Myoglobin oxygenation and autoxidation in three reptilian species

Comparative Biochemistry and Physiology, Part A 187 (2015) 8–12

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Myoglobin oxygenation and autoxidation in three reptilian species Signe Helbo ⁎, Amanda G. Bundgaard, Angela Fago Zoophysiology, Department of Bioscience, Aarhus University, C.F. Møllers allé 3, Bldg. 1131, DK-8000 Aarhus C, Denmark

a r t i c l e

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Article history: Received 13 January 2015 Received in revised form 9 April 2015 Accepted 13 April 2015 Available online 18 April 2015 Keywords: Alligator Allosteric effect Autoxidation Comparative Oxygen affinity Reptiles S-nitrosation Tortoise Turtle Vertebrates

a b s t r a c t Differences between species in the oxygen (O2) affinity (P50) of myoglobin (Mb) may serve to fine tune O2 supply to cardiac and skeletal muscle in ectotherms. In support of this view, it has been shown that fish Mb O2 affinities differ between species when measured at the same temperature, but are in fact similar when adjusted for in vivo muscle temperatures, most likely to maintain intracellular O2 delivery in species adapted to different environments. It is unknown whether similar adaptations exist in the O2 affinity of Mb from reptiles, despite this group of ectothermic vertebrates displaying great variation in the tolerance to both temperature and hypoxia. In this study, we have purified Mb from muscle tissues of three reptilian species (turtle, tortoise and alligator) with different lifestyles. We have measured O2 binding characteristics and autoxidation rates of the three Mbs and measured the effects of temperature, lactate and blocking of reactive thiols on the O2 affinity of turtle Mb. Our data show that, at a constant temperature, reptilian Mbs have similar O2 affinities that are lower than those of mammalian Mbs, which may optimize intracellular O2 transport at lower body temperatures. Reptilian Mbs have lower autoxidation rates than both mammalian and fish Mbs, which may be beneficial during oxidative stress. Furthermore, the O2 affinity of turtle Mb is without allosteric control and independent of either lactate or thiol covalent modification. This study reveals some common adaptive patterns in the temperature-dependent regulation of Mb oxygenation in vertebrates. © 2015 Elsevier Inc. All rights reserved.

1. Introduction The main physiological role of the oxygen (O2) binding hemeprotein myoglobin (Mb) is to facilitate O2 diffusion in heart and skeletal muscle during hypoxia (Wittenberg and Wittenberg, 2003; Gros et al., 2010) and to function as an O2 store, a property which contributes to prolong aerobic diving in marine mammals and birds (Kooyman and Ponganis, 1998). Consistent with its dual role in intracellular O2 delivery, Mb has an O2 affinity (P50, the oxygen tension (PO2) at 50% saturation) that lies between that of hemoglobin (Hb) in the blood and cytochrome c oxidase in the mitochondria and that is close to the PO2 within the muscle cell so that Mb exists in a partially deoxygenated state in vivo (Wittenberg and Wittenberg, 1989). Differences between species in the Mb P50 may thus serve to fine tune O2 delivery according to tissue PO2 and O2 demand. P50 values of vertebrate Mbs decrease at lower temperatures and the dependency on temperature (reflecting the heat of oxygenation, ΔH) is very similar in Mbs from mammals and fish. Mammalian Mb P50 values lie invariably close to ~1 Torr at 25 °C and ~2.3 Torr at 37 °C (Nichols and

Abbreviations: O2, oxygen; Mb, myoglobin; P50, O2 tension at 50% saturation; PO2, oxygen tension; Hb, hemoglobin; ΔH, heat of oxygenation; NO, nitric oxide; Cys, cysteine; Mb-SNO, S-nitrosated Mb; DTT, dithiothreitol; FPLC, fast protein liquid chromatography; IEF, isoelectric focusing; n50, cooperativity value; NEM, N-ethylmaleimide. ⁎ Corresponding author. Tel.: +45 61660382. E-mail address: [email protected] (S. Helbo).

http://dx.doi.org/10.1016/j.cbpa.2015.04.009 1095-6433/© 2015 Elsevier Inc. All rights reserved.

Weber, 1989; Helbo et al., 2013), most likely reflecting that mammals maintain a constant, high body temperature in order to sustain an active lifestyle. In contrast, greater diversity is found in fish Mbs with P50 values ranging between ~1 and ~5 Torr at 25 °C (Helbo et al., 2013), suggesting that Mb must function efficiently over the wider range of in vivo O2 tensions and body temperatures of fish compared to mammals. In support of this hypothesis, Marcinek et al. (2001) showed that fish Mb O2 affinities differ between species when measured at the same temperature, but that they are indeed similar when adjusted for in vivo muscle temperatures, most likely to maintain a constant intracellular O2 delivery in species adapted to different environments. There is general agreement that both fish and mammalian Mbs are insensitive to allosteric cofactors such as H+ and lactate (see Helbo et al., 2013 for references) that bind non-covalently to the protein. However, we have recently shown that covalent S-nitrosation by the signaling molecule nitric oxide (NO) of a specific cysteine (Cys) residue (most likely in position 107) of salmonid Mbs (generating S-nitrosated Mb, Mb-SNO) allosterically increases the O2 affinity, a mechanism that may aid in the release of protective NO from Mb to the hypoxic heart (Helbo and Fago, 2011; Howes et al., 2012; Helbo et al., 2014). Reptilian Mbs contain a conserved Cys in position 109 (Helbo et al., 2013 and references herein), but it is unknown whether allosteric regulation by Snitrosation and in addition whether allosteric non-covalent regulation by lactate and H+ is present in this group. In general, knowledge about the functional properties, stability towards oxidation, temperature sensitivity and allosteric regulation of

S. Helbo et al. / Comparative Biochemistry and Physiology, Part A 187 (2015) 8–12

reptilian Mbs is to our knowledge restricted to very few studies (Okotore and Aboderin, 1979; Weber et al., 1981; Okotore and Brown, 1983; Livingston et al., 1986) despite the fact that this large group of vertebrates shows great variation in lifestyle and adaptations to surviving in extreme environments. In fact, the most hypoxia-tolerant species known are found among the reptiles and within this group, the semiaquatic turtles of the subfamily Deirochelyinae, including the painted turtle (Chrysemys picta) and the closely related red-eared slider (Trachemys scripta), are by far the most tolerant of severe and prolonged hypoxia or even anoxia among all vertebrates (Storey, 1996; Hermes-Lima and Zenteno-Savin, 2002; Bickler and Buck, 2007). To expand our knowledge on Mb functional characteristics in reptiles, we have selected three species with different lifestyles and tolerances to hypoxia. The red-eared slider (T. scripta elegans) is a semiaquatic turtle that is among the most hypoxia-tolerant vertebrates known as it can survive several months in total anoxia at freezing temperatures during winter hibernation (Milton and Prentice, 2007). In contrast, the steppe tortoise (Agrionemys (Testudo) horsfieldii) from Central Asia, is a land-living species that may rarely encounter hypoxia, but can withstand great temperature variations (Lagarde et al., 2002). The American alligator (Alligator mississippiensis) is a semi-aquatic species that inhabits more constantly warm climates and performs dives regularly (Andersen, 1961). In this study, we report O2 binding characteristics and autoxidation rates of Mb purified from muscle tissues of the three reptilian species. Furthermore we show the effects of temperature, lactate and blocking of reactive thiols on the O2 affinity of turtle Mb. 2. Materials and methods 7 hearts from juvenile American alligators (A. mississippiensis) (designated alligator onwards) kept at −80 °C were kindly donated by Tobias Wang, Aarhus University, in relation with another investigation (Skovgaard et al., 2008). 2 adult red-eared sliders (T. scripta elegans) (designated turtle onwards) were obtained from Lemberger (Oshkosh, WI, USA) and sent by air-freight to Aarhus University (Denmark). 6 steppe tortoises (A. horsfieldii) (designated tortoise onwards) were kindly donated by Copenhagen Zoo where they were predestined for euthanization due to chronic mycoplasmosis. Turtles and tortoises were kept at 21 °C in large aquaria with free access to dry platforms under infrared lamps and with access to food and water ad libitum at the department of Bioscience, Aarhus University. For functional comparisons, commercially purified horse heart Mb (Sigma-Aldrich) was used as representative for a mammalian Mb. 2.1. Purification of myoglobin Turtles and tortoises were euthanized by injection of 200 mg/kg of sodium pentobarbital into the bloodstream where after hearts (tortoise) and skeletal muscle (turtle) were quickly dissected out, frozen in liquid nitrogen and stored at −80 °C. Animal care and sampling protocol were performed in compliance with the EU legislation directive for the treatment of laboratory animals (2010/63/EU). Mb from each of the three reptilian species was purified as described in detail for trout Mb (Helbo and Fago, 2011). In brief, Mbs were purified from heart (alligator and tortoise) and skeletal muscle homogenates (turtle) by ammonium sulfate fractionation (40 and 80%), desalted on a PD10 column (GE-Healthcare) equilibrated with gel filtration buffer (50 mM Tris, 0.5 mM EDTA, 5 mg/mL dithiothreitol (DTT), 0.15 M NaCl, pH 8.2) and finally separated from contaminating Hb by fast protein liquid chromatography (FPLC) gel filtration using a Tricorn Superdex 75 10/300 GL column (GE-Healthcare) equilibrated with gel filtration buffer. All Mbs were then desalted on a PD10 column (GEHealthcare) equilibrated with 50 mM Hepes, 0.5 mM EDTA pH 7.4 and stored at − 80 °C for use in further experiments. Conversion of partly ferric (met) alligator Mb to the ferrous (oxy) form was obtained by

9

standard procedures after adding solid dithionite and immediate desalting at 4 °C on a PD10 column (GE-Healthcare) equilibrated with 50 mM Hepes, 0.5 mM EDTA, pH 7.4. The same procedure was used to convert commercially available ferric horse Mb to the oxy form (oxyMb). Turtle and tortoise Mbs were already present as oxyMb after purification. Mb purity was assessed by SDS and isoelectric focusing (IEF) on polyacrylamide gels (Phast System, GE Healthcare) stained with Coomassie blue. Heme oxygenation/oxidation state was assessed by UV–vis absorption spectroscopy in the range of 400–700 nm by using known absorption peaks for mammalian Mbs (Antonini and Brunori, 1971). 2.2. Oxygen equilibria O2 binding curves were determined using a modified diffusion chamber technique previously described (Sick and Gersonde, 1969; Weber et al., 2000; Helbo and Fago, 2011). Briefly, water-saturated gas mixtures of O2 or air and ultrapure (N 99.998%) N2 created by Wösthoff gas mixing pumps were used to equilibrate a thin smear (4 μL, ~100 μM heme, extinction coefficient ε543 = 13.6 mM−1 cm−1 (Antonini and Brunori, 1971)) of oxyMb solution in 50 mM Hepes, 0.5 mM EDTA pH 7.4 with stepwise (4–5 steps) increases in PO2. Changes in absorbance upon oxygenation were recorded continuously at 436 nm by a photomultiplier (model RCA 931-A) and an Eppendorf model 1100 M photometer. The absorbance signal was measured using a laptop computer with the in-house made data acquisition software, Spectrosampler. P50 (PO2 at half-saturation) and n50 (cooperativity) values were calculated from the zero intercept and slope, respectively, of Hill plots: log(Y) / (1 − Y) vs. logPO2, where Y is the fractional saturation of Mb. Experiments were carried out in triplicate at 15, 20 and 25 °C. The effect of temperature on the P50 values was assessed by calculating the heats of oxygenation (ΔH) from the slope of the van't Hoff plots of logP50 as a function of 1/T as described previously (Helbo et al., 2012). Effects of sodium lactate (100 mM, 20 °C), low pH (6.5, 20 °C) and blocking of reactive thiols (see below) on the O2 affinity of turtle Mb were also determined. 2.3. Blocking reactive thiols in tortoise Mb To measure a possible effect of blocking reactive Cys on the O2 equilibria and kinetics, turtle oxyMb was reacted with N-ethylmaleimide (NEM) at a 3:1 NEM/heme molar ratio for 1 h at room temperature (in 50 mM Hepes, 0.4 mM EDTA pH 7.4) to generate Mb-NEM, which is functionally equivalent to Mb-SNO but more stable and not photolabile (Helbo et al., 2014). Excess NEM was removed using a PD-10 desalting column (GEHealthcare) equilibrated with 50 mM Hepes, 0.5 mM EDTA, pH 7.4 and O2 binding curves were determined at 20 °C (n = 3). 2.4. Autoxidation rates To determine oxyMb (Fe2+) stability, the rate of spontaneous oxidation of MbO2 to metMb (Fe3+) was measured for all Mbs in air at 22 °C in 50 mM Hepes, 0.5 mM EDTA pH 7.4 (n = 2). The decrease in absorbance over time was monitored in quartz cuvettes at 543, 562 and 580 nm every hour for ~48 h using a HP 8543 UV–visible diode array spectrophotometer. Mb concentration in the cuvette was ~10 μM. Due to the long incubation time, to correct autoxidation rates for baseline changes, the autoxidation rate was calculated by plotting (A543 + A580) − 2 × A562, where A = absorbance at the given wavelength, as a function of time (t) and fitting according to a first-order exponential decay function. 3. Results Tortoise and alligator Mbs were successfully purified from heart muscle as judged by IEF and SDS PAGE, with strong bands

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4. Discussion

0.8

0.6

Alligator Tortoise Turtle Horse

0.4

0.2

0.0 0

2

4

6

8

10

PO2 (torr) 0.6

B

0.4

0.2

0.0

-0.2

A major finding of this study is that reptilian species adapted to different environments have Mbs with similar O2 affinities that are lower than those of mammalian Mbs and with enthalpy of oxygenation similar to mammalian Mbs (Table 1). Furthermore, we discovered that in the reptiles here investigated, Mbs are more stable than mammalian Mbs as they are less prone to spontaneous oxidation (Table 1), and that in turtle, Mb was without allosteric control and independent of either lactate or thiol covalent modification by NEM. Ectothermic animals, like reptiles, in general cannot maintain a constant body temperature; instead their internal temperature more or less follows that of their surroundings. This implies that Mb must be able to

Arbitrary absorbance

Turtle Tortoise Alligator

300

A

1.0

Fractional O2 saturation

corresponding to ~17 kDa (not shown). Additional bands were present in the purified turtle Mb, but as they did not display a red color prior to staining, they were assumed not to interfere with the following experiments. All purified reptilian Mbs showed visible absorbance spectra with peaks characteristic for mammalian oxygenated Mbs (Antonini and Brunori, 1971) (Fig. 1), either as purified (turtle and tortoise) or after reduction with dithionite (alligator) as described in Materials and methods. Reptilian Mb O2 equilibrium curves were hyperbolic (Fig. 2A) and P50 values were all higher (and thus the O2 affinities lower) than that of horse Mb measured at the same temperature (Fig. 2A). Measured P50 values were similar to other published values for reptilian Mbs at 20 °C (Table 1). Slopes of Hill plots were ~ 1 for all Mbs (values in Table 1) confirming a monomeric protein structure and that no contaminating Hb or other globins with different O2 affinities were present. Overall heats of oxygenation, ΔH (kcal/mol O2) derived from the slopes of vant' Hoff plots (Fig. 2B) were similar for all reptilian Mbs (Table 1) and also similar to previously published values for reptilian and horse Mbs (Table 1). The O2 affinity of turtle Mb showed no sensitivity to low pH or lactate with P50 values of 1.55 ± 0.12 Torr at pH 6.5 and 1.33 ± 0.4 Torr at pH 6.5 + 100 mM lactate, n = 3. Blocking of reactive thiols with NEM had no effect on turtle Mb O2 affinity (P50 = 1.51 ± 0.18 Torr, n = 3). Autoxidation experiments showed that reptilian Mbs are very stable in the ferrous state compared with horse Mb under the experimental conditions used (20 °C, pH 7.4) with estimated half-times for the conversion of oxyMb to oxidized, ferric Mb in the range of ~ 40–160 h (rate ~0.004–0.017 h−1) (Table 1) (Fig. 3).

Log P50

10

400

500

600

700

Wavelength (nm) Fig. 1. Visible absorbance spectra of purified reptilian oxy Mbs. Absorbance peaks were 417, 543 and 581 nm for turtle, 418, 543 and 580 nm for alligator and 418, 543 and 580 nm for tortoise. Measured at room temperature in 50 mM Hepes, 0.5 mM EDTA, pH 7.4.

3.36

3.39

3.42

3.45

3.48

1/T×103 Fig. 2. Oxygen binding equilibria and temperature dependence of reptilian Mbs. A: representative O2 equilibrium curves of reptilian Mbs, showing hyperbolic fitting to the data. B: temperature dependence (van't Hoff plot) of oxygen affinity in reptilian Mbs. The slope of the plots is proportional to the heat of oxygenation (ΔH). Values are means ± SD. Measurements were performed in 50 mM Hepes, 0.5 mM EDTA pH 7.4 at 20 °C.

maintain its physiological function as O2 carrier over a much broader temperature range and often at lower temperatures than in mammals. In this study we found that all three reptilian Mbs have lower O2 affinities than mammalian Mbs, but highly similar temperature dependencies (Table 1), which is comparable to previous results for fish Mbs (Marcinek et al., 2001; Helbo et al., 2012). Thus, at a fairly constant heat of heme oxygenation (ΔH) of Mb in vertebrates (Table 1, Antonini and Brunori, 1971; Helbo et al., 2012), Mb appears to have evolved slightly different O2 affinities across vertebrate groups living at low and variable (i.e. ectotherms) or high and constant (i.e. endotherms) temperatures in order to perform its function as intracellular O2 carrier. For both turtle and tortoise Mb, the P50 is conserved around 2.5 Torr at the respective preferred mean body temperatures, which is very similar to the P50 of horse Mb at 37 °C (see Fig. 4) and to other mammalian Mbs (Nichols and Weber, 1989). This feature is analogous to previous conclusions for fish Mbs (Marcinek et al., 2001) and to the conservation of the enzyme-substrate apparent affinity constant, Km (comparable to the P50 of Mb), for a given biochemical reaction, in differently adapted ectotherms and mammals, reflecting the necessity to maintain correct biochemical functions at cellular and molecular levels (Graves and Somero, 1982; Somero and Suarez, 2005). Surprisingly, alligator Mb has a higher P50 value (4.1 Torr, Fig. 4) at the preferred body temperature (~30 °C), suggesting that other factors play a role in determining the optimum O2 affinity of Mb in different species. We found only relatively small differences in both Mb O2 affinity and autoxidation rates between the three species of reptiles examined in

S. Helbo et al. / Comparative Biochemistry and Physiology, Part A 187 (2015) 8–12

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Table 1 Oxygen affinities (P50), cooperativity coefficients (n50), heats of oxygenation (ΔH) and autoxidation rates of reptilian myoglobins compared with literature data. Conditions: pH 7.4, 20 °C (P50, n50) and 22 °C (autoxidation). P50 (Torr)

Turtle Tortoise Alligator Horse Chelonia mydas Amphisbaena alba

ΔH (kcal/mol O2)

n50

1.53 ± 0.13 1.36 ± 0.08 1.76 ± 0.20 0.62 ± 0.01 1.0 1.4

Autoxidation

−13.04 ± 2.40 −12.82 ± 1.28 −15.12 ± 0.87 −13.71 −15.0 −13.4

1.04 ± 0.01 1.03 ± 0.05 0.96 ± 0.03 0.99 ± 0.27 ~1 ~1

this study despite the semi-aquatic turtle most likely experiencing more extreme hypoxic events than the terrestrial tortoise and the diving alligator, although little is known about the hypoxia tolerance of these reptile species (Bickler and Buck, 2007). Thus, our data indicate that reptilian Mbs show virtually identical oxygenation properties despite interspecific differences in lifestyle, as we have also previously found for Mbs from whales with different diving capacities (Helbo and Fago, 2012). Perhaps this lack of variation in Mb O2 affinity among reptiles and among whales indicates that the most important factor determining Mb O2 affinity is the body temperature of the animal and not the tolerance to hypoxia, which is controlled by other adaptive responses (Bickler and Buck, 2007). Interestingly, the reptilian Mbs here investigated have very low autoxidation rates (~0.004–0.017 h−1) compared with both mammalian (~ 0.08 h−1) (Table 1) and fish Mbs (~ 0.026 h− 1) (Madden et al., 2004; Helbo and Fago, 2011) measured under identical conditions. At present, we cannot distinguish whether these variations in heme stability can be ascribed to differences in ligand (i.e. water or O2) accessibility or in heme reducing potential or to a combination of both. Regardless of its origin, in the tissue, Mb stability towards oxidation would limit the consumption of reducing equivalents to keep the heme iron in the reduced state. In addition, it would prevent the formation of protein radicals generated when ferric heme reacts with hydrogen peroxide (Alayash et al., 2001), a major product of tissue oxidative stress during cycles of hypoxia and reoxygenation (Bickler and Buck, 2007). In freshwater turtles, lactate levels increase considerably and pH drops (H+ concentration increases) during prolonged hypoxic events (Penney, 1974) and these could therefore be potential non-covalent allosteric effectors of Mb. However, according to our study the O2 affinity of turtle Mb shows no sensitivity towards changes in either pH or lactate concentration, which is in agreement with other studies on mammalian and fish Mbs (Helbo and Fago, 2011; Helbo et al., 2012, 2013). Also, blocking of

Reference

t½ (h)

h−1

39 66 157 9 n.a. n.a.

0.017 0.011 0.004 0.077 n.a. n.a.

reactive thiols in turtle Mb with NEM causes no changes in the O2 affinity, confirming that this covalent allosteric effect is linked to a specific Cys present at position 108 in salmonid Mbs and absent in other Mbs (Helbo and Fago, 2011; Helbo et al., 2014). The Mb sequence of C. picta, a freshwater turtle species closely related to T. scripta, has been determined and it contains a single Cys at position 109, similarly to most other reptilian Mbs (Helbo et al., 2013 and references herein). Position 107 in fish and 109 in reptilian Mbs may seem close to each other, but as fish and reptilian Mbs differ in the number of amino acids residues, these two Cys residues are positioned at opposite sides of the G-helix. Despite the lack of allosteric control, Cys residues may still be important in regulating NO homeostasis and in various redox-reactions that are important in the protection against hypoxia (Giles et al., 2003). Alligator Mb does not contain Cys residues (Dene et al., 1980) similarly to Mbs from birds and most mammals (Enoki et al., 2008; Helbo et al., 2013).

5. Conclusions We found that reptilian Mbs have lower O2 affinities than mammalian Mbs, which may optimize Mb function at lower body temperatures in these animals, and oxidize less easily than mammalian and fish Mbs, which may be beneficial during tissue oxidative stress. Furthermore, the Mb O2-affinity of turtle Mb is without allosteric control and independent of either lactate or thiol covalent modification, similarly to mammalian Mbs. Our data expand the knowledge on Mb functional properties and reveals some common adaptive patterns in the temperature-dependent regulation of Mb oxygenation in vertebrates.

10.0

Alligator Tortoise Turtle Horse

7.5

P50(torr)

Relative absorbance (AU)

1.00

This study This study This study This study, 1 Antonini, 1965 Livingston et al. (1986) Weber et al. (1981)

5.0

0.95 2.5

Alligator Tortoise Turtle Horse

0.90

0.0 15

10

25

30

35

40

Temperature (°C)

0.85 0

20

20

30

40

Time (h) Fig. 3. Autoxidation rates of reptilian Mbs with single exponential fitting to the data. Measurements were performed in 50 mM Hepes, 0.5 mM EDTA pH 7.4 at 20 °C.

Fig. 4. Oxygen affinities (P50) of reptilian and horse Mbs at different temperatures. Curves are generated by calculating Mb P50 values at a range of temperatures using the van't Hoff equations for the different species. (⋆) indicates the average preferred optimal temperature under normoxia of the different species (or very closely related species in the case of tortoise (Testudo hermanii) and turtle (Chrysemys picta)) taken from Brattstrom, 1965. Data on alligator preferred temperature is from http://icwdm.org/handbook/reptiles/ Alligators.asp. Grey bar indicates P50 values similar to that of horse Mb at 37 °C.

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