Mechanisms of the Plurality of Scorpaena Porcus L. Serum Albumin

doi:10.4236/ojms.2011.12003

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Open Journal of Marine Science, 2011, 1, 31-35

doi:10.4236/ojms.2011.12003 Published Online July 2011 (http://www.SciRP.org/journal/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

E-mail: [email protected]

Received March 18 2011; revised May 10, 2011; accepted May 20, 2011

Abstract

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-

32 A. M. ANDREEVA

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 (www.ma-

trixscience.com). 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

LMP

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.

A. M. ANDREEVA

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).

(a)

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.

(b)

6 M3

(c)

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-

00954a.

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.

34 A. M. ANDREEVA

Table 1. ММ of scorpaena albumins and their tryptic cleavage products.

MM of albumin

Da MM of products after albumin tryptic cleavage

73214

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

76128

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.

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