Biochemical Systematics and Ecology 50 (2013) 240–247

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Biochemical Systematics and Ecology

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Biochemical characterization of the putative isoformsof myoglobins from mollusks of the Biomphalaria genus

Kádima N. Teixeira a,*, Karyne N. Souza a, Fabrício F. Melo b, Jamil S. Oliveira a,Bruna Drabowski a, Alexandre M.C. Santos c, Marcelo M. Santoro a

a Laboratório de Enzimologia e Físico-química de Proteínas, Departamento de Bioquímica e Imunologia, Universidade Federal de MinasGerais, MG, Brazilb Laboratório de Pesquisa em Bacteriologia, Departamento de Microbiologia, Universidade Federal de Minas Gerais, MG, Brazilc Laboratório de Bioquímica e Biofísica Molecular de Proteínas, Departamento de Ciências Fisiológicas, Universidade Federal do EspíritoSanto, ES, Brazil

a r t i c l e i n f o

Article history:Received 26 November 2012Accepted 10 April 2013Available online 22 May 2013

Keywords:Autoxidation rateBiomphalariaChromatofocusingMyoglobin isoforms

* Corresponding author. Tel.: þ55 31 409 2627.E-mail addresses: kadnayt@yahoo.com.br, kadbio

0305-1978/$ – see front matter � 2013 Elsevier Ltdhttp://dx.doi.org/10.1016/j.bse.2013.04.006

a b s t r a c t

Although gastropod myoglobin has been extensively studied, there are knowledge gaps inits biological characteristics. We describe for the first time the presence of a myoglobin inthe triturative stomach of Biomphalaria gastropods. We compared biochemical parametersof myoglobins of stomach and radular muscles of Biomphalaria glabrata and Biomphalariatenagophila. Apomyoglobin and holomyoglobin were obtained. Myoglobin was the mostabundant protein in the stomach (85.0%) and radular muscles (80.0%) of the two Bio-mphalaria species evaluated. The Molecular mass and isoeletric point of stomach myo-globins were 16,124.93 Da and 7.98 and 16.095.28 Da and 7.77 for B. glabrata and B.tenagophila, respectively. Stomach myoglobins of B. glabrata and B. tenagophila rateautoxidation were equal to 8.0 � 10�4 h�1 � 0.0002 and 1.0 � 10�4 h�1 � 0.00142,respectively. Analysis of N-terminal amino acid sequencing revealed that stomach myo-globins are blocked in this region for a chemical group. Concluding, the differences weobserved in the biochemical properties of stomach and radular myoglobins of B. glabrataand B. tenagophila suggest they may be isoform representing an evolutionary event relatedto the adaptation of these proteins.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Myoglobin is a protein with a single polypeptide chain consisting of 145–155 amino acids that form eight a-helices foldedaround a hydrophobic core that contains the heme prosthetic group, a protoporphirin bound to a central iron atom. Theprotein is found in mammals and some mollusks, annelids and arthropods and has numerous functions including oxygenstorage in muscles, oxygen supplier, acts as an intracellular scavenger of nitric oxide, protecting NO-sensitive respiratoryenzymes such as cytochrome c oxidase, biochemical catalyst in oxidative phosphorylation and signal transducer (Suzuki andImai, 1998; Wittenberg and Wittenberg, 2003). The physiological functions of myoglobin are based on its reversible oxygen-binding capability and a scavenger bioactive of NO (Flögel et al., 2001) and to become an oxidase in vitro (Osawa et al., 1993).

Myoglobin concentration in tissues varies according to the animal species being closely linked to the work performed bythe tissue. The protein is abundant in certain mollusks tissues such as in the radular muscle of Aplysia in which the

nayt@hotmail.com (K.N. Teixeira).

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K.N. Teixeira et al. / Biochemical Systematics and Ecology 50 (2013) 240–247 241

concentration of myoglobin is two-fold or three-fold higher than that observed in human cardiac and skeletal muscles (Rossi-Fanelli and Antonini, 1957).

In gastropods, the presence of myoglobin has been reported either in radular muscle (Takagi et al., 1983; Terwillinger andRead, 1971; Suzuki et al., 2003) or triturative stomach (Suzuki, 1986; Suzuki and Furukohri, 1990). In respect to Biomphalariagenus, myoglobin was described in radular muscle (Dewilde et al., 1998). The genus includes species of snails that act asintermediate hosts of Schistosoma mansoni, the etiologic agent of the schistosomiasis (Teles et al., 1991). Schistosomiasis is awater-borne disease considered the second most important parasitic infection after malaria in terms of public health andeconomic impact (WHO, 2012).

Although myoglobin is a well studied protein, there are still some points of its biological functions that need to beelucidated. In a previous study, we evaluated several biochemical parameters of radular muscle myoglobin of Biomphalariaglabrata, Biomphalaria tenagophila and Biomphalaria straminea (Teixeira et al., 2011). We hypothesized that myoglobins fromdifferent tissues of the same Biomphalaria species have different biochemical parameters; they may represent isoforms.Therefore the aim of this study was to evaluate biochemical characteristics such as molecular mass, isoeletric point andkinetic parameters of the triturative stomach and radular muscle myoglobins of B. glabrata and B. tenagophila. In the presentstudy, myoglobin was detected for the first time in the stomach of two Biomphalaria species, which provides new data for acomparative biochemical analysis between the stomach and radular myoglobins and their characterization as isoforms.

2. Materials and methods

2.1. Snails

B. tenagophila and B. glabrata strains were maintained in controlled laboratory surviving conditions in a glass aquariumwith filtered and dechlorinated circulating water supplemented with calcium carbonate oxygenated by a pump. Animalswere daily fed with lettuce. For dissection (sacrifice), the animals were anesthetized by immersion in a solution of 0.05% v/vsodium pentobarbital (Hypnol�) for 5 min.

2.2. Purification of native stomach myoglobins

Specimens of B. tenagophila and B. glabrata were dissected to obtain the triturative stomachs. The crude extracts and thesize exclusion chromatography (SEC) procedures were done according to Teixeira et al., (2011). Fractions of 1.5 mL werecollected and monitored at 280 and 405 nm. The fractions with absorbance at 405 nm were pooled and dialyzed against20 mmol L�1 Tris–HCl, pH 8.5 for 12 h at 4 �C (dialysis factor: 100,000�). The dialyzed samples were submitted to twodifferent processes of ion exchange chromatography (IEX).

2.2.1. IEX IThe dialyzed samples were applied into a Mono Q 5/5 cationic exchange column (Amersham Pharmacia�) coupled to a FPLC

system.Thecolumnwaspreviouslyequilibratedwith0.1mol L�1Tris–HCl, pH8.0. Theelutionwasperformedwitha lineargradient0–1.0 mol L�1 NaCl for 30 min. Fractions of 1.5mL were collected at flow rate of 1.0mLmin�1 andmonitored at 280 and 405 nm.

2.2.2. IEX IIThe dialyzed samples were incubated at 25 �C for up to 2 h in 50 mmol L�1 potassium phosphate, pH 7.4 containing

6.0 mmol L�1 sodium dithionite to convert reduced form into oxymyoglobin; these samples were subsequently applied intoMono Q 5/5 cationic exchange column (Amersham Pharmacia�) coupled to a FPLC system. The elution and monitoringprocedures were the same used in the IEX II.

The third purification step of was performed in high performance liquid chromatography (HPLC) equipment (Shimadzu)by reversed phase chromatography (RPLC). Fractions from IEX I showing concomitant absorbance at 280 and 405 nm wereapplied into C18 Shim-pack 4.6/250 column (Shimadzu) and the chromatographic procedures were performed according toTeixeira et al., (2011). All peaks were collected and tested for the presence of myoglobin by spectral analysis in the range from200 to 800 nm. Peaks showing absorbance at 405 nm were considered the holo form of myoglobin (Ochiai et al., 2009).Calculation of chromatographic resolution was performed according to Harris (2001) using the Origin� 5.0 software.

2.3. Purity and molecular mass determination

In order to analyze the efficiency of the purification, aliquots of each chromatographic step were collected and analyzed by12.5% SDS-PAGE performed according to Laemmli (1970) at 120 V for 4 h and the proteins were silver-stained. The experi-mental molecular mass was determined by electrospray ionization mass spectrometry (ESI-Q-TOF Micro, Micromass, UK) inthe positive ion mode. Mass spectrometer calibrationwas carried out with sodium iodide in the 100–2500m/z range. Proteinsamples (20–25 mg) were solubilized in 50 mL of 50% (v/v) acetonitrile in aqueous solution of 0.2% (v/v) formic acid and appliedto the mass spectrometer by a syringe pump system at a flow rate of 10 mL min�1. The capillary and cone voltage were 2500 Vand 40 V, respectively. The final spectrum was the result from 20 combined scans. Original data (m/z) were treated andanalyzed by the Masslynx 4.0 software (Santos et al., 2008).

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2.4. Amino acid sequencing

N-terminal amino acid sequencing was performed by Edman degradation in an automatic PPSQ-21A Protein Sequencer(Shimadzu, Kyoto, Japan) coupled to a reversed-phase separation of PTH-amino acids in a WAKOSIL-PTH 4.6/250 column(Wako, Osaka, Japan) at 1.0 mL min�1, with detection at 235 nm. The samples were solubilized in 0.1% (v/v) TFA aqueoussolution and submitted to sequencing.

2.5. Isoelectric point

The isoelectric point of the stomach myoglobin was determined by chromatofocusing in the pH range from 6 to 9. Themyoglobins from B. glabrata and B. tenagophila were used in their apo form (without heme group). Briefly, approximately100–200 mg of each protein was dialyzed against the start buffer (25.0 mmol L�1 diethanolamine-HCl buffer, pH 9.5) for 12 h(dialysis factor: 100.000�). TheMono P 5/5 column (Amersham Pharmacia�) coupled in an FPLCwas usedwith a pH gradientformedwith 5.0mol L�1 NaOH and start buffer according to themanufacturer’s recommendations. The proteins were applied,separately, with Polybuffer 96 pH 6.0 (Amersham Pharmacia�) and eluted with a flow rate of 0.5 mL min�1. The pH found inthe elution of each protein was measured by potentiometer of the FPLC apparatus and checked by isolated equipment.

2.6. Autoxidation rate and myoglobin content

The autoxidation rate of oxymyoglobin to metmyoglobin was conducted according to Dosi et al. (2006), with minormodifications. The experiment was performed by monitoring the changes in the absorption spectrum at 400–750 nm of thestomach myoglobins obtained in the purification step IEX II. Data were collected every 15 min for 5 h. Absorption spectrum

Fig. 1. Chromatogram profiles of the purification steps of the stomach myoglobins of Biomphalaria species. A,. B glabrata; B, B tenagophila; 1. SEC (void volumewas not represented), 2. IEX, 3. RPLC, 4. Scanning spectrum (200–800 nm) of the peaks 1 and 2 from RPLC. In this graphics, � shows absorbance atw280 nm of thepeak 2 (apoprotein) and * shows absorbance at w405 nm of the peak 1 (holoprotein). Arrows show myoglobin peak.

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was recorded on an UV-160A UV–visible recording spectrophotometer (Shimadzu), equipped with a thermostaticallycontrolled cell holder, set at 25 �C. Autoxidation rate (k) was calculated by the gradual decrease in the absorbance at 575 nm,thus k¼ ln{[oxymyoglobinA575nm]t/[oxymyoglobinA575nm]0} where t is time (h) for incubation at 25 �C according to Chow et al.(2009). The measurements were performed in triplicate for each point.

The myoglobin content was determined spectroscopically according to Krzywicki (1982), with minor modifications.Radular and stomach muscles were carefully dissected, cleaned and dried in a vacuum concentrator. After, radular andstomach muscles wereweighed and the samemass of tissue for each organ and species of Biomphalariawas macerated in thesame volume of 50 mmol L�1 potassium cyanide for conversion of oxymyoglobins into metmyoglobins. Then, the sampleswere analyzed at 215, 280, 405 and 540 nm to calculate the amount of myoglobin per milligram of tissue. Horse heartmyoglobin was used as standard. This experiment was performed in triplicate.

3. Results

3.1. Liquid chromatography

Chromatographic procedures employed in our laboratory proved to be useful to obtain radularmyoglobins from B. glabrataand B. tenagophila with high resolution and yield (Teixeira et al., 2011). Thus, the three chromatographic steps were used toobtain pure stomach myoglobin. The profile of stomach and radular myoglobins of the two Biomphalaria species evaluatedwas similar in the three chromatography steps (data not shown). In the second step the absorbance at 405 nmwas observedin the fractions 10 and 11 for both Biomphalaria species, indicating the presence of the hemeprotein (Fig. 1A and B).

Because anion exchange chromatography showed high resolution and efficiency, themolecule of interest was eluted in thestationary phase as unbound fraction in a single and sharp peak (Fig. 1A-2). The absorbance at 405 nm was observed in thefraction that protein did not bind to resin.

The RPLC resulted in two sharp and symmetrical peaks with retention time ranging from 34.57 min (peak 1) to 35.26 min(peak 2) and from 34.25 min (peak 1) to 90 min (peak 2) for B. tenagophila and B. glabrata, respectively (Fig. 1A-3 and B-3).Absorbance at 405 nm was only observed in the peak 1 (Fig. 1A-4 and B-4).

Fig. 2. SDS-PAGE with silver stain (A) and mass spectrometer profile (B and C) from B tenagophila and B glabrata myoglobins. SDS-PAGE: lane 1. Crude extract ofthe triturative stomach from B tenagophila, lane 2. Crude extract of the radular muscle from B tenagophila, MW. Molecular weight marker (kDa), lane 3. Crudeextract of the triturative stomach from B glabrata, lane 4. Crude extract of the radular muscle from B glabrata, lane 5. Pure stomach myoglobin from B tenagophila(apomyoglobin-peak 2 from RPLC), lane 6. Pure stomach myoglobin from B glabrata (apomyoglobin-peak 2 from RPLC), Mass profiles B and C show deconvolutedand calculated mass of 16,095.28 Da for B glabrata and 16,124.92 Da for B tenagophila, respectively.

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The homogeneity of the proteins obtained in the RPLC was tested using SDS-PAGE that showed a single band for eachsample (Fig. 2, Lanes 5 and 6) and ESI-MS that showed a single distribution in the ionic form Gaussian, revealing that therewas only one molecule in the aliquot analyzed (Fig. 2B and C). The pool of proteins of crude stomach and radular muscleextract was very similar between the two Biomphalaria species studied (Fig. 2). Comparative analysis between stomach andradular proteins of the same Biomphalaria species showed that myoglobin is the most abundant protein in these tissues ofboth species. Using software tools (Quantity one� – Bio Rad) we found that myoglobin corresponds to about 85% and 80% ofthe total protein of stomach and radular muscles, respectively.

3.2. Edman degradation

The N-terminal sequencing yielded no detectable phenylthiohydantoin (PTH) amino acids after 20 cycles, which indicatesthat the stomach myoglobins from B. glabrata and B. tenagophila could be blocked at N-terminal by a chemical group.

3.3. Purity and molecular mass

SDS-PAGE and spectrophotometry analyses of the two peaks observed at RPLC showed that these peaks correspond tothe same type of protein, myoglobin. The SDS-PAGE also showed that myoglobin is a predominant protein in the stomachof two Biomphalaria species (Fig. 2). The ESI-mass spectrometry of the stomach myoglobins revealed a single ionicGaussian distribution with a molecular mass of 16,124.92 Da and 16,095.28 Da for B. tenagophila and B. glabrata,respectively (Fig. 2).

3.4. Isoelectric point

The pI values of stomach myoglobins of B. tenagophila and B. glabrata were, 7.98 and 7.77, respectively.

Fig. 3. Autoxidation rate of the stomach myoglobins from Biomphalaria species. The spectra are showed and isosbestic points are pointed by arrows. The first-order plots for the autoxidation of myoglobins at 25 �C based on the absorbance at 575 nm are inserted in the spectra. A. B glabrata (R2 ¼ 0.9998); B. B tenagophila(R2 ¼ 0.9999). Each data point indicates the average with standard deviation of triplicate measurements. Autoxidation rates are showed (k).

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3.5. Autoxidation rate and myoglobin content

The decrease in the absorbance values (at 575 nm) of themolar concentration of myoglobin and in its autoxidation rate areshown in Fig. 3. Autoxidation proceeded as a first-order reaction, and the measurement was quite reproducible as demon-strated by the small standard deviation (SD). The correlation coefficients (R2) ranged from 0.998 to 999 for all samplesexamined. The autoxidation rate was calculated by plotting the decreasing of oxymyoglobin concentration versus time; twoisosbestic points at 530 and 590 nm were observed (Fig. 3). Stomach myoglobin from B. glabrata and B. tenagophila showedautoxidation rate equal to 8.0 � 10�4 h�1 � 0.0002 and 1.0 � 10�4 h�1 � 0.00142, respectively. The content of myoglobin permass unit of tissue was 1.73 mg/mg and 1.68 mg/mg in the radular muscle and 1.51 mg/mg and 1.47 mg/mg in the triturativestomach muscle of B. glabrata and B. tenagophila, respectively.

4. Discussion

Myoglobins are present in several phyla. In the phylum Mollusca they seem to be more abundant in species exposed toenvironment with low oxygen pressures (Weber, 1980). These proteins are distributed in some tissues and in the hemolymphof invertebrates. Globins are commonly found in the radular muscle, nerves and heart of many gastropods. In bivalvemollusksmyoglobin can be found in the adductor muscle, cardiac muscle and feet (Weber and Vinogradov, 2001).

In the present study by using several purification steps, we detected, for the first time, the presence of myoglobin in themuscle of two Biomphalaria species, which were obtained as pure holomyoglobin and apomyoglobin. We evaluated thebiochemical characteristics of the stomach myoglobin and compared with those of radular muscle. The two Biomphalariaspecies evaluated showed different retention times.

In order to obtain pure myoglobin, the analysis of the stomach and radular muscles of Biomphalaria included severalpurification steps as described previously (Teixeira et al., 2011). At the end of the three purification steps we obtained pureholomyoglobin and apomyoglobin of stomach of the two Biomphalaria species evaluated, which showed different retentiontimes. Holomyoglobins showed the lowest retention time and revealed absorbance at Soret band (405 nm) indicating thepresence of hemeproteins. The later peaks corresponded to apomyoglobins that lost its prosthetic group, probably due to theorganic solvent used in the chromatography. The heme group is bound to myoglobin in a cavity named hydrophobic pocketconsisting of many amino acids with non polar properties (Suzuki and Imai, 1998). Thus, during reversed-phase chroma-tography when the acetronitrile concentration increases, losing of the heme group of the protein exposes these amino acidsthat could increase the interaction of molecules with stationary phase, retarding the protein elution. All peaks were eluted inmore than 60% acetonitrile suggesting the hydrophobic nature of these proteins.

The behavior of both stomach and radular myoglobins during purification process indicates that these proteins possessvery similar biochemical properties as electric charge, hydrophobicity and molecular size, that however are not enough tosupport that these proteins are the same ones.

The result of themass analysis (SDS-PAGE and ESI-MS) indicated that stomachmyoglobins of B. glabrata and B. tenagophilaare monomeric proteins in accordance with universal globin category that usually consists of 145–155 amino acid residues.Most of the myoglobins distributed from bacteria to mammals, fall under this category, which led Suzuki and Imai to proposethe term globin superfamily to define these proteins (Suzuki and Imai, 1998). Similar results were found for radular myo-globins of B. glabrata and B. tenagophila, which are monomeric proteins also in agreement with a previous study of our group(Teixeira et al., 2011). The molecular mass difference between the stomach and the radular myoglobins observed in the samespecies of Biomphalaria could be explained by differences in the amino acid sequence.

The pI values of stomach and radular myoglobins were similar between the Biomphalaria species analyzed (Teixeira et al.,2011) and they are in accordance with the range values described for other myoglobins, such as horse heart (7.3) and spermwhale myoglobin (8.3) (Westermeier et al., 1985). In proteins, the change of pI varies with the number of chemical groupsinserted in their sequences. Moreover, the change is greatest when inserted groups have electrically charged moieties(Legendre et al., 1998). Thus, the difference in the pI between the stomach and the radular myoglobins we observed in thesame Biomphalaria species may be explained by difference in the primary amino acid sequence as well as by difference inmolecular mass.

The N-terminal sequencing indicated that the stomach myoglobins of Biomphalaria species examined were blocked. Thisblockade has also been observed in radular myoglobin of B. glabrata (Dewilde et al., 1998), B. tenagophila and B. straminea(Teixeira et al., 2011). Probably, these proteins may have undergone post-translational modifications, acetylation or another.The biological role of acetylated protein is diverse, reflecting the different acetyltransferases that have evolved to meet in-dividual proteins or protein family (Polevoda and Sherman, 2002). Proteins containing serine or alanine residues as the firstN-terminal amino acid, as occurs in radular myoglobin of B. glabrata (Dewilde et al., 1998) and Aplysia limacina (Cutruzzolaet al., 1996) are frequently acetylated. These sequences along with methionine, glycine and threonine represent more than95% of the N-terminal acetylated residues (Driessen et al., 1985; Persson et al., 1985).

In invertebrates myoglobin has been reported in some tissues including body wall of Platyhelminthes (Tuchsmid et al.,1978), Nematoda (Blaxter et al., 1994) and Annelida (Garlick and Terwilliger, 1977), adductor, radular muscle and tritu-rative stomachmuscles of Mollusca (Suzuki, 1986; Terwillinger and Read,1971; Smith et al., 1988). The presence of myoglobinwas firstly described in the radular muscle of the mollusk B. glabrata, a fresh water snail, by Dewilde et al. (1998). Thebrownish-red color observed in the stomach of these animals was supposed to be due to the presence of myoglobin.

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It is known that myoglobin is the most important determinant of the color of meat (Dosi et al., 2006). In live animals, thereis a balance between the purple-red myoglobin (deoxy form) and the cherry-red (oxy form). During storage of meat, thesetwo forms of reduced myoglobins quickly become oxidized (Faustman and Cassens, 1990). In its ferrous state the iron of theheme group of myoglobin binds to the molecular oxygen to form the oxymyoglobin. Otherwise, when iron binds to water thedesoxymyoglobin is formed. In both cases myoglobin is responsible for the red color to the tissues. The ferrous ion can beconverted into ferric ion, and consequently metmyoglobin is formed (oxidation) that has a brownish-red color being unableto bind to the oxygen (Guilherme et al., 2008).

The prevalence of myoglobin in the stomach indicates that the color is brownish-red on account of their presence in themet form, while the radular muscle in situ has a bright red color that indicates the predominance of myoglobin in the oxyform. The values of the autoxidation rates of stomachmyoglobin from B. glabrata and B. tenagophila, obtained experimentally,indicate that they are slow if compared with the autoxidation rates of myoglobins from other mollusks (Suzuki et al., 1993).Comparatively, themyoglobin of B. glabrata is less stable thanmyoglobin from B. tenagophila being its autoxidation rate eight-fold greater. Fast autoxidation rate is caused by instability of the globin, but the specific amino acid residues responsible forthe instability of the molluscan myoglobins remain unknown.

The radularmuscle of Biomphalaria has an intense red color, while the stomachmuscle presents a brownish-red color, exceptin albine individuals, inwhich thehemoglobinof thehemolymphconfers a characteristic red color to tissues. According toWarrisand Rhodes (1977) in very red tissues such as those found in adult bovine, myoglobin can represent 90% or more of the totalpigment. In pale muscle tissues such as chicken breast, myoglobin can represent up to half of the total pigment.

In respect to the stomach muscle color, autoxidation rate from myoglobin is closely related to the brownish-red colorobserved as result of the metmyoglobin presence (Dewilde et al., 1998). In the present study, analysis of the myoglobincontent per mass unit of tissue in the radular and stomach muscles ruled out the possibility of the difference in color to beassociated to concentration of myoglobin. The methodology used to quantify the content of myoglobin after conversion intometmyoglobin was satisfactory because the amount of other proteins containing heme group, such as cytochromes, flavinsand catalases is relatively small in these tissues, which probably not interfere with the tissue color (Ledward, 1984).

The autoxidation rate of stomach myoglobin of B. tenagophila is 12-fold lower than its equivalent radular myoglobin.However, for B. glabrata the stomach myoglobin revealed an autoxidation rate 2.5-fold lower than the radular myoglobin.Carver et al. (1992) constructed a recombinant sperm whale myoglobin in which a leucine (Leu 29 - B10) was replaced by aphenylalanine. The resultant protein in its oxygenated form proved to be identical to the wild protein in structure however,the former had 15-fold increase in oxygen affinity but, its autoxidation rate decreased. This finding indicates that theautoxidation rate is associated to the primary sequence of the protein. Because in the present study the autoxidation rate wasdifferent for the stomach and radular myoglobins of Biomphalaria, we may hypothesize that difference in primary amino acidsequences of the proteins are responsible for the divergent values found.

Because the experiments of autoxidation rate were performed in vitro, under the same conditions, we can rule out thepossibility of the difference observed between the values of the autoxidation rate of myoglobins from the same species ofBiomphalaria be due to variation in physiological conditions or chemical microenvironment of the animals. The difference inthe autoxidation rate of stomach and radular myoglobins between Biomphalaria species and also between the myoglobins ofthe same species may be explained by differences in the tertiary structure or change of position of the amino acids close to theheme group.

Structural differences could affect the heme group position or the environment around them, and thus influence the speedbywhich the oxygen binds to and leaves the iron atom of the prosthetic group. A global alignment of primary sequences of theradular myoglobins from B. glabrata and B. tenagophila showed that although the mollusk species belong to the same genustheir myoglobins had five different amino acid residues, which could be enough to generate structural change.

Chow (1991) showed that high values of autoxidation rate is related to the molecule instability, thus molecules with lowvalues of autoxidation rate are resistant to metmyoglobin formation; in this way the low values observed for radular andstomach myoglobins of Biomphalaria species indicate resistance to oxidation. Concerning the color, the results disagree withautoxidation data obtained for Biomphalaria. However, at physiological conditions, the stomach brownish-red color could beexplained by the interference of the stomach pH in autoxidation ratewhich is accelerated by acidic conditions usually presentin the stomach (Matsuura et al., 1962).

The biochemical data obtained in this study suggest that stomach myoglobins of Biomphalaria mollusks are probablydifferent in their primary sequence because the values provided by experimental mass, pI and differences observed in theautoxidation rates. The differences observed between stomach and radular myoglobins of the same Biomphalaria species alsosuggest that these proteins are supposedly isoforms. The presence of these putative isoforms should be considered anevolutionary event that could be related to the adaptation of these proteins to bind to oxygen according to the tissue needs.

Acknowledgments

We thank Dr. Gifone A. Rocha, professor at the Faculty of medicine – UFMG for linguistic correction contribution.

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