Open Journal of Marine Science, 2011, 1, 31-35
doi:10.4236/ojms.2011.12003 Published Online July 2011 (
Copyright © 2011 SciRes. OJMS
Mechanisms of the Plurality of Scor paena porcus
L. Serum Albumin
A. M. Andreeva
I. D. Papanin Institute for Biology of Inland Waters RAS
Received March 18 2011; revised May 10, 2011; accepted May 20, 2011
The proteins, which bind albuminspecific dye Evans blue, are revealed in the low-molecular protein fraction
of the blood serum from Scorpaena porcus L. and identified as serum albumin. They were represented by
three bands in 2D-SDS-PAAG. MALDI-TOF-analysis revealed the fundamental similarity of the mass spec-
trum of the fragments of tryptic cleavage of proteins with molecular weight 73 and 76 kDa. The role of du-
plications and intragenic reconstructions in the creation of the plurality of scorpaena albumins is discussed.
Keywords: Scorpaena, Blood, Tissue Fluids, Low-Molecular Prote ins, A lbum in, Ma ss Spectr um , Dupl icat ions
1. Introduction
Serum albumins accomplish important functions in the
organism of vertebrates, participating in the filtration of
tissue fluid, in the transport of biomolecules and in the
plastic metabolism. Mammalian albumins are simple
monomeric proteins with the molecular weight about 67
kDa; they are represented, as a rule, by one component
on the electrophoregram [1]. Fish albumins differ from
mammalian ones in the diversity of organization ways,
physical and chemical properties: there are simple pro-
teins and glycoproteins, monomers, oligomers and ag-
gregates among them. Usually their electrophoretic mo-
bility does not co incid e with that of mamma lian alb umin,
and they are often represented by the plural forms in the
electrophoresis [2-20]. The ability of fish albumins to
bind albuminspecific dyes, palmitic acid, incapacity to
bind nickel, molecular weight and other characteristics
are used for the fish albumin identification [9,11,14,
17,21,22]. We identified serum albumin of scorpaena
Scorpaena porcus by the molecular weight, the ability to
bind albuminspecific dyes and by means of MALDI-
TOF-analysis; the results obtained were used to reveal
the mechanisms of albumin plurality.
2. Material and Methods
2.1. Objects of Study
The objects of this study were Scorpaenas Scorpaena
porcus L. from the Black Sea. For comparison we used
Mullus barbatus L., Uranoscopus scaber L., Symphodus
tinca L., Gaidropsarus mediterraneus L., Neogobius
melanostomus P. and Mesogobius batrachocephalus P.
from the Black Sea and also roach Rutilus rutilus L. and
perch Perca fluviatilis L. from the Rybinsk Reservoir.
For the work we used proteins from blood serum and
plasma and tissue fluids from the peritoneum, brain and
white muscles.
2.2. Methods of Analysis
The biological fluids obtaining. The blood was ob-
tained from the caudal artery, tissue fluids were taken by
pipetting or by the impregnation of the strip (0.5 × 4.0
mm) of the chromatographic paper Watmann 3 MM [23].
Protein concentration measurement. We used micro-
biuret method to estimate the concentration of total pro-
tein [24].
Electrophoresis methods. We analysed the albumins
by disk- and 2D-electrophoresis (in gradient of PAGE
concentration 5% - 40%, in PAGE with 8 M urea [17]
and SDS [25]. For calculation of the molecular weights
(MM) of proteins we used: myoglobin and the polymeric
forms of human serum albumin HSA and ovalbumin
OVA; the markers Fermentas PageRulerTM Prestained
Protein Ladder Plus (11, 17, 28, 36, 55, 72, 95, 130, 250
kDa). Results were processed statistically with program
package OneDscan.
The binding of proteins by albuminspecific dyes. We
studied the binding of proteins by Evans blue and brom-
cresol purple BCP by recording the formation of the pro-
tein-dye complexes in PAGE and, in the case BCP, spec-
trophotometrically. The formation of specific complex is
accompanied by λ max shift from 590 t o 603 nm [26] .
MALDI-TOF-analysis. This method was used for the
precise determination of protein MM and for compara-
tive analysis of the mass-spectrum (MS) of the fragments
of tryptic cleavage of proteins, which bind Evans blue
under the native conditions. Data obtained data were
used to determine homology of the scorpaena proteins
and for scorpaena albumin identification. Analysis was
performed on the base of Scientific Research Institute of
Physical-Chemical Medicine in the laboratory of pro-
teomic analysis. Proteins for the MALDI-analysis were
obtained from 2D-SDS-PAGE. Mass-spectra MS and the
fragmentation spectra MS/MS were obtained by the
mass-spectrometer Ultraflex II BRUKER (Germany),
equipped by UV laser (Nd). The accuracy of the meas-
ured masses of fragments was 1Da.
Albumin identification. The binding of proteins by
the albuminspecific dyes, the value of MM and MALDI-
TOF-analysis data were used for scorpaena albumin
identification. The proteins were identified by means of
“peptide fingerprint” and the fragmentation spectra
MS/MS by means of the Mascot program ( The search was carried out in the NCBI
database among the proteins of all organisms with pre-
scribed accuracy, the possible oxidation of methionine
by atmospheric oxygen and possible modification of
cysteines by acrylamide were taken into consideration.
The cumulative search on the basis of MS + MS/MS was
carried out by means of a program BioTools of v.3
(Bruker, Germany). Only those proteins with the test of
significance score > 85 (r < 0.05 ) were con sidered as the
reliable candidates.
3. Results and Discussion
Differentiation of low-molecular proteins from fishes
extracellular fluids in electrophoresis. The low-mo-
lecular fraction of the scorpaena plasma contained 6-10
proteins with MM from 20 to 90 kDa, the relative con-
tent of this fraction was 28% (Figure 1).
The same proteins were also presented also in the tis-
sue fluids of scorpaena, however, their relative content in
the peritoneal fluid was above (39.9%), and in brain tis-
sue fluid it was lower (22.3%), than in the plasma (28%).
The subunit repertoire of the proteins from tissue fluids
coincides with that of plasma proteins, this fact confirms
the identical composition of the proteins in all extracel-
lular fluids of organism (Figure 2).
Scorpaena had 15 low-molecular serum and tissue
fluid proteins in the 2D-electrophoresis in the PAGE
concentration grad ient, 24 LM-pro tein in PAGE with 8M
urea and 34 LM-protein s in SDS-PAGE (Figure 3). And
we detected only 3 macrocomponents with MM about 60
- 70 kDa under the denaturing conditions (Figure 3).
The binding of low-molecular proteins by albumin-
specific dyes. The low-molecular fraction in the disk-
electrophoresis of scorpaena and fresh-water perch
plasma contains 1-2 proteins, which bind the Evans blue,
(Figure 4).
Unlike Evans blue, the BCP dye did not bind scor-
paena proteins, but it bound all roach serum proteins un-
specifically, shifting λmax from 590 to 593 nm [27,28].
BCP did not bind scorpaena proteins and binds all roach
blood proteins in PA GE as well.
Scorpaena proteins, which bind Evans blue in the
disk-electrophoresis, were represented in the 2D-elec-
trophoresis by the large number of protein spots, among
which there were only three macrocomponents with MM
1 2 3 4 5 6 7
Figure 1. Disk- electrophoresis of the blood plasma proteins
of Mullus barbatus L. 1. Gaidropsarus mediterraneus L.; 2.
Mesogobius batrachocephalus P.; 3. and Neogobius melano-
stomus P.; 4. Uranoscopus scaber L.; 5. scorpaena; 6. and
roach; 7. LMP—low-molecular proteins. Vertical arrow
shows the electrophoresis direction.
1 2 3 M 4 5
(a) (b)
Figure 2. Electrophoresis of blood and tissue fluid proteins
of scorpaena and Mesogobius batrachocephalus P.: (a)
Disk-electrophoresis of peritoneal fluid 1, brain tissue fluid
2 and plasma 3 from scorpaena; (b) SDS—electrophoresis
of brain tissue fluid (4) and plasma (5) from Mesogobius
batrachocephalus P.; M—the marker Fermentas. Vertical
arrow shows the electrophoresis direction.
Copyright © 2011 SciRes. OJMS
Copyright © 2011 SciRes. OJMS
64, 69 and 70 kDa, which bind the dye (Figure 4). These
very proteins are supposed to be albumins, because they
bind albuminspecific dye and have MM most similar to
HSA. The results obtained revealed the plurality of
scorpaena albumins.
M1 1 2 3 4
Scorpaena albumins mass-spectra. We obtained the
mass-spectra for those albu mins, which have MM 64 and
69 kDa in SDS-electrophoresis. Calculation of MM for
these albumins by means of MALDI-TOF gave higher
values—73.2 and 76.1 kDa. The MM comparison for the
tryptic cleavage products of these two proteins revealed
their almost perfect match (Table 1). These proteins dif-
fered only in three fragments (Table 1).
M1 M2 5
4. Conclusions
The results obtained show the plurality of scorpaena al-
bumin and make it possible to assume that these proteins
are the products of the different genes, which are united
by the same origin. It is possible to explain the set of the
identical amino-acid fragments in these proteins by the
fact that one gene appeared as a result of the duplication
of another initial (ancestral) gene. The presence of the
amino-acid fragments in one protein, while they are ab-
sent in other protein, can arise from subsequent in-
tra-genetic reconstructions—deletions or insertions. The
search for the homologues of these scorpaena proteins in
the NCBI database gave no results. However, data ob-
tained made it possible to conclude that scorpaena has
serum albumin, which differ from mammalian albumin.
6 M3
Figure 3. 2D-electrophoresis of plasma and tissue fluid pro-
teins from scorpaena: in the PAGE concentration gradient
(a), in PAGE with 8M urea (b) and SDS-PAGE (c).
(1—scorpaena plasma; 2, 3, 4—tissue fluids from peritoneal,
white muscles and the brain. Marker proteins: M1—HSA
and OVA; M2—myoglobin, M3—the Fermentas marker.
Horizontal arrow shows the disk-electrophoresis direction,
vertical—gradient-electrophoresis direction, electrophore-
sis with urea and SDS-electrophoresis directions respec-
tively. Two small vertical arrows show the paths of proteins
ith MM 60—70 kDa.)
Work is executed with the suppor t of Russian Founda-
tion of Basic Research (RFBR) grant number 10-04-
5. Acknowledgements
I would like to thank my colleagues from Institute of
1 2 3 4 5 6 7 8 9 10 M
(a) (b) (c)
Figure 4. The binding of Evans blue by the blood proteins from: (a) Scorpaena 1, human 2, perch 3, HSA 4; controls: Evans
blue 5, bromphenol blue 6 in the disk-electrophoresis; (b) The staining of proteins from scorpaena 7, human 8, perch 9 and
HSA 10 by Coomassie R-250 in the disk-electrophoresis; small horizontal arrows show the areas of Evans blue binding; Ver-
tical arrow shows the disk-electrophoresis direction; (c) 2D-SDS-electrophoresis of scorpaena plasma proteins; the proteins,
which bind Evans blue, are outlined by the frame. M—the Fermentas marker. Vertical arrow shows SDS-electrophoresis
direction, horizontal—disk-electrophoresis direction.
Table 1. ММ of scorpaena albumins and their tryptic cleavage products.
MM of albumin
Da MM of products after albumin tryptic cleavage
723.38; 733.32; 778.40; 851.49; 901.43; 927.41; 949.57; 1011.51; 1050.53; 1083.52; 1125.6 2; 1197.59; 1214.65;
1243.6; 1248.57; 1256.64; 130 9 .63; 1310.60; 1317.73; 1 38 6 .78; 1420.69; 1466.74; 1509.80; 1636.81; 1658.83;
1660.69; 1680. 8 6 ; 1682.79; 1698.78; 1704.88; 1717.86; 1739 .85; 1785.89; 1808.95; 186 6 .83; 1908.86; 1 954.88;
2165.02; 2540. 0 6; 2629.26; 2805.20; 2933.31; 3054.37; 3121.7*; 3399.54
723.38; 733.32; 778.40; 851.49; 901.43; 927.41; 949.57; 1011.51; 1050.53; 1083.52; 1125.6 2; 1197.59; 1214.65;
1243.6; 1248.57; 1256.64; 130 9 .63; 1310.60; 1317.73; 1 38 6 .78; 1420.69; 1466.74; 1509.80; 1636.81; 1658.83;
1660.69; 1680. 8 6 ; 1682.79; 1698.78; 1704.88; 1717.86; 1739 .85; 1785.89; 1808.95; 186 6 .83; 1908.86; 1 954.88;
2165.02; 2540. 0 6; 2629.26; 2805.20; 2933.31; 3005.54*; 3054.37; 3070.37*; 3399.54
*albumin tryptic cleavage products, which MM doesn, t match.
Biology of South Seas (U kraina) I. I. Rudnava and V. G.
Shayda for the blood of marine fishes.
6. References
[1] E. A. Tinaeva, L. G. Markovich, V. V. Konkina and E. A.
Semikrasova, “About Possibility of Blood Proteins Poly-
morphism Using as the Index of Selection in Fur Farm-
ing,” Vestnik, Vol. 11, No. 1, 2007, pp. 122-130.
[2] F. A. Robey, T. Tanaka and T. Y. Liu, “Isolation and
Characterization of Two Major Serum Proteins from the
Dogfish, Mustelus Canis, C-Reactive Protein and Amy-
loid P Component,” The Journal of Biological Chemistry,
Vol. 258, No. 6, 1983, pp. 3889-3894.
[3] V. S. Kirpichnikov, “Genetika I Selektsija Ryb,” Nauka,
Leningrad, 1987, p. 520.
[4] L. Byrnes and F. Gannon, “Atlantic Salmon (Salmo Salar)
Serum Albumin: cDNA Sequence, Evolution, and Tissue
Expression,” DNA Cell Biology, Vol. 9, No. 9, 1990, pp.
647-565. doi:10.1089/dna.1990.9.647
[5] P. J. Bentley, “A High-Affinity Zinc-Binding Plasma
Protein in Channel Catfish (Ictalurus Punctatus),” Com-
parative Biochemistry and Physiology Part C: Compara-
tive Pharmacology, Vol. 100, No. 3, 1991, pp. 491-494.
[6] W. Nunomura, “C-Reactive Protein In Eel: Purification
and Agglutinating Activity,” Biochimica et Biophysica
Acta, Vol. 1076, No. 2, 1991, pp. 191-196.
[7] L. Vazquez-Moreno, J. Porath, S. F. Schluter and J. J.
Marchalonis, “Purification of a Novel Heterodimer from
Shark (Carcharhinus plumbeus) Serum by Gel-Immobi-
lized Metal Chromatography,” Comparative Biochemistry
and Physiology Part C: Comparative Pharmacology, Vol.
103, No. 3, 1992, pp. 563-568.
[8] V. Metcalf, S. Brennan, G. Chambers and P. George,
“The Albumins of Chinook Salmon (Oncorhynchus
tshawytscha) and Brown Trout (Salmo trutta) Appear to
Lack a Propeptide,” Archives of Biochemistry and Bio-
physics, Vol. 350, No. 2, 1998, pp. 239-244.
[9] V. J. Metcalf, S. O. Brennan, G. K. Chambers and P. M.
George, “The Albumin of the Brown Trout (Salmo trutta)
is a Glycoprotein,” Biochimica et Biophysica Acta, Vol.
386, No. 1, 1998, pp. 90-96.
[10] V. J. Metcalf, S. O. Brennan, G. K. Chambers and P. M.
George, “High Density Lipoprotein (HDL), and Not Al-
bumin, is the Major Palmitate Binding Protein in New
Zealand Long-Finned (Anguilla dieffenbachii) and
Short-Finned Eel (Anguilla australis schmidtii) Plasma,”
Biochimica et Biophysica Acta, Vol. 1429, No. 2, 1999,
pp. 467-475. doi:10.1016/S0167-4838(98)00260-X
[11] V. J. Metcalf, S. O. Brennan and P. M. George, “The
Antarctic Toothfish (Dissostichus mawsoni) Lacks
Plasma Albumin and Utilises High Density Lipoprotein
as Its Major Palmitate Binding Protein,” Comparative
Biochemistry and Physiology Part B: Biochemistry and
Molecular Biology, Vol. 124, No. 2, 1999, pp. 147-155.
[12] V. Metcalf, S. Brennan and P. George, “Using Serum
Albumin to Inter Vertebrate Phylogenies,” Applied Bio-
informatics, Vol. 2, 2003, pp. 97-107.
[13] V. J. Metcalf, P. M. George and S. O. Brennan, “Lung-
fish Albumin is More Similar to Tetrapod than to Teleost
Albumins: Purification and Characterization of Albumin
from Australian Lungfish, Neocaratodus Forsteri,” Com-
parative Biochemistry and Physiology Part B: Biochem-
istry and Molecular Biology, Vol. 147, No. 3, 2007, pp.
[14] A. M. Andreeva, “Structural and Functional Organization
of Albumin System of Fish Blood,” Journal of Ichthyol-
ogy, Vol. 39, No. 9, 1999, pp. 788-794.
[15] A. M. Andreeva, “Serum Peroxidases of Fish,” Journal of
Ichthyology, Vol. 41, No. 1, pp. 104-111.
[16] A. M. Andreeva, “The Structure of Serum Albumins of
Fishes,” Zhurnal Evolyutsionnoi Biokhimii i Fiziologii,
Vol. 46, No. 2, 2010, pp. 111-118.
[17] A. M. Andreeva, “The Role of Structural Organization of
Blood Plazma Proteins in the Stabilization of Water Me-
tabolism in Bony Fish (Teleostei),” Journal of Ichthyol-
ogy, Vol. 50, No. 7, 2010, pp. 552-558.
Copyright © 2011 SciRes. OJMS
[18] C. Szebedinszky and K. M. Gilmour, “The Buffering
Power of Plasma in Brown Bullhead (Ameiurus nebulo-
sus),” Comparative Biochemistry and Physiology Part B:
Biochemistry and Molecular Biology, Vol. 131, No. 2,
2002, pp. 171-183. doi:10.1016/S1096-4959(01)00492-4
[19] Y. Xu and Z. Ding, “N-Terminal Sequence and Main
Characteristics of Atlantic Salmon (Salmo salar) Albu-
min,” Preparative Biochemistry and Biotechnology, Vol.
35, No. 4, 2005, pp. 283-290.
[20] A. M. Andreeva and R. A. Federov, “Features of the Or-
ganization of Low-Molecular Weight Proteins from the
Blood and Tissue Fluid of the Common Stingray Dasyatis
Pastinaca L. (Chondroichthyes: Trigonidae),” Russian
Journal of Marine Biology, Vol. 36, No. 6, 2010, pp.
[21] L. L.Sulya, B. E. Box and G. Gordon, “Plasma Proteins
in the Blood of Fishes from the Gulf of Mexico,” Ameri-
can Journal of Physiology, Vol. 200, No. 1, 1961, pp.
[22] H. De Smet, R. Blust and L. Moens, “Absence of Albu-
min in the Plasma of the Common Carp Cyprinus Carpio
Binding of Fatty Acids to High Density Lipoprotein,”
Fish Physiology and Biochemistry, Vol. 19, No. 1, 1998,
pp. 71-81. doi:10.1023/A:1007734127146
[23] A. M. Andreeva, I. P. Ryabtseva and V. V. Bolshakov,
“Analysis of Permeability of Capillaries of Different De-
partments of Microcirculatory System for Plasma Pro-
teins in Some Representatives of Bony Fishes,” Zhurnal
Evolyutsionnoi Biokhimii i Fiziologii, Vol. 44, No. 2,
2008, pp. 212-214.
[24] R. F. Itzhaki and D. M. Gill, “A Micro-Biuret Method for
Estimating Protein,” Analytical Biochemistry, Vol. 9, No.
4, 1964, pp. 401-410. doi:10.1016/0003-2697(64)90200-3
[25] U. K. Laemmli, “Cleavage of Structural Proteins during
the Assembly of the Head of Bacteriophage,” Nature, Vol.
227, No. 5259, 1970, pp. 680-685.
[26] A. E. Pinnel and B. E. Northam, “New Automated
Dye-Binding Method for Serum Albumin Determination
with Bromcresol Purple,” Clinical Chemistry, Vol. 24,
No. 1, 1978, pp. 80-86.
[27] A. M. Andreeva, “Identification of Serum Albumin and
the Study Some of Its Physical Chemistry Properties in
the Representatives of the Families Acipenseridae and
Cyprinidae,” Inf Bull IBII AS USSR, Vol. 69, 1986, pp.
[28] A. M. Andreeva, “Physical Chemical Properties of Serum
Albumin of the Blood from Acipenseridae and Cyprini-
dae on the Example to Sterljad and Bream. Physiology
and the Biochemistry of the Hydrobionts,” Yaroslavl,
1987, pp. 108-114.
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