actin, alpha 2, smooth muscle, aorta 42374 5.23 Structural 2
Proteomic analysis of snakehead fish muscle tissue 29
SWISS-PROT
Protein Name Mw pI Function Band number
Accession number
The protein profile of the aqueous soluble protein and 38 cm fishs length, respectively. The protein profiles
extracted from various sizes snakehead fish muscle tissues of fish with different lengths and month of catches did
from B2 and B3 is shown in figure 3 (protein profile of not differ significantly. Upon Coomassie Blue staining,
B1 is not shown; there was no variation between the protein bands, which were evenly distributed in the range
three batches). Each of the lanes was loaded with similar of molecular masses from 10 kDa to 205 kDa were
amounts (50 g) of protein extracts from fish of different detected. The relative intensity of the protein band in
lengths. Lanes 1 to 6 represent the protein profiles from each lane was evaluated using densitometry analysis
B2 snakehead fish at 23, 24, 25, 28, 29 and 30 cm fishs (Figure 4). In addition to the similar protein profile
length, respectively. Lanes 7 to 14 represent the protein displayed by all the fish, the relative intensity of the
profiles of B3 snakehead fish at 16, 23, 24, 25, 28, 29, 30 proteins is also similar. Thus, the non-variable features
(protein profiles and bands intensity) shown by wild type
snakehead fish is beneficial for monitoring of the protein
composition of cultured snakehead fish.
protein, calcium ion binding protein, DNA/RNA-binding In addition to sarcoplasmic proteins, myofibrillar
protein and signal transduction protein, which made up a protein or structural protein is also made up the major
minor constituent that consist of less than 2.4 % of the group of protein identified in snakehead fish. There were
total protein detected. Moreover, a series of hypothetical a total of six different myofibrillar proteins detected;
proteins or unknown gene products (about 8.2 % of the they were actin (alpha 1, skeletal muscle), actinin (alpha
total proteins) were also identified in this study. Generally, 2), actin (alpha 2, smooth muscle, aorta), fast skeletal
hypothetical proteins are still considered as a group of myosin light chain 3, myosin heavy chain (fragment) and
proteins that have no indication about their existence at skeletal muscle actin (fragment). Other than these major
the protein level. Most of them have been only described proteins, some minor proteins such as Complement factor
at the nucleic acid level as well as predicted from cDNA Bf-1, Brain glycogen phosphorylase Pygb, Pol-like
sequences but were never been identified by protein protein, Platelet-activating factor acetylhydrolase (isoform
chemical method so far [12,13]. Ib, alpha subunit b), Pygb protein (Fragment), Replicase/
helicase/endonuclease, Reverse transcriptase, Solble
The major group of enzymes identified belonged to guanylyl cylase alpha2 subunit, ST7 protein and many
sarcoplasmic proteins, which is mainly composed of more (as listed in Table 1) were also found in snakehead
enzymes associated with energy-producing metabolism fish muscle tissue. These proteins were detected as low
[14]. The identified sarcoplasmic proteins were found to abundant proteins in snakehead fish muscle tissue.
responsible for the glycolysis activity and ATP hydrolysis.
Among the enzymes, kinases are the most frequently The list of protein identified in snakehead fish muscle
identified proteins. It was revealed that twenty-seven tissue (Table 1) shown that the glycolytic and ATP
proteins or 31.8 % of the total identified proteins were metabolism are the main activities of the fish muscle
categorized as kinases. The proportional of major tissue. Both of these metabolic specializations are
enzymes found in snakehead fish muscle tissue is shown essentially required for power locomotor in fish. The
in Figure 5. These enzymes include kinases, aldolase, high abundance of these two groups of enzyme together
dehydrogenase, isomerase, enolase and others. By with myofibrillar proteins suggests that snakehead fish
number, six proteins (7.1 %) were responsible for aldolase muscle is composed mainly of white muscle tissue.
activity. Ten proteins (11.8 %) were classified as
dehydrogenase and another six proteins (7.1 %) were
known as isomerase. Three proteins (3.5 %) were derived
Conclusion
from enolase family. A series of glycolytic enzymes The protein profiles of snakehead fish of different
were identified in this study. These proteins were sizes that were caught in different months of the year
Phosphoglucose isomerase-2, Aldolase (also known as were compared. The results showed that all the fishes
Fructose 1,6-biphosphate aldolase), Triosephosphate have similar protein profiles, where each protein band
isomerase, Glyceraldehyde-3-phosphate dehydrogenase, consisted of identical proteins. Furthermore, the relative
Phosphoglycerate kinase, Enolase, Pyruvate kinase and intensity of protein bands of all the fishes analyzed is
L-lactate dehydrogenase. also similar. In view of high demands of snakehead fish,
culturing of the fish is the only solution. The present
30
data can be used as a reference for obtaining cultured
snakehead fish most similar in fish muscle protein
Number of Identified
25
composition to the wild type fish.
20
Proteins
15
10 Acknowledgements
5 We would like to thank Universiti Sains Malaysian
0 short term grant for providing financial support to carry
out this project. We also want to extend our gratitude
se
e
se
s
e
se
er
as
as
na
la
na
th
er
ol
do
O
ge
En
om
Al
ro
Is
yd
Enzymes
D
References
1. Qasim. The growth of the freshwater murrel, Ophiocephalus 9. Perkin DN, Pappin DJC, Creasy DM and Cottrell JS.
punctatus Bloch. Hydrogiologica 1966; 27: 289-316. Probability-based protein identification by searching
2. Bowman WC and Rand MJ. Textbook of Pharmacology, sequence databases using mass spectrometry data.
2nd Ed. Blackwell Sci. Pub., Oxford, London. 1980. Electrophoresis. 1999; 20: 3551-3567.
3. Mat Jais AM, McCulloch R and Croft K. Fatty Acid and 10. Ng PKL, and Lim KKP. Snakeheads (Pisces: Channidae):
amino acid composition in Haruan as a Potential Role in Natural history, biology and economic importance. In
Wound Healing. Gen. Pharmac1994; 25: 947-950. Essays in Zoology. Paper Commemorating the 40 th
4. Baie S Hj, Sheikh KA. The wound healing properties of Annivesary of the Department of Zoology, National
Channa striatus-cetrimide cream-tensile strength University of Singapore (ed. Chou L. M. and. Ng P. N. L),
measurement. J. Ethnopharm. 2000a; 71: 93-100. pp. 127-152. Department of Zoology, National University
5. Baie S Hj, Sheikh KA. The wound healing properties of Of Singapore, Singapore. 1990.
Channa striatus-cetrimide cream-wound contraction and 11. Folkvord A. and Ottera H. Effects of initial size distribution,
glycosaminoglycan measurement. J. Ethnopharm. 2000b; day length, and feeding frequency on growth, survival and
73: 15-30. cannibalism in juvenile Atlantic cod (Gadus morhua L.).
6. Bradford, MM. A rapid and sensitive method for the Aquaculture. 1993; 114: 243-260.
quantification of microgram quantities of protein utilizing 12. Fountoulakis M, Tsangaris G, Oh J, Maris A and Lubec G.
the principle of protein-dye binding. Analyt. Biochem. Protein profile of the HeLa cell line. J. Chromato. A.
1976; 72: 248-254. 2004;1038: 247-265.
7. Laemmli UK. Cleavage of Structural Proteins during the 13. Afjehi-Sadat L, Shin J, Felizardo M, Lee K, Slavc I and
Assembly of the Head of Bacteriophage T4. Nature. 1970; Lubec G. Detection of hypothetical proteins in 10 individual
227: 680-685. human cell lines. Biochim. Biophy. Acta. 2005; 1747: 67-80.
8. Shevchenko A, Matthias W, Vorm O and Mann M.. Mass 14. Nakagawa T, Watabe S and Hanshimoto K. Identification
spectrometric sequencing of protein from silver-stained of three major components in fish sarcoplamic proteins.
polyacrylamide gels. Anal. Chem. 1996;68: 850-858. Nippon Suisan Gakkaishi. 1988; 54 (6): 999-1004.