Isolation of heat-tolerant myoglobin from Asian swamp eel Monopterus ...

Fish Physiol Biochem (2012) 38:1533–1543 DOI 10.1007/s10695-012-9644-y

Isolation of heat-tolerant myoglobin from Asian swamp eel Monopterus albus Chatrachatchaya Chotichayapong • Kittipong Wiengsamut Saksit Chanthai • Nison Sattayasai • Toru Tamiya • Nobuyuki Kanzawa • Takahide Tsuchiya

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Received: 26 December 2011 / Accepted: 9 April 2012 / Published online: 27 April 2012 Ó Springer Science+Business Media B.V. 2012

K. Wiengsamut e-mail: [email protected]

peptide fragments of this protein identified by LC– MS/MS were homologous to Mbs of sea raven Hemitripterus americanus, yellowfin tuna Thunnus albacores, blue marlin Makaira nigicans, common carp Cyprinus carpio, and goldfish Carassius auratus. According to the Mb denaturation, the swamp eel Mb had thermal stability higher than walking catfish Clarias batrachus Mb and striped catfish Pangasius hypophthalmus Mb, between 30 and 60 °C. For the thermal stability of Mb, the swamp eel Mb showed a biphasic behavior due to the O2 dissociation and the heme orientation disorder, with the lowest increase in both Kdf and Kds. The thermal sensitivity of swamp eel Mb was lower than those of the other Mbs for both of fast and slow reaction stages. These results suggest that the swamp eel Mb globin structure is thermally stable, which is consistent with heat-tolerant behavior of the swamp eel particularly in drought habitat.

N. Sattayasai Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected]

Keywords Denaturation rate LC–MS/MS MALDI-TOF–MS Myoglobin Peptide fragment Swamp eel Monopterus albus Thermal stability

Abstract Myoglobin from Asian swamp eel Monopterus albus was purified from fish muscle using salt fractionation followed by column chromatography and molecular filtration. The purified Mb of 0.68 mg/g wet weight of muscle was determined for its molecular mass by MALDI-TOF–MS to be 15,525.18 Da. Using isoelectric focusing technique, the purified Mb showed two derivatives with pI of 6.40 and 7.12. Six C. Chotichayapong K. Wiengsamut S. Chanthai (&) Department of Chemistry, Faculty of Science, Center of Excellence for Innovation in Chemistry, Khon Kaen University, 123 Mittrapab Road, T. Ni-Muang, A. Muang, Khon Kaen 40002, Thailand e-mail: [email protected] C. Chotichayapong e-mail: [email protected]

T. Tamiya N. Kanzawa T. Tsuchiya Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo 102-8554, Japan e-mail: [email protected] N. Kanzawa e-mail: [email protected] T. Tsuchiya e-mail: [email protected]

Introduction Myoglobin (Mb) is a favorable model heme protein that has been intensively studied for many years in relation to structure–function relationship and the reaction it concerns (Kendrew et al. 1960; Wittenberg

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et al. 1975; Jones et al. 1979; Watts et al. 1980; Nichols and Weber 1989; Seidel and Adkins 1989; Chow 1991; Chanthai et al. 1996, 1998; Kooyman and Ponganis 1998; Wen and Chau 2001; Ueki and Ochiai 2004; Stewart et al. 2004; Ueki et al. 2005; Dosi et al. 2006; Chow et al. 2009). The emphasis is largely on those from mammalian sources particularly in deep diving mammals, which are able to submerge for a long time (Kendrew et al. 1960; Wittenberg et al. 1975; Kooyman and Ponganis 1998). Study on the physico-chemical properties, the oxygen affinity level of Mb is higher than that of hemoglobin. The distribution and concentration of Mb in these animals are also a reflection of its physiological function (Perutz et al. 1965; Fosmire and Brown 1976; Nichols and Weber 1989; Marleen et al. 1998; Birnbaum et al. 1994; Marcinek et al. 2001; Polasek and Davis 2001). The Mb structure and content vary widely among animal species, and among muscle tissues with different activities (Jones et al. 1979; Ueki et al. 2005; Stewart et al. 2004; Dosi et al. 2006; Fraser et al. 2006; Cossins et al. 2009; Helbo et al. 2011). The Mb of lower vertebrates particularly fish species is interesting to study because their genetic evolution is different from mammalian ones. Fish species also provide an excellent system in which to study the natural variation of Mb function and to understand the interrelationship between structure and function of Mb. A number of Mb structures of marine and freshwater fish species have been solved (Fosmire and Brown 1976; Watts et al. 1980; Nichols and Weber 1989; Seidel and Adkins 1989; Birnbaum et al. 1994; Chanthai et al. 1996, 1998; Marleen et al. 1998; Wen and Chau 2001; Marcinek et al. 2001; Ueki and Ochiai 2004; Stewart et al. 2004). The results showed that the structure of fish Mbs maintain the same characteristic globin-fold structure as found in mammal but difference in primary structure. Fish Mbs have fewer amino acid residues and contain a much lower percentage of charge residues than mammalian Mbs. In addition, fish Mbs contain a cysteine residue, which is not present in mammalian Mbs. Fish and mammalian Mbs share the same globin-fold tertiary structure. However, mammalian Mbs have eight helical segments labeled A through H, but fish Mbs lack D helix in their structure (Perutz et al. 1965; Watts et al. 1980). There are also many reports on the stability of Mb under heat (Awad and Deranleau 1968; Chow 1991; Chen and Chang 1992; Chanthai et al. 1996; Chen

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2003; Chen et al. 2004; Ueki et al. 2005; Shareghi et al. 2009; Ochiai 2011). The studies showed the differences in stability exist among fishes under thermal denaturation. It has been reported that at the temperature of 40–50 °C is high enough to accelerate the denaturation of many fish Mb (Chen and Chang 1992; Chen 2003). These results are thermally attributed to low conformation stability of Mb in fish species. Compared to mammalian Mbs, fish Mbs are tend to aggregate at high temperature and show more rapidly released O2 over the wide-range temperature. Thus, fish Mbs are more unstable than the mammalian counterpart when autooxidation rate and aggregation profile are compared (Chanthai et al. 1996, 1998; Wen and Chau 2001; Ueki and Ochiai 2004; Stewart et al. 2004). As far as freshwater fish concerned, there are some reports on Mb from hypoxia-tolerant fishes. The results showed that some Mb isoforms of common carp Cyprinus carpio and Zebrafish Danio rerio are expressed in non-muscle tissue, where it probably effected on the regulation of capillary function, the metabolic activity levels of kidneys and brain function (Cossins et al. 2009). Furthermore, Mb isoforms in the hypoxia-tolerant common carp and goldfish Carassius auratus were studied to understand the functional roles of these diverged isoforms. The results demonstrated that two isoforms of Mbs possessed different functions. One of these, Mb1, which is presents in oxidative muscle and also several other tissues, plays an important role in O2 supply and in generation of NO during hypoxia. Mb2, an isoform specific to brain neurons, may protect carp brain during reoxygenation following long hypoxia period by elimination of H2O2 and other reactive oxygen species. (Cossins et al. 2009; Helbo et al. 2011). The Asian swamp eel Monopterus albus, one of hypoxic-tolerant freshwater fish in Thailand, is thought to be interesting fish. The swamp eel is highly adaptable fish; it can survive both in hot and cold climates (Schofield and Nico 2009). It has been reported that the eel can tolerate varying degrees of salinity (Tay et al. 2003; Siang et al. 2007). Moreover, Asian swamp eel is largely fossorial and tend to be opportunistic predators. If the skin kept moist, it can breathe atmospheric air and persists multiple days out of water by burrowing in mud (Schofield and Nico 2009). Thus, the swamp eel Mb may have some interesting characteristics and relative high structural stability. From these backgrounds, it is


important to investigate its structure and properties, of which no report is available at present. In the present study, in order to establish the physico-chemical properties of swamp eel Mb, attempt has been done to purify Mb from the fish muscle by using column chromatography combined with molecular filtration. Some characteristic properties including molecular mass, spectral properties, and partial peptide sequences of the purified Mb are described. Thermal stabilities of the swamp eel Mb compared with walking catfish Mb and striped catfish Mb have also been reported.

Materials and methods Materials Six- to eight-month-old swamp eel Monopterus albus (body length: 40.5 ± 5.0 cm; weight: 400 ± 50 g) was randomly chosen from a public market in Khon Kaen province, Thailand. After removing its head and blood, fresh muscle was dissected free of connective tissue and fat and used immediately for Mb extraction. Walking catfish Clarias batrachus and stripped catfish Pangasius hypophthalmus were also bought from the market. Their fresh muscles were obtained by the method used for the eel. Commercially available horse hearts Mb and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). Sephadex G-75 and DEAE-cellulose were the product of Amersham Biosciences Corp., NJ and SigmaAldrich, respectively. Isolation and purification of myoglobin Mb was purified from fish muscle according to the following process. Briefly, the muscle was chopped coarsely and soaked in 10 mM Tris–HCl buffer, pH 6.8 (1:2 w/v) for 24 h at 4 °C, and homogenized in the buffer using blender at low speed. After the homogenate was centrifuged for 20 min at 10,0009g, 4 °C, the supernatant was collected and then was subjected to salting out using ammonium sulfate precipitation in the range of 45–75 % (w/v). The salt precipitate (75 % w/v ammonium sulfate) was dissolved in a small volume of 2 mM Tris–HCl buffer, pH 6.8, and dialyzed overnight against the same buffer. The dialyzate was concentrated using Centricon Ym 10

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filtration unit (Amicon, Germany), referred to as the crude protein extract, and stored at -70 °C until used. The crude protein extract of Mb was then loaded onto a Sephadex G-75 column (90 9 1.75 cm, i.d.) equilibrated with 10 mM Tris–HCl buffer, pH 6.8. The proteins were eluted with the same buffer with a flow rate of 0.3 mL/min and 2 mL fractions were collected. The elution pattern was monitored by A280 and A408 nm. The Mb-containing fractions, which had high absorbance ratio of 408/280 nm, were pooled and applied to Centricon Ym 30 filtration unit (Amicon, Germany). After centrifugation at 7,5009g, 10 °C for 10 min, the filtrate was collected. The pooled filtrate was then concentrated in Centricon Ym 10 filtration unit. The retainate fraction from Centricon Ym 10 filtration unit was then chromatographed on a DEAE-cellulose column (50 9 1.5 cm, i.d.), which was equilibrated with 10 mM Tris–HCl buffer, pH 6.8. The Mb was eluted with an isocratic elution using the same buffer at a flow rate of 0.3 mL/min. Each of 2 mL fractions was collected, and Mb-containing fractions were determined for their purity using SDS-PAGE (Laemmli 1970; Mclellan 1984). Walking catfish Mb and striped catfish Mb were extracted and purified by the same manner. Determination of molecular mass The protein samples were analyzed for their purities and relative molecular mass by using 15 % SDS– polyacrylamide gel electrophoresis as previously described (McLellan 1984). The molecular mass standard marker mixture (Amersham Bukinghamshire, UK): phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa), and alpha-lactalbumin (14.4 kDa) were used for molecular mass estimation. An accurate molecular mass of the purified eel Mb was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF–MS; an AXIMA PerforTM mance mass spectrometer, Shimadzu Biotech, Japan) with 3,5-dimethoxy-4-hydroxycinamic acid (Sigma–Aldrich) as a matrix. The spectrum was recorded in linear mode with an acceleration voltage of 377 mV. To prepare sample for mass spectrometry, 1 lL of matrix solution was spotted on the MALDI plate. The purified Mb solution (1 lL) was then spot quickly onto a drop of the matrix solution and then air dried at room temperature to allow sample

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crystallization prior to insertion into the instrument (Beavis and Chait 1990). Determination of isoelectric point Isoelectric focusing (IEF) technique was used for isoelectric point (pI) determination. This method was carried out on a thin-layer 5 % polyacrylamide gel with a pH range 3–10 (Mclellan 1984). Electrophoresis was performed according to Runglawan et al. (Sudmoon et al. 2008). Coomassie Brilliant blue R 250 (Fluka Chemika, Switzerland) was used to stain the proteins. Isoelectric point (pI) of the protein was estimated from linear extrapolation of the calibration curve relating electrophoretic mobility (Rf) from cathode to anode against pIs of isoelectric point calibration standard :trypsinogen (9.30), lentil lectin basic band (8.65), lentil lectin middle band (8.45), lentil lectin acidic band (8.15), horse myoglobin basic band (7.35), horse myoglobin acidic band (6.85), human carbonic anhydrase B (6.55), bovine carbonic anhydrase B (5.85), blactoglobin (5.20), soybean trypsin inhibitor (4.55), and amyloglucosidase (3.50) (Phamacia Pitcataway, USA). Spectroscopic analysis of myoglobin Absorption spectra from 250 to 700 nm for the purified swamp eel Mb, horse heart Mb, and BSA were taken on Agilent 8453 UV–Visible spectrophotometer at room temperature. The absorbance ratio between A408 and A280 was calculated. Intrinsic tryptophan fluorescence spectra were measured on Shimadzu RF-5301PC spectrofluorophotometer. The excitation wavelength was set at 280 nm for tryptophan residue. Both fluorescence intensity and the maximum emission wavelength were recorded. Peptide fragments analysis The purified Mb band obtained from SDS-PAGE with Coomassie Brilliant blue staining was excised from the gel. The excised band was washed twice with 100 lL of 50 mM ambic/50 % methanol, dehydrated in 100 lL of 75 % ACN for 20 min, and then dried. Plug was then rehydrated in 20 mM ambic and digested with 10 lL of trypsin solution at 37 °C for 60 min. Peptides were extracted twice with 60 lL of 50 % ACN/ 0.1 %TFA for 20 min and with 40 lL of the same mixture solution for 20 min. Then, the two extracts

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were pooled and dried. Peptides were resuspended in 3 lL of 50 % ACN/0.1 %TFA, mixed with equal volumes of a-cyano-4-hydroxycinnamic acid in 50 % ACN/0.1 %TFA and 2 % w/v ammonium citrate and then analyzed by LTQ Linear Ion Trap Mass Spectrometer (ThermoFinnigan, San Jose, USA). Based on the LC–MS/MS results, a search in nr.FASTA by BioworkTM 3.1 SR1 (ThermoFinnigan) was performed to identify the protein band. The in-gel tryptic digestion, LC–MS/MS and database search were done at the Bioservice Unit, National Science and Technology Development Agency, Bangkok, Thailand. Determination of thermal stability Two mL of purified swamp eel Mb solutions in 5 mL test tubes were heated at 30, 40, 50, 60 or 70 °C in a water bath. The percentage of Mb denaturation (PMD) at each temperature was determined after heating for 5, 10, 30, 40, 50, 60 and 120 min, respectively. Absorbance of the Mb solution was scanned from 450 to 700 nm using Agilent 8453 UV–Visible spectrophotometer. Mb concentration was determined with five replicates, and the PMD was calculated as followed (Chen and Chang 1992). Mb ðmg=gÞ ¼ ðA525 A700Þ 2:303 dilution factor PMD ¼ 100 ðMbr Mbh Þ=Mbr where Mbr and Mbh represent Mb concentration before and after heating, respectively. The denaturation rate of Mb was determined from the slope of the plot between the natural logarithm of residual Mb (Mbr) and heating time according to Chen and Chang (Chen et al. 2004; Shareghi et al. 2009). The denaturation rate constant (Kd) was calculated as followed. Kd ¼ ðln Mbr0 ln Mbrt Þ=Dt where Mbr0 and Mbrt represent the residual Mb (%) in Mb solution before and after t min heating, respectively.

Results Purification of the swamp eel Mb After the protein precipitate of 75 % (w/v) ammonium sulfate salting out was separated on Sephadex G-75


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Fig. 1 Sephadex G-75 elution profile of the crude protein extract obtained from Asian swamp eel (Monopterus albus) muscle

column, two main peaks were obtained for both A280 and A408 (Fig. 1). The fraction No. 13–15 of the second peak was judged to contain mainly Mb because of their high UV–Visible absorption ratio of 408/280 nm. Based on the protein composition determined by SDS-PAGE, these fractions contained a protein that was most likely Mb (about 15 kDa) and other high molecular mass proteins (data not shown). To further purify Mb, the fraction No. 13–15 were pooled and subjected to molecular filtration units through a 30 kDa MW cutoff membrane in order to remove the proteins with molecular mass of 30 kDa or higher. Subsequently, the brown color filtrate was concentrated using a 10 kDa MW cutoff membrane and considered to contain Mb. The protein fractions separated by molecular filtration were then analyzed using SDS-PAGE (Fig. 2). The 10 kDa retainate showed three major bands with molecular mass of less than 20 kDa. These three bands were also found in the 30 kDa retainate. The 10 kDa retainate was then applied to DEAE-cellulose column, the elution profile showed only one peak with a brown color (Fig. 3a). Using SDS-PAGE, the brown peak was found to contain only one protein band with molecular mass of 15.3 kDa (Fig. 3b). This protein band was expected to be Mb. The yield of eel Mb was 0.68 mg/g wet weight of muscle. Characterization of the swamp eel myoglobin To be certain that the purified protein was Mb, the other properties were determined. UV–Visible spectrum of the purified Mb in the ranges of 250–600 nm was measured compared to those of horse heart Mb

Fig. 2 Protein compositions of the protein extract partially purified by molecular filtration. After purification using molecular filtration, the protein samples were subjected to SDS-PAGE. M molecular mass standard marker, 1 crude protein extract, 2 horse heart Mb, 3 Centricon YM 30 unit retainate, 4 Centricon YM 10 unit retainate

and BSA (Fig. 4). The spectrum of swamp eel Mb exhibited maximal absorption at 280, 408, and 510 nm with strong absorption at the Soret band (408 nm). The spectrum was similar to that of horse heart Mb but different from BSA (Fig. 4). In addition, the tryptophan fluorescence spectra of these proteins excited at 280 nm were observed. The fluorescence spectrum of purified swamp eel Mb gave less intensity at 330 nm than the spectrum of standard BSA but agreed with that of horse heart Mb (Fig. 5). Since the swamp eel Mb exhibited its molecular mass of about 15.3 kDa on SDS-PAGE, which was calculated by interpolation using a standard plot of log molecular mass versus relative migration distance, its molecular mass was then confirmed using MALDITOF–MS analysis. Normally, the obtained ions in this technique have just one charge (z = 1). Thus, the m/z value that showed in mass spectrum is numerically equal to the molecular ionic mass in Da. The molecular mass of the swamp eel Mb obtained by this method was 15,525.18 Da (Fig. 6). IEF analysis showed two protein bands on IEF slab gel with pI of 6.4 as the major band and pI of 7.12 as the minor band denoted the microheterogeneity of eel Mb (Fig. 7). Six sequence tags of the swamp eel Mb obtained from LC– MS/MS are shown in Table 1. From database search, the tags were homologous to parts of Mb from some marine and freshwater fish (Table 2).

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Fig. 3 a The elution profile of eel Mb obtained from DEAECellulose ion-exchange chromatography. b SDS-PAGE of the fractions eluted from DEAE-Cellulose column. M molecular


mass standard marker, 1 Centricon YM 10 unit retainate, 2 horse heart Mb, 3 fraction No. 19 eluted from DEAE-Cellulose column

Fig. 4 UV–Visible absorption spectrum of the purified eel Mb comparing with those of horse heart Mb and BSA Fig. 6 The molecular ion produced from purified eel Mb using MALDI-TOF mass spectrometry

Thermal stability

Fig. 5 Tryptophan fluorescence spectrum of the purified eel Mb comparing with those of horse heart Mb and BSA

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Thermal stability of swamp eel Mb, walking catfish Mb, and stripped catfish Mb at 30, 40, 50, 60 and 70 °C was expressed as the PMD. The plot of the PMD versus heating time is shown in Fig. 8. The PMD of these Mbs increased rapidly at the first 20 min and increased slowly between 20 and 120 min. After heating for 120 min, the PMD of the Mbs kept higher than 90 % at 60 and 70 °C. However, at the temperature of 40 °C, the PMD of swamp eel Mb was less than 10 % but walking catfish Mb and stripped catfish Mb was higher than 60 and 70 %, respectively. Similar results were obtained at 30 and 50 °C; PMD


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Fig. 7 Determination of isoelectric point (pI) of the eel Mb using isoelectric focusing. M pI marker, 1 horse heart Mb, 2 the purified eel Mb

Table 1 Sequence tags of the 15.3 kDa protein and their cross-correlation Sequence tags from LC–MS/MS data

Cross-correlation scorea (Xcorr)

AKGSHAAILKPLANSHATK

5.94

GSHAAILKPLANSHATK

4.55

LFTEHPETQK

3.19

LFTEHPETQKLFPK

3.16

HKIPINNFR

2.44

IPINNFR

2.20

The brown fraction obtained from DEAE-cellulose column chromatography was subjected to SDS-PAGE. The 15.3 kDa protein band was excised. After in-gel digestion, the peptide fragments were analyzed by LC–MS/MS a

Cross-correlation score (Xcorr): an alignment score between the observed and theoretical peptide spectra and used to produce the final ranking of the candidate peptide fragments in the search. XCorr values above 2.0 are usually indicative of a good correlation

of swamp eel Mb was less than those of walking catfish Mb and striped catfish Mb. Because the PMD of the Mbs were noticeably different at 30–60 °C, the natural logarithm of residual Mb (Mbr) versus heating time was plotted in this temperature range as in Fig. 9. The slope of the straight line represents the denaturation rate constant (Kd). The Kd of Mbs showed the two stage of thermal denaturation reaction. The first stage that was regarded as the fast reaction stage occurred at the beginning of heating for up to 20 min,

showing that the Mbr decreased readily in this period. The second stage occurred during heating of 20–120 min where the Mbr decreased slowly and was regarded as the slow reaction stage (Fig. 9). Therefore, the denaturation rate constant (Kd) of Mb can define as Kdf and Kds for fast and slow stage reaction, respectively (Chen et al. 2004; Shareghi et al. 2009). It was found that the straight line became steeper when the heating temperature increased. The slope of the swamp eel Mb at fast reaction stage (Kdf) rather unchanged during 30–50 °C (Fig. 9a) and the slope particularly steepens at 60 °C. In contrast, the slopes of walking catfish Mb and striped catfish Mb were clearly observed since the heating temperature was only 40 °C (Fig. 9b, 9c). Therefore, their Kdf was higher than the Kdf of the eel Mb at nearly all heating temperature. To further realize the stability of swamp eel Mb to heat denaturation, the common logarithm of denaturation rate constant (Kd) versus reciprocal of absolute temperature was plotted as Fig. 10. The slope of the plot between common logarithm of Kd and absolute temperature reflects the thermal sensitivity of Mb; the thermal sensitivity of swamp eel Mb was 8.68 9 103 and 4.92 9 103/s per K for fast and slow reaction stage, respectively. Compared with walking catfish and striped catfish Mb, the swamp eel Mb showed the lowest thermal sensitivity (Table 3).

Discussion This work is the first report on Mb from Asian swamp eel. The purification of the swamp eel Mb was done by using combination of salt precipitation, chromatographic method, and molecular filtration. The brown fraction that had high ratio of 408/280 nm and contained small molecular mass protein was pursued in every purification step. We finally obtained a purified protein that exhibited molecular mass of 15.3 kDa on SDS-PAGE. A more accurate molecular mass of this protein was determined to be 15,525.18 Da by using MALDI-TOF–MS. The molecular mass of the protein was closed to Mbs from some animals such as mackerel, sardines, and tuna species (Watts et al. 1980; Wen and Chau 2001; Ueki et al. 2005; Beavis and Chait 1990; Ochiai et al. 2010; Liong et al. 2001). To be certain that this protein was swamp eel Mb, characteristic properties of Mb

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Table 2 Comparisons of the six sequences tags of the Asian swamp eel 15.3 kDa protein with full amino acid sequences of fish myoglobins by using database search (ExPASy proteomics server) Myoglobin source

Amino acid sequence

Sea raven (Hemitripterus americanus)

LVLKCWGPVE ADYAAYGSLV LTRLFTEHPD TQKLFPKFAG IAQGDMAADA RKLGELLKAK GSHAAILKPL ANSHATKHKI PINNFRLITE VIGKVMGEKT GLDAAGQQAL RNVMAIVVAD MEADYKLLGF TG

Yellowfin tuna (Thunnus albacores)

MADFDAVLKC WGPVEADYTT MGGLVLTRLF KEHPETQKLF PKFAGIAQAD IAGNAAISAH GATVLKKLGE LLKAKGSHAA ILKPLANSHA TKHKIPINNF KLISEVLVKV MHEKAGLDAG GQTALRNVMG IIIADLEANY KELGFSG

Blue marlin (Makaira nigicans)

MADFEMVLKH WGPVEADYAT HGNLVLTRLF TEHPETQKLF PKFAGIAKAD MAGNAAISAH GATVLKKLGE LLKAKGSHAAIIKPMANSHA TKHKIPIKNF ELISEVIGKV MHEKAGLDAA GQKALKNVMT TIIADIEANY KELGFTG

Common carp (Cyprinus carpio)

MHDAELVLKC WGGVEADFEG TGGEVLTRLF KQHPETQKLF PKFVGIASNE LAGNAAVKAH GATVLKKLGE LLKARGDHAA ILKPLATTHA NTHKIALNNF RLITEVLVKV MAEKAGLDA

Goldfish (Carassius auratus)

GGQSALRRVM DVVIGDIDTY YKEIGFAG MADHELVLKC WGVVEADFEG TGGEVLTRLF KQHPETQKLF PKFVGIAQSD LAGNAAVNAH GATVLKKLGE LLKARGDHAA ILKPLATTHA NKHKIALNNF RLITEVLVKV MAEKAGLDAA GQTALRKVME AVIGDIDTYY KEFGFAG

All residues corresponding to the sequences of the sequence tags are italicized. The underlined residues are those different from the sequence tags Accession number of cited sequence as follows: sea raven; AY029587; yellowfin tuna, P00205.2; blue marlin, Q6DGJ1.3; common carp, P02204.2; goldfish, B3CJI6

were then determined. UV–Visible spectrum and intrinsic tryptophan fluorescence spectrum showed that it was a heme-containing protein. Six peptide fragments of the protein obtained from LC–MS/MS were identified as parts of Mbs from some marine fishes, such as sea raven Hemitripterus americanus, yellowfin tuna Thunnus albacores, blue marine Makaira nigicans, and some freshwater fishes such as common carp Cyprinus carpio and goldfish Carassius auratus (Table 2). Therefore, it was certain that this purified protein was swamp eel Mb. From Table 2, the peptide sequences were more consistent with the marine fish Mbs than freshwater fish Mbs. It might reflect some relation between the swamp eel and marine fishes. This suggestion may be supported by the tolerance to saline condition of the swamp eel (Tay

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et al. 2003; Siang et al. 2007). Using IEF technique, the swamp eel Mb displays microheterogeneity during IEF with apperent pI at pH 6.4 and 7.12, which should be metMb and oxyMb, respectively. It has been suggested that the presented oxyMb was caused by the reduction of a small amount of metMb to oxy derivatives during IEF run (Satterlee and Snyder 1964). By comparison, The pI values of swamp eel Mb higher than pI of some fish species, for examples, mackerel and sardine Mb have pI of 5.8 and 5.9, respectively (Shiraki et al. 2002) and carp Mb has pI of 5.3 (Hamoir and Konosu 1965). Interestingly, the pI values of the swamp eel Mb were close to the pIs of horse heart Mb (Fig. 7) and some turtle species Chelydra serpentina (Seidel and Adkins 1989). It could be suggested that swamp eel Mb may have some


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Fig. 8 Changes of percentage Mb denaturation (PMD) of a asian swamp eel Mb, b walking catfish Mb, and c stripped catfish Mb

Fig. 9 Changes of residual Mb (Mbr) of a asian swamp eel Mb, b walking catfish Mb, and c stripped catfish Mb

Fig. 10 Changes of Mb denaturation rate constant (Kd) of a asian swamp eel Mb, b walking catfish Mb, and c stripped catfish Mb, heated at various temperatures circle. Fast reaction stage, white circle; slow reaction stage, black circle

structural characteristic similar to Mb of vertebrate species. For analysis of thermal stability, we wanted to compare the eel Mb with Mb of ordinary fresh-water fish. Walking catfish and striped catfish were used since the fishes have similar behavior to most of fresh

water fishes. The eel Mb exhibits lower PMD at 30 °C to 60 °C than walking catfish Mb and striped catfish Mb (Fig. 8). It means that swamp eel Mb is the most thermostable. The Kd of all Mbs showed biphasic firstorder reaction (Fig. 9) (Ochiai 2011). The fast stage assigned to oxygen dissociation and the oxidation of

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Table 3 The sensitivity of Asian swamp eel Mb to thermal denaturation in fast and slow reaction stages Species

Thermal sensitivity (9103/s per K) Fast reaction stage

Slow reaction stage

Asian swamp eel

8.68

4.92

Walking catfish

9.13

5.52

Stripped catfish

9.25

7.65

swamp eel Mb, while the slow stage indicates the heme orientation disorder (Light et al. 1987; Cutruzzolaa et al. 1996). Swamp eel Mb exhibits the lowest increase in both of Kdf and Kds when the temperature increase suggesting that Mb of this fish can bind and release oxygen more slowly at high temperature than walking catfish Mb and striped catfish Mb. Therefore, swamp eel Mb has high tolerance against heat denaturation when compared to the other two. The suggestion was confirmed by analysis of thermal sensitivity; the eel Mb showed a lower sensitivity to heat compared with walking catfish Mb and striped catfish Mb. The thermostability differences exist among Mbs is caused by the differences of the helical content and the affinity between the helices and heme (Tay et al. 2003; Liong et al. 2001). The stability in terms of thermal sensitivity among these Mbs for fast reaction stage was shown to be similar (Table 3). However, swamp eel Mb was likely to be more stable to heat at this stage than walking catfish and striped catfish Mb. At low temperature or short heating incubation time, the stability of Mb may be affected by a minor region alteration in the tertiary structure, which may occur to changes in the interaction of helices and heme (Liong et al. 2001), while the heme that is responsible to bind oxygen molecule did not suffer from thermal structural change and some helices still was not completely disturbed or remains unchanged (Tay et al. 2003). Thus, the thermal sensitivity among these Mbs is not much different. On the other hand, at high temperature or long heating incubation times, the extensive alteration take place in the secondary and tertiary structure of the Mb molecule (Chen and Chang 1992). Moreover, the interaction of globin and heme of swamp eel Mb particularly with proximal and distal His residues, which contribute to the structural stability of Mb, was perturbed and the loss of heme from Mb occurred

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(Liong et al. 2001). The slow reaction stage of thermal denaturation showed clear differences in thermal sensitivity. It was thus found that the swamp eel Mb was more heat stable than the other inland fishes. It might suggest that the swamp eel Mb structure may bind and hold on oxygen better than Mb from other inland fishes in hot environment. The results of thermal sensitivity indicate the heat tolerance of the swamp eel Mb under hot environment. This may be one factor that involved in adaptable behavior of this Asian swamp eel. However, additional characterizations are required to get better understanding for its structure–function relationship and the relation between Mb function and the eel behavior. Acknowledgments The research funding supported by Center of Excellence for Innovation in Chemistry (PERCH-CIC) and Rajamangala University of Technology Isan, Thailand, was gratefully acknowledged. Thanks were also extended to the Hitachi Scholarship Foundation, Tokyo, Japan.

References Awad ES, Deranleau DA (1968) Thermal denaturation of myoglobin. I. Kinetic resolution of reaction mechanism. Biochem 7:1791–1795 Beavis RC, Chait BT (1990) High accuracy molecular mass determination of protein using matrix assisted laser desorption mass spectrometry. Anal Chem 62:1836–1840 ˚ Birnbaum GI, Evans SV, Przybylska M, Rose DR (1994) 1.70 A resolution structure of myoglobin from yellowfin tuna. An example of a myoglobin lacking the D helix. Acta Cryst D50:283–289 Chanthai S, Ogawa M, Tamiya T, Tsuchiya T (1996) Studies on thermal denaturation of fish myoglobins using differential scanning calorimetry, circular dichroism, and tryptophan fluorescence. Fish Sci 62:927–932 Chanthai S, Nieda H, Ogawa M, Tamiya T, Tsuchiya T (1998) Effect of heating on autoxidation rate of fish holo- and reconstituted myoglobins. Fish Sci 64:574–577 Chen HH (2003) Effect of cold storage on the stability of chub and horse mackerel myoglobins. J Food Sci 68:1416–1419 Chen JR, Chang HM (1992) Studies on some shark actomyosins denature factors—I. Food Sci 19:324–335 Chen LC, Lin SB, Chen HH (2004) Thermal stability and denaturation rate of myoglobin from various species of fish. Fish Sci 70:293–298 Chow CJ (1991) Relationship between the stability and autoxidation of myoglobin. J Agric Food Chem 39:22–26 Chow CJ, Wu JC, Lee PF, Ochiai Y (2009) Characterization and kinetics studies of water buffalo (Bubalus bubalis) myoglobin. Comp Biochem Physio Part B 154:274–281 Cossins AR, Williams DR, Foulkes NS, Berenbrink M, Kipar A (2009) Diverse cell-specific expression of myoglobin isoforms in brain, kidney, gill and liver of the hypoxia-tolerant carp and zebrafish. J Exp Biol 212:627–638

Fish Physiol Biochem (2012) 38:1533–1543 Cutruzzolaa F, Travagliniallocatelli C, Brancaccio A, Brunori M (1996) Aplysia limacina myoglobin cDNA cloning: an alternative mechanism of oxygen stabilization as studied by active-site mutagenesis. Biochem J 314:83–90 Dosi R, Maro DA, Chambery A, Colonna G, Costantini S, Geraci G, Parente A (2006) Characterization and kinetics studies of water buffalo (Bubalus bubalus) myoglobin. Comp Biochem Physiol Part B 145:230–238 Fosmire GJ, Brown WD (1976) Yellowfin tuna (Thunnus albacares) myoglobin: characterization and comparative stability. Comp Biochem Physiol 55B:293–299 Fraser J, Mello LV, Ward D, Rees HH, Williams DR, Fang Y, Brass A, Gracey AY, Cossins AR (2006) Hypoxic-inducible myoglobin expression in nonmuscle tissues. Proc Natl Acad Sci USA 103:2977–2981 Hamoir G, Konosu S (1965) Carp Myogens of white and red muscles: general composition and isolation of lowmolecular-weight components of abnormal amino acid composition. Biochem J 96:85–96 Helbo S, Dewilde S, Williams DR, Berghmans H, Berenbrink M, Cossins AR, Fago A (2011). Functional differentiation of myoglobin isoforms in the hypoxia-tolerant carp indicates tissue-specific protective roles. Am J Physiol Regul Integr Comp Physiol 302(6):R693–701 Jones NB, Wang CC, Dwulet EF, Lehman DL, Meuth LJ, Bogardt AR, Gurd RNF (1979) Complete amino acid sequence of the myoglobin from the pacific spotted dolphin, Stenella attenuata graffmani. Biochi Biophys Acta Protein Struct 577:454–463 Kendrew JC, Dickerson RE, Strandberg BE, Hart RG, Davies DR, Phillips DC, Shore VC (1960) Structure of myoglobin. Nature 185:422–427 Kooyman GL, Ponganis PJ (1998) The physiological basis of diving to depth: birds and mammals. Annu Rev Physiol 60:19–32 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 Light WR, Rohlfs RJ, Palmer G, Olson JS (1987) Functional effects of heme orientational disorder in sperm whale myoglobin. J Biol Chem 262:46–52 Liong EC, Dou Y, Scott EE, Olson JS, Phillips GN Jr (2001) Waterproofing the heme pocket. Role of proximal amino acid side chains in preventing hemin loss from myoglobin. J Biol Chem 276:9093–9100 Marcinek DJ, Bonaventura J, Wittenberg JB, Block BA (2001) Oxygen affinity and amino acid sequence of myoglobins from endothermic and ectothermic fish. Am J Physiol Regul Integr Comp Physiol 280:1123–1133 Marleen BE, Groot L, Tombe LA, Van der Laarse JW (1998) Calibrated histochemistry of myoglobin concentration in cardiomyocytes. J Histochem Cytochem 46:1077–1084 McLellan T (1984) Molecular charge and electrophoretic mobility in cetacean myoglobins of known sequence. Biochem Genet 22:181 Nichols JW, Weber LJ (1989) Comparative oxygen affinity of fish and mammalian myoglobins. J Comp Physiol B 159:205–209 Ochiai Y (2011) Temperature dependent structural perturbation of tuna myoglobin. WASET 74:731–735

1543 Ochiai Y, Watanabe Y, Ozawa H, Ikegami S, Uchida N, Watanabe S (2010) Thermal denaturation profile of tuna. Biosci Biotechnol Biochem 74:1673–1679 Perutz MF, Kendrew JC, Watson HC (1965) Structure and function of haemoglobin II. Some relations between polypeptide chain configuration and amino acid sequence. J Expt Bio J Mol Biol 13:669–678 Polasek LK, Davis RW (2001) Heterogeneity of myoglobin distribution in the locomotory muscles of five cetacean species. J Exp Biol 204:209–215 Satterlee LD, Snyder HE (1964) Isoelectric fractionation of bovine metmyoglobin. J Chromatogr 41:417–422 Schofield JP, Nico GL (2009) Salinity tolerance of non-native Asian swamp eels (Telostei: Synbranchidae) in Florida, USA: comparison of three populations and implication for dispersal. Environ Biol Fish 85:51–59 Seidel ME, Adkins MD (1989) Variation in turtle myoglobins (subfamily emydinae: testudines) examined by isoelectric focusing. Comp Biochem Physiol B 94:569–573 Shareghi H, Nazem Z, Dehkordi KZ, Dehkordi JA (2009) Study on the stability of myoglobin in the presences of sodium dodecyl sulfate (SDS) and temperature. Comp Clin Pathol 19:141–145 Shiraki K, Kudou M, Fujiwara S, Imanakab T, Takagi M (2002) Biophysical effect of amino acids on the prevention of protein aggregation. J Biochem 132:591–595 Siang YH, Yee ML, Seng TC (2007) Acute toxicity of organochlorine insecticide endosulfan and its effect on behaviour and some hematological parameters of Asian swamp eel (Monopterus albus, Zuiew). Pestic Biochem Phys 89:46–53 Stewart JM, Blakely JA, Karpowicz PA, Kalanxhi E, Thatcher BJ, Martin BM (2004) Unusually weak oxygen binding, physical properties, partial sequence, autooxidation rate and a potential phosphorylation site of beluga whale (Delphinapteris leucas) myoglobin. Comp Biochem Physiol B 137:401–412 Sudmoon L, Sattayasai N, Bunyatratchata W, Chaveerach A, Nuchadomrong S (2008) Thermostable mannose-binding lectin from Dendrobium findleyanum with activities dependent on sulfhydryl content. Acta Biochim Biophys Sin 40:811–818 Tay SLA, Chew ST, Ip KY (2003) The swamp eel Monopterus albus reduces endogenous ammonia production and detoxifies ammonia to glutamine during 144 h of aerial exposure. J Exp Biol 206:2473–2486 Ueki N, Ochiai Y (2004) Primary structure and thermostability of bigeye tuna myoglobin in relation to those of other scombridae fish. Fish Sci 70:875–884 Ueki N, Chaujen C, Ochiai Y (2005) Characterization of bullet tuna myoglobin with reference to the thermostabilitystructure relationship. J Agric Food Chem 53:4968–4975 Watts DA, Rice RH, Brown WB (1980) The primary structure of myoglobin from yellowfin tuna (Thunnus albacares). J Biol Chem 255:10916–10924 Wen LC, Chau JC (2001) Studies on the physicochemical properties of milkfish myoglobin. J Food Biochem 25:157–174 Wittenberg BA, Wittenberg JB, Caldwell PB (1975) Role of myoglobin in the oxygen supply to red skeletal muscle. J Biol Chem 250:9038–9043

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