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forez v razdelenii biologicheskikh makromolekul. (Electrophoresis in Separating Biological Macro molecules), Moscow, 1982. 16. Fyhn Unni, E.H. and Bolling, S.
ISSN 00220930, Journal of Evolutionary Biochemistry and Physiology, 2006, Vol. 42, No. 6, pp. 678—685. © Pleiades Publishing, Inc., 2006. Original Russian Text © A. M. Andreeva, 2006, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2006, Vol. 42, No. 6, pp. 537—542.

COMPARATIVE AND ONTOGENIC BIOCHEMISTRY

Effect of Destabilizing Factors on the StructuralFunctional Hemoglobin Parameters in Euryhaline and Diadromous Fish A. M. Andreeva Institute of Biology of Internal Waters, Borok, Russian Academy of Sciences, Yaroslavl Province, Russia Received June 14, 2006

Abstract—A comparative analysis of stability of the hemoglobin structuralfunctional organization to effect of some experimental factors modeling action of internal medium and habitat was per formed in representatives of Chondrostei and Teleostei belonging to euryhaline and diadromous fish. Differentiation of hemoglobins by stability parameters was shown to coincide to division of the studied fish into two main ecological groups: euryhaline fish with unstable hemoglobin and diadro mous fishes with stable hemoglobin. In turn, the euryhaline fish by the character of destruction of hemoglobins under effects of destabilizing factors are differentiated into three subgroups: cartilagi nous fish, cartilaginous ganoids, and bony fish. Thus, differentiation of hemoglobins by parameters of resistance to destabilizing factors is determined by peculiarities of the fish mode of life and is of adaptive nature. The described in the present study method of determination of critical precipita tion concentration (CPC) of hemoglobin may be used as an expressmethod for estimation of he moglobin resistance to salt effects, while the CPC values—as one of criteria of fish belonging to various ecological groups (diadromous, euryhaline). DOI: 10.1134/S0022093006060032

INTRODUCTION In hydrobionts, the habitat is extremely variable by its hydrological and hydrochemical parameters, the most important of which are temperature, sat uration of water with oxygen and carbon dioxide, mineralization, and pH. Biochemical adaptations of hemoglobin (Hb) to changes of environmental conditions are based on use of numerous different mechanisms described in detail in literature [1–6]. All of them are strategically aimed at adjustment of hemoglobin function to the habitat conditions. Such adjustment is due to the fact that the oxygen affinity of Hb is under an intensive effect of selec tion of Hb binding and releasing oxygen under the temperature and oxygenation conditions that are characteristic of habitat of a particular species [2].

As for adaptation of the hemoglobin structure, with the peculiar to Pisces general tendency for estab lishment and stabilization of the hemoglobin tet ramer, there takes place a significant variability in the primary structure of polypeptide protein chains, the main function of this variability being to provide maximum efficiency of protein func tioning [7]. The main requirement to all structural hemoglobin modifications is to preserve protein structural integrity at fluctuations of the internal medium composition, which are fixed by natural selection and occur in the course of fish adapta tion to the habitat. Thus, at changes of the habitat salinity, adaptation of the internal medium com position in marine cartilaginous fish is based on a compensatory change of urea concentration in body fluids, while in the sturgeons, of urea and salt

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concentrations [8]. The changes of concentration of the internal medium components (urea and salts), which are produced by external factors, can produce a destabilizing effect on the hemoglobin structure to cause its destruction [3]. The effects of destabilizing factors on hemoglobins are ex pressed to the greatest degree in migratory fish that are regularly exposed to abrupt shifts in hydrolog ical and hydrochemical environmental conditions during anadromous and catadromous spawning migrations [9]. Therefore, the hemoglobin struc ture in migratory fish should be sufficient stable in order not only to endure the environmental factor fluctuations, but also to perform the main func tion on oxygen supply of tissues [3, 10]. The goal of this study was the comparative anal ysis of structuralfunctional stability of organiza tion of hemoglobins to action of some experimen tal factors modeling effects of the internal medi um and habitat in cartilaginous and bony fish be longing to euryhaline and diadromous forms. MATERIALS AND METHODS Hemoglobins (Hb) were studied in cartilaginous fish: marine euryhaline spiny dogfish Squalus acan thias L., buckler skate Raja clavata L., Atlantic stin gray Dasyatis pastinaca L. (10, 3 and 2 specimens, respectively); bony fish: cartilaginous ganoids— euryhaline freshwater sterlet Acipenser ruthenus L. and semidiadromous Russian sturgeon Acipenser güldenstädti B., as well as migratory starred sturgeon A. stellatus L. and beluga Huso huso L. (50 individ uals from each species); bony fish: euryhaline freshwater bream Abramis brama L., and zander Stizostedion lucioperca L., (200 and 30 individual, respectively). Cartilaginous fish were caught in the Black Sea (the region of Batumi), sturgeons—in the Volgograd reservoir and Volga estuary, bream and zander—in the Rybinsk and Volgograd reservoirs. Hb of sexually mature fish were analyzed. Hb from erythrocytes was isolated as described previously [11]. The Hb concentration was determined by absorption changes at the region of γband of oxy Hb [11]. To estimate stability of the Hb structuralfunc tional parameters, protein was subjected to action of factors destabilizing protein structure. The salt (ammonium sulfate, 5–75% of saturation) and

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freezing–thawing were used as experimental de stabilizing factors modeling effects of habitat; urea (5–8 M solution) was used as a factor modeling effect of internal medium. Effect of ammonium sulphate on Hb was evalu ated by changes of protein solubility and of Hb spectral characteristics (λmax in the Soret’s band area). After adding ammonium sulphate (5–75% saturation) to the Hb solution, the sample was incubated for 30 min and centrifuged; then absorp tion spectra were recorded in the range from 405 to 430 nm. The maximal extinction value and the corresponding ammonium sulfate concentration were considered the ordinate and abscissa, respec tively, on the Hb saltingout curve. On passing the maximal extinction point on the Hb absorption curve, Hb lost its stability and was precipitated. The salt concentration value at which Hb started pre cipitation was considered the critical precipitation concentration (CPC). Before and after exposure to destabilizing fac tors the structural (the ability to form regular crys tals, level of Hb organization—monomer, dimer, tetramer, aggregate) and functional Hb parame ters (the λmax absorption value in the Soret’s band area for identification of oxy, deoxy, and met Hb) were determined [3]. Hb was considered stable if its CPC values were high and after exposure to destabilizing factors it was able to form regular crystals and to remain in the working oxyform with λmax about 413–414 nm. Hb with signs of structural degradation was con sidered unstable when the following took place: (1) decomposition of molecules into subunits or into heme and globin revealed electrophoretically (chromatographically); (2) inability of Hb to form regular crystals; (3) Hb precipitation from the so lution at low ammonium sulphate concentrations; (4) predominance of nonworking metHb with λmax about 406 nm. Hb crystals were obtained by adding two volumes (2V ) of saturated ammonium sulphate solution to one volume (V ) of fish Hb solution (Hb concen tration of 0.19–2.0 g%). The solution was kept in a closed vessel on cooling for 24 h [10]. The crystal size was estimated from diameter of the prism base D (μm). Aggregation of Hb and its decomposition was recorded using column gel chromatography on

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Molecular weight of native structural variations was determined in PAAG gradient under non denaturating conditions, with use as markers of ferritin (440 kDa), bovine serum albumin (BSA, 67 kDa), ovalbumin (OA, 45 kDa), cytochrome c (12 kDa), troponins T, I, and C (38, 24, and 18.5 kDa, respectively). Molecular weight of subunits was determined in SDSPAAG, with use as mark ers of BSA, OA, cytochrome c, and troponins T, I, and C. Hemin was stained with Muller’s reagent [15]. RESULTS Fig. 1. Scheme of fish electrophoretic spectra. (1 ) Spiny dogfish, (2 ) thornback ray, (3 ) Common stingray, (4 ) beluga, (5 ) sterlet, (6 ) Russian sturgeon, (7 ) stellate sturgeon, (8 ) zander, (9 ) bream. Vertical arrow from cathode (–) to anode (+) indicates direction of disk electrophoresis. Vertical scale of electrophoretic mobil ity (Rf) is graduated from 0.0 to 1.0.

Fig. 2. Elution profile of spiny dogfish hemoglobin from Sephadex G100 column. Abscissa: volume of fractional yield V (ml); ordinate: absorption at 280 nm. (1 ) High molecular aggregate; (2 ) Hb, tetramer; (3 ) Hb, dimer; (4 ) Hb, monomer.

Sephadex G100 and G200 and electrophoreti cally: by the method of diskelectrophoresis in polyacrylamide gel (PAAG) [12], in PAAG gradi ent concentrations [13], in SDSPAAG [14] in twodimensional SDS and PAAGgradients. Hb stained by Coomassie R250 and benzidine [15].

Cartilaginous euryhaline marine fish. Using disk electrophoresis, 6–8 components of Hb were found in the spiny dogfish and buckler skate (Rf 0.038 –0.85), while 10–12—in the Atlantic stin gray (Rf 0.04–0.5) (Fig. 1). In the spiny dogfish, the Hb elution from the Sephadex column was rep resented by peaks of monomer, dimer, tetramer, and the hemefree high molecular aggregate (HMA) (Fig. 2), an aggregated globin. The heme free protein inclined to polymerization has also been found in Hb of other Elasmobranch fish [16, 17]. No aggregated Hb forms were detected in skates. From the data of column gelchromatog raphy the Hb molecular weights in spiny dogfish and skates were 66–68 and 34–36 kDa for the Hb tetramer and dimer, respectively. When using SDSelectrophoresis, the molecular weights of Hb subunits were identical in spiny dogfish and buck ler skate (13.5–15 kDa) and differed from those of less mobile Hb subunit in chuco (14.5–16 kDa). In the gradient of PAAG concentrations under nondenaturating conditions the spiny dogfish Hb was destroyed in the course of electrophoresis. Addition of 5 M urea to PAAG and electrode buffer has made it possible to prevent the Hb destruction and to stabilize it. In this case the electrophoretic spectrum was represented by the single structural variant, Hb dimer. Unlike Hb of the spiny dogfish, the skate Hb was stable under nondenaturating conditions. The cartilaginous fish Hb was rapidly dehydrat ed by ammonium sulphate. The spiny dogfish Hb was precipitated at once at all ammonium sulphate concentrations (5–75%) (Fig. 3, 1 ), the CPC val ue amounting to 5%.

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The spiny dogfish Hb crystals formed in ammo nium sulphate were represented by short hexago nal prisms with the base angles repeating every two ones, with the base diameter D of approximately 100 μm (Fig. 4a). Freezing–thawing caused destruction of the spiny dogfish Hb molecules in the form of distur bances of the crystallization process. The tetramer Hb of sterlet and sturgeon were separated into 3–4 basic and 3–5 minor fractions; teleost Hb were represented by only one fraction (Fig. 1). After diskelectrophoresis under condi tions of twodimensional electrophoresis in PAAG concentration gradient, each Hb fraction of stur geons was divided into several Hb components with identical charges and different, but close by mo lecular weights corresponding to tetramer (Fig. 5), which distinguished this protein from Hb of bream and zander represented by only one band after disk electrophoresis and electrophoresis in PAAG con centration gradient [18]. Under conditions of SDSelectrophoresis, Hb in all fish was represent ed by the single band of monomer with molecular mass of 16–18 kDa. The Hb crystals in ammonium sulfate were fine short tetraand hexagonal prisms, about 100 μm in the D base diameter (Fig. 4), often with struc tural defects (skewed prisms, dual growth, etc.). 5 M urea caused a dissociation of the Hb tetram ers into dimers. The curves of Hb saltingout in (NH4)2SO4 in sterlet and zander had similar CPC values—from 20% to 25% and in the Russian sturgeon—from 25% to 30% (Fig. 3). In the case of the ammonium sulfate saturation from 5 to 25–30% the Hb oxy form still was detected, but MetHb predominat

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Fig. 3. Saltingout curves of fish hemoglobins by sul phate ammonium. Abscissa: sulfate ammonium satura tion (%), ordinate: absorption of hemoglobin (ABS, ar bitrary units). (1 ) Spiny dogfish, (2 ) zander, (3 ) bel uga, (4 ) Russian sturgeon, (5 ) stellate sturgeon, (6 ) sterlet.

ed. If the ammonium sulfate saturation exceeded 25–30%, Hb was represented solely as the met form. The bream Hb the only form was metHb at all salt saturation values. The bream Hb was pre cipitated at all (NH4)2SO4 saturation values (from 5 to 75%), the CPC value amounting to 5%. The bream Hb reminded the spiny dogfish by the CPC value and by shape of the saltingout curve. Freezing–thawing and storage of Hb solution for

Fig. 4. Hemoglobin crystals. (a) Spiny dogfish (scheme), (b) and (c) sterlet (photo). Magnification × 800. JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 42 No. 6 2006

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Fig. 5. Scheme of twodimensional electrophoresis of sterlet hemoglobin. Direction: (a)—diskelectrophore sis, (b)—electrophoresis in PAAG concentration gra dient.

2 weeks at 4°C led to Hb destruction in all fish spe cies. The Hb destruction was also detected from disturbance of crystallization. Two main types of destruction were revealed: the type I—rupture of bonds between heme and globin in the Hb mole cule (such destruction was found by hemin accu mulation on the Kohlrausch’s border in the course of electrophoresis under nondenaturating condi tions); the type II—the breakdown of Hb tetramer into dimers and then monomers (such destructions were identified by the disappearance of the tetram er band and the appearance of the dimer and/or monomer band on electrophoregrams). Transformation of oxyHb to metHb was found to facilitate a more effective decomposition of Hb molecules. In bony fish, Hb was quickly trans formed into metHb under effects of salts, urea and freezing–thawing. The degraded structural Hb variants formed defective crystals during crystalli zation. The Hb destruction of the type I is the most char acteristic of teleosts, whereas of the type II—of ster let and sturgeon. The hemeglobin bond was firm in Hb of sterlet and sturgeon, whereas unstable in Hb of bream and zander to the extent that Hb un derwent a comparatively fast destruction even without use of destabilizing factors. Thus, 2 h after blood sampling, additional Hb bands appeared in PAAG (one in bream and two in zander); the bands were stained for protein, but were not stained with benzidine; the released hemin was accumulated on the Kohlrausch’s border. Thus, action of experimental destabilizing fac

tors on Hb in euryhaline fresh water teleosts and semidiadromous Russian sturgeon resulted in Hb destruction, but its character was different in car tilaginous ganoids and in teleosts. By CPC values the studied species could be differentiated into groups (in the ascending order of Hb stability): euryhaline bream → euryhaline sterlet, zander → semidiadromous sturgeon. Diadromous bony fishes. The migratory forms were represented by cartilaginous ganoids—starred sturgeon and beluga. By Hb charge these species were differentiated into 2–4 main fractions (Fig. 1), each of which, as in the case of sterlet (Fig. 5), contained several Hb components with identical charge and different, but close values of molecular weights corresponding to tetramer. Un der conditions of SDS electrophoresis, differenti ation by the molecular weights disappeared: the molecular weight of the Hb subunits amounted to 17–18 kDa. The Hb crystals in ammonium sulphate were represented by large, short and long tetra and hex agonal prisms with bases about 1200 μm in diame ter that exceeded 12 times D of Hb crystals in eu ryhaline freshwater teleosts (sterlet, bream, and zander), euryhaline cartilaginous marine (spiny dogfish), and semidiadromous fish (Russian stur geon). Treatment with 5 M urea led to decompo sition of Hb tetramers into dimers. In Hb saltingout curves in beluga and starred sturgeon the CPC value amounted to 35–40% ammonium sulphate saturation (Fig. 3). At salt saturation from 5 to 35–40%, hemoglobins were predominantly present as oxy and deoxyforms. At salt saturation above 35–40%, the beluga and starred sturgeon Hb were precipitated. Formation of the sediment began no less than 2 h after addi tion of salt and ended 1–2 days later. The beluga and starred sturgeon hemoglobins were highly re sistant: even after two freezing–thawing, Hb of anadromous fish did not lose the ability to form regular large crystals. DISCUSSION The revealed differences in stability of the Hb structuralfunctional parameters to actions of de stabilizing factors (urea, salts, freezing–thawing) in the studied fish can be explained by peculiari

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ties of internal medium and of conditions of spe cies habitation. Thus, in cartilaginous (euryhaline marine) fish, both Hb stabilization (spiny dogfish) and Hb insensitivity (skates) took place under ef fect of high urea concentrations. Similar results were obtained in Dasyatis salina [19] and other Elasmobranchii species, whose Hb is insensitive to high urea concentrations (to 5 M) [20]. Both the insensitivity of Hb to urea and the stabilizing urea action on spiny dogfish Hb can be explained only by taking into account formation of the blood pro tein system in cartilaginous fish under conditions of high urea concentrations (2–2.6% in sharks and 1.42–2% in skates). Cartilaginous fish are known to respond to re duced environmental salinity by a decrease of urea reabsorption in renal tubules and an increase of its urinary excretion. Owing to these regulatory mech anisms, some sharks endure considerable changes of water salinity when appearing even in fresh wa ters. Since the water salinity fluctuations in ma rine cartilaginous fish are compensated by varia tions in urea concentrations in their internal me dium, it can be suggested that the elasmobranch Hb (due to the absence of urea gradient in tissues) also should endure significant fluctuations of urea concentration and be resistant to them. The dif ferent effect of urea on Hb structure of spiny dog fish and in skates might be due to peculiarities of osmoregulatory mechanisms in sharks and skates belonging to different ecological groups. Under effect of high urea concentrations in bony fish (euryhaline, freshwater, semi and diadromous fish) the Hb tetramers were destroyed into dimers. In this case the Hb destruction also can be ex plained by peculiarities of the internal medium in cartilaginous ganoids and bony fish whose urea concentration is 10–50 times lower than that in marine cartilaginous species. Thus, adaptation of the Baikal sturgeon and the Amu Darya big shov eler to water with 10.5‰ salinity was associated with a twofold increase of the urea blood concen tration [8]. However, the reached threshold of the blood urea concentration in sturgeons all the same was significantly lower than the stationary blood urea concentrations in marine elasmobranches. In teleosts and some sturgeons, unlike shark fish, the blood salt content fluctuates [8, 21]. Analysis of Hb saltingout parameters in the studied fish has

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shown that the CPC values of hemoglobins are ar ranged as follows (in the ascending order of resis tance): spiny dogfish, → sterlet, → sturgeon → beluga, bream zander starred sturgeon (euryhaline) (euryhaline) (semiana (anadromous) dromous)

The differences in teleosts Hb stability to salt action between bream and zander can be explained by that these species belong to different ecological groups: predator pelagophages (zander) and me sobenthophilous benthophages (bream). Differen tiation of the sturgeon fish Hb by stability to ac tion of salts coincides clearly with fish differentia tion into euryhaline, semianadromous, and anadromous species. Thereby, for the fish studied by the Hb stability to saltingout, the above scheme may be presented in the following form: euryhaline → semianadromous → anadromous. Different action of all destabilizing factors on the Hb structuralfunctional parameters in the stud ied fish can may be presented the most illustratively by the example of sturgeons including anadromous (starred sturgeon, beluga), euryhaline (sterlet), and semianadromous forms (Russian sturgeon). The anadromous sturgeon Hb proved to be the most resistant to high ammonium sulphate concentra tions that had no saltingout and denaturating ef fects on Hb for a long time. Unlike Hb of euryha line sterlet and the semianadromous Russian stur geon as well as euryhaline cartilaginous and bony fish, Hb of anadromous fish endured twofold freezing–thawing and long storage without loss of the capability for crystallization and with preser vation of the working oxyform. Such pronounced differences seem to be due to the mode of life pe culiarities of anadromous fish and were formed as adaptation to sharp hydrological and hydrochem ical variations at migrations from sea to river and vice versa. The absence of such migrations in eu ryhaline marine and freshwater fish determined a low resistance level of their Hb. Thus, according to different stability to experimental destabilizing factors the sturgeon Hb can be arranged as follows (in the ascending order of resistance): euryhaline → semianadromous → anadromous. By the CPC value the Russian sturgeon Hb is in an intermediate place between euryhaline and

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anadromous fish forms; however, by the total pa rameters, we still ascribe it to the group of unstable hemoglobins of euryhaline fish. Thus, Hb differentiation by parameters of stabil ity to action of experimental destabilizing factors does not coincide with taxonomic fish differentia tion (cartilaginous, teleosts; bony fish, cartilaginous ganoids). With differences in structural parameters, hemoglobins of cartilaginous and bony fish have resemblance of stability to saltingout (spiny dog fish, bream) and freezing–thawing (spiny dogfish, bream, zander, sterlet, sturgeon), whereas hemo globins of cartilaginous ganoids and teleosts (bony fish), with similarity in structural parameters (form of Hb crystals), can be divided by CPC values, sta bility to freezing–thawing and crystal sizes—into two groups: stable and unstable hemoglobins. The obtained data allow suggesting that the dif ferences in the Hb stability to destabilizing factors are due to peculiarities of the fish mode of life and have adaptive character. The Hb differentiation by stability coincides with differentiation of the stud ied species into two main ecological groups: eury haline fish with unstable Hb and diadromous fish with stable Hb. In turn, the euryhaline fish group is differentiated by the character of Hb destruction into three subgroups: (1) elasmobranches, (2) car tilaginous ganoids, and (3) bony fish. The method described in this paper for determi nation of the Hb CPC value can be used as an ex pressmethod for estimation of Hb stability to salt action, while the CPC values—as one of criteria of the fish belonging to different ecological groups (anadromous, euryhaline), as well as for control of the fish physiological status.

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