Structural and kinetic characterization of myoglobins from eurythermal and stenothermal fish species

Teleost myoglobin (Mb) proteins from four fish species inhabiting different temperature environments were used to investigate the relationship between protein function and thermal stability. Mb was isolated from yellowfin tuna (homeothermal warm), mackerel (eurythermal warm), and the Antarctic teleost Notothenia coriiceps (stenothermal cold). Zebrafish (stenothermal tropical) myoglobin was expressed from cloned cDNA. These proteins differed in oxygen affinity, as measured by O2 dissociation rates and P50 values, and thermal stability as measured by autooxidation rates. Mackerel Mb had the highest P50 value at 25 degrees C (3.7 mmHg), corresponding to the lowest O2 affinity, followed by zebrafish (1.0 mmHg), yellowfin tuna (1.0 mmHg), and N. coriiceps (0.6 mmHg). Oxygen dissociation rates and Arrhenius plots were similar between all teleost species in this study, with the exception of mackerel myoglobin, which was two-fold faster at all temperatures tested. Myoglobin from the Antarctic teleost had the highest autooxidation rate (0.44 h(-1)), followed by mackerel (0.26 h(-1)), zebrafish (0.22 h(-1)), and yellowfin tuna (0.088 h(-1)). Primary structural analysis revealed residue differences distributed throughout the polypeptide sequences, making it difficult to identify, which, if any, residues contribute to structural flexibility. However, analysis of molecular dynamics trajectories indicates that Mb from the eurythermal mackerel is predicted to be the most flexible protein within the D loop and FG turn. At the same time, it has the lowest O2 affinity and the highest O2 dissociation rates when compared to myoglobins from teleosts that appear to be less flexible in our dynamics simulations.     

Identification of small juvenile scombrids from northwest tropical Australia using mitochondrial DNA cytochrome <Emphasis Type="Italic">b</Emphasis> sequences

Small juveniles of the nine species of scombrids in Australian waters are morphologically similar to one another and, consequently, difficult to identify to species level. We show that the sequence of the mitochondrial DNA cytochrome b gene region is a powerful tool for identification of these young fish. Using this method, we identified 50 juvenile scombrids collected from Exmouth Bay, Western Australia. Six species of scombrids were apparent in this sample of fish: narrow-barred Spanish mackerel (Scomberomorus commerson), Indian mackerel (Rastrelliger kanagurta), frigate tuna (Auxis thazard), bullet tuna (Auxis rochei), leaping bonito (Cybiosarda elegans), and kawakawa (Euthynnus affinis). The presence of Indian mackerel, frigate tuna, leaping bonito, and kawakawa is the first indication that coastal waters may be an important spawning habitat for these species, although offshore spawning may also occur. The occurrence of small juvenile S. commerson was predicted from the known spawning patterns of that species, but other mackerel species (Scomberomorus munroi, Scomberomorus queenslandicus, Scomberomorus semifasiciatus) likely to be spawning during the sampling period were not detected among the 50 small juveniles analyzed here.     

Temperature and Ca2+ dependence of [3H]ryanodine binding in the burbot (Lota lota L.) heart

Opening and closing of the cardiac ryanodine (Ry) receptor (RyR) are coordinated by the free intracellular Ca2+ concentration, thus making the Ca2+ binding properties of the RyR important for excitation-contraction coupling. Unlike mammalian cardiac RyRs, which lose their normal function at low temperatures, RyRs of ectothermic vertebrates remain operative at 2-4 degrees C, as indicated by Ry sensitivity of contractile force. To investigate the mechanisms of low temperature adaptation of ectothermic RyRs, we compared Ca2+-dependent kinetics of [3H]ryanodine binding in cardiac preparations of a fish (burbot, Lota lota) and a mammal (rat). The number of ventricular [3H]ryanodine binding sites determined at 20 degrees C was 1.54 times higher in rat than burbot heart (0.401 +/- 0.039 and 0.264 +/- 0.019 pmol/mg protein, respectively) (P < 0.02), while the binding affinity (Kd) for [3H]ryanodine was similar (3.38 +/- 0.63 and 4.38 +/- 1.14 nM for rat and burbot, respectively) (P = 0.47). The high-affinity [3H]ryanodine binding to burbot and rat cardiac preparations was tightly coordinated by the free Ca2+ concentration at both 20 degrees C and 2 degrees C and did not differ between the two species. Half-maximal [3H]ryanodine binding occurred at 0.191 +/- 0.027 microM and 0.164 +/- 0.034 microM Ca2+ for rat and at 0.212 +/- 0.035 microM and 0.188 +/- 0.039 microM Ca2+ for burbot (P = 0.65), at 2 degrees C and 20 degrees C, respectively. In two other fish species, rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius carassius), the Ca2+-binding affinity at 20 degrees C was 4.4 and 5.9 times lower, respectively, than in the burbot. At 20 degrees C, the rate of [3H]ryanodine binding to the high-affinity binding site was similar in rat and burbot but was drastically slowed in rat at 2 degrees C. At 2 degrees C, [3H]ryanodine failed to dissociate from rat cardiac RyRs, and at 10 degrees C and 20 degrees C, the rate of dissociation was two to three times slower in rat than burbot preparations. The latter finding is compatible with a channel gating mechanism, where the closing of the Ca2+ release channel is impaired or severely retarded by low temperature in rat but less so in burbot preparations. The stronger effect of low temperature on association and dissociation rate of [3H]ryanodine binding in rat compared with burbot suggests that RyRs of the ectothermic fish, unlike those of endothermic rat, are better able to open and close at low temperatures.     

Electrophysiological properties of the L-type Ca(2+) current in cardiomyocytes from bluefin tuna and Pacific mackerel

Tunas are capable of exceptionally high maximum metabolic rates; such capability requires rapid delivery of oxygen and metabolic substrate to the tissues. This requirement is met, in part, by exceptionally high maximum cardiac outputs, opening the possibility that myocardial Ca(2+) delivery is enhanced in myocytes from tuna compared with those from other fish. In this study, we investigated the electrophysiological properties of the cardiac L-type Ca(2+) channel current (I(Ca)) to test the hypothesis that Ca(2+) influx would be large and have faster kinetics in cardiomyocytes from Pacific bluefin tuna (Thunnus orientalis) than in those from its sister taxon, the Pacific mackerel (Scombe japonicus). In accordance with this hypothesis, I(Ca) in atrial myocytes from bluefin tuna had significantly greater peak current amplitudes and faster fast inactivation kinetics (-4.4 +/- 0.2 pA/pF and 25.9 +/- 1.6 ms, respectively) than those from mackerel (-2.7 +/- 0.5 pA/pF and 32.3 +/- 3.8 ms, respectively). Steady-state activation, inactivation, and recovery from inactivation were also faster in atrial myocytes from tuna than from mackerel. In ventricular myocytes, current amplitude and activation and inactivation rates were similar in both species but elevated compared with those of other teleosts. These results indicate enhanced I(Ca) in atrial myocytes from bluefin tuna compared with Pacific mackerel; this enhanced I(Ca) may be associated with elevated cardiac performance, because I(Ca) delivers the majority of Ca(2+) involved in excitation-contraction coupling in most fish hearts. Similarly, I(Ca) is enhanced in the ventricle of both species compared with other teleosts and may play a role in the robust cardiac performance of fishes of the family Scombridae.     

Influence of swimming speed on metabolic rates of juvenile pacific bluefin tuna and yellowfin tuna

Bluefin tuna are endothermic and have higher temperatures, heart rates, and cardiac outputs than tropical tuna. We hypothesized that the increased cardiovascular capacity to deliver oxygen in bluefin may be associated with the evolution of higher metabolic rates. This study measured the oxygen consumption of juvenile Pacific bluefin Thunnus orientalis and yellowfin tuna Thunnus albacares swimming in a swim-tunnel respirometer at 20 degrees C. Oxygen consumption (Mo2) of bluefin (7.1-9.4 kg) ranged from 235+/-38 mg kg(-1) h(-1) at 0.85 body length (BL) s(-1) to 498+/-55 mg kg(-1) h(-1) at 1.80 BL s(-1). Minimal metabolic rates of swimming bluefin were 222+/-24 mg O(2) kg(-1) h(-1) at speeds of 0.75 to 1.0 BL s(-1). Mo2 of T. albacares (3.7-7.4 kg) ranged from 164+/-18 mg kg(-1) h(-1) at 0.65 BL s(-1) to 405+/-105 mg kg(-1) h(-1) at 1.8 BL s(-1). Bluefin tuna had higher metabolic rates than yellowfin tuna at all swimming speeds tested. At a given speed, bluefin had higher metabolic rates and swam with higher tailbeat frequencies and shorter stride lengths than yellowfin. The higher M dot o2 recorded in Pacific bluefin tuna is consistent with the elevated cardiac performance and enhanced capacity for excitation-contraction coupling in cardiac myocytes of these fish. These physiological traits may underlie thermal-niche expansion of bluefin tuna relative to tropical tuna species.     

Temperature dependence of the Ca2+-ATPase (SERCA2) in the ventricles of tuna and mackerel

Recent physiological studies on the cardiovascular performance of tunas suggest that the elevated heart rates of these fish may rely on increased use of intracellular sarcoplasmic reticulum (SR) Ca2+ stores. In this study, we compare the cellular cardiac performance in endothermic tunas (bluefin, albacore, yellowfin) and their ectothermic sister taxa (mackerel) in response to acute temperature change. The cardiac sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) plays a major role during cardiac excitation-contraction (E-C) coupling, transporting Ca2+ from the cytosol into the lumen of the SR and thus promoting the relaxation of the muscle. Measurements of oxalate-supported Ca2+ uptake in SR-enriched ventricular vesicles indicated that tunas were capable of sustaining a rate of Ca2+ uptake that was significantly higher than the mackerel. Among tunas, the cold-tolerant bluefin had the highest rates of SR Ca2+ uptake and ATPase activity. The differences among Ca2+ uptake and ATP hydrolysis rates do not seem to result from intrinsic differences between the SERCA2 present in the different tunas, as shown by their similar temperature sensitivities and similar values for activation energy. Western blots reveal that increased SERCA2 protein content is associated with the higher Ca2+ uptake and ATPase activities seen in bluefin ventricles compared with albacore, yellowfin, and mackerel. We hypothesize that a key step in the evolution of high heart rate and high metabolic rate in tunas is increased activity of the SERCA2 enzyme. We also suggest that high levels of SERCA2 in bluefin tuna hearts may be important for retaining cardiac function at cold temperatures.     

ATP-induced reverse temperature effect in isohemoglobins from the endothermic porbeagle shark (Lamna nasus)

The evolutionary convergence of endothermic tunas and lamnid sharks is unique. Their heat exchanger-mediated endothermy represents an interesting example of the evolutionary pressure associated with this specific characteristic. To assess the implications of endothermy for gas transport and the possible contribution of hemoglobin (Hb), we investigated the effect of temperature on the oxygen equilibria of purified isohemoglobin components V and III from the porbeagle shark (Lamna nasus). In the absence of ATP the effect of temperature on oxygen affinity is normal in both Hb III (P50 = 0.9 and 2.2 torr at 10 and 26 degrees C, respectively) and Hb V (P50 = 1.5 and 2.5 torr at 10 and 26 degrees C, respectively). In the presence of this effector P50 decreases with increasing temperature in both components (P50 at 10 and 26 degrees C = 9.9 and 8.4 torr (Hb III), respectively, and 9.6 and 7.4 torr (Hb V), respectively. The reverse temperature effect in the presence of ATP will reduce the risk of oxygen loss from the arterial to the venous blood by lowering the oxygen tension gradient between the blood vessels. The mechanism behind the reverse temperature effect resembles that found in the bluefin tuna (Thunnus thynnus), an endothermic teleost, thus evidencing further convergent evolution.     

Quantification and speciation of mercury and selenium in fish samples of high consumption in Spain and Portugal

Mercury (Hg) and selenium (Se) determinations were carried out to evaluate human exposure to those elements through fish consumption in Spain and Portugal. Atomic fluorescence spectroscopy (AFS) was applied in a cold vapor mode for total mercury quantification and was also hyphenated to gas chromatography (GC) to achieve the speciation of organomercurial species in fish samples. The results obtained show the highest concentration of Hg in swordfish and tuna (0.47+/-0.02 and 0.31+/-0.01 microg g-1, respectively), and a much lower concentration in sardine, mackerel shad, and octopus (0.048+/-0.002, 0.033+/-0.001, and 0.024+/-0.001 microg g-1, respectively). The determination of alkyl mercury compounds revealed that 93-98% of mercury in the fish samples was in the organic form. Methylmercury (MeHg) was the only species found in the three fish species with higher mercury content.Total selenium concentration was high in sardine, swordfish, and tuna (0.43+/-0.02, 0.47+/-0.02, and 0.92+/-0.01 microg g-1, respectively), but low in mackerel shad and octopus (0.26+/-0.01 and 0.13+/-0.01 microg g-1, respectively). Speciation of selenium compounds was done by high-performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (LC-ICP-MS). Selenomethionine (SeMet) was the only selenium compound identified in the fish samples with higher selenium content.Among the fish species studied, sardine had the most favourable Se:Hg and SeMet:MeHg molar ratios; therefore, its consumption seems to be preferable.     

Red blood cell function and haematology in two tropical freshwater fishes from Australia

Salmon catfish and tarpon occur in habitats that periodically become deficient in oxygen resulting in high mortalities of other fish species. The water-breathing catfish, Arius leptaspis, and the facultative air-breathing tarpon, Megalops cyprinoides, both have high haemoglobin and haematocrit, and the oxygen carrying capacity in the air-breather is exceptionally high (15.6+/-1.2 vol%). Iso-pH oxygen equilibria of the red blood cells at 25 degrees C revealed high affinity (P(50)=9 mmHg, pH 7.4) and co-operativity (n(50)>2.2, pH 7.4) in the catfish, and contrasted with low affinity (P(50)=32 mmHg, pH 7.4) and co-operativity (n(50) approximately 1) in the air-breathing tarpon. Oxygen binding was further distinguished by relative pH insensitivity (Bohr factor, ?=Deltalog P(50)/Deltalog pH=-0.22) in the catfish, compared with a significant Bohr effect in the tarpon (?=-0.96). The potential for modulation of haemoglobin-oxygen affinity was indicated by a high ratio of GTP to ATP in the erythrocytes of the catfish, whereas regulation in the tarpon appeared due to ATP alone. Differences in blood respiratory functions between the two species are likely to reflect reduced opportunity for activity under extreme hypoxia in the catfish.     

Potential hazards associated with the consumption of Scombridae fish: Infection and toxicity from raw material and processing

Scombridae fish (tuna, bonito and mackerel) have significant ecological and economic values. They are very appreciated by consumers worldwide for their high-quality flesh and for their high nutritional value. However, consumption of Scombridae fish is potentially hazardous. Indeed, several cases of infections and toxicity linked to the consumption of Scombridae fish as raw, or processed food products have been reported worldwide. In this review, we presented the most common health risks associated with Scombridae fish consumption. Diseases associated with the consumption of these fish are generally infectious or toxic and are caused by biological hazards, such as bacteria, viruses, parasites or chemicals hazards that enter the body through contaminated fish (polycyclic aromatic hydrocarbons, histamine) or by physical contaminants, such as heavy metals. The risks of contamination exist throughout the food chain, from primary production to the preparation of products for consumption.     

Sodium o-iodobenzoate and hemoglobin-oxygen affinity: in vivo effects.

Sodium o-iodobenzoate (OISB) was given intravenously to 15 dogs to test the in vivo effect of this drug on the oxyhemoglobin dissociation curve. Administration of a single dose of 500 mg/kg was followed by an average increase in P50 (PO2 at 50% oxyhemoglobin saturation) of 3.6 mmHg from 26.8 +/- 0.5 to 30.4 +/- 1.8 mmHg (corrected to pH 7.4). This elevation was sustained for 7 days. During intravenous infusions of 200 mg/kg every other day for 3 wk, there was a sustained increase in P50 of 2.6 mmHg from 27.8 +/- 1.1 to 30.4 +/- 0.9 mmHg. All dogs survived the experiment and no ill effects of the drug were noted. An increase in serum lactate and pyruvate occurred in all animals following acute or chronic exposure to the drug. There was no significant change in whole blood pH, 2,3-diphosphoglycerate concentrations, intracellular pH, or serum total phosphate. Multiple infusions of sodium cyanate (50 mg/kg per day) reduced P50 by an average of 12.2 +/- 0.3 mmHg. A subsequent single infusion of OISB (500 mg/kg) failed to increase P50. Our preliminary data indicate that pharmacological manipulation of hemoglobin O2 affinity is possible with organic compounds unrelated to erythrocyte metabolism.     

Hematology of three deep-sea fishes: a reflection of low metabolic rates

Blood was collected from three species of fish, Antimora rostrata (Moridae), Lycodes esmarkii (Zoarcidae), Macrurus berglax (Macrouridae), caught at depths ranging from 280 to 2300 m. Hemoglobin concentrations were low in all three species, ranging from 4.4 to 5.4 g/100 ml. Mean erythrocyte volumes were relatively large, and ranged from 277 micron3 in M. berglax to 672 micron3 in A. rostrata. Blood oxygen dissociation curves were hyperbolic, with relatively low Hill constants (0.95-1.26). Mean P50 values ranged from 10 mmHg in M. berglax to 28 mmHg in L. esmarkii. It is concluded that the hematology and oxygen-binding characteristics of the blood of these three deep-sea fish reflects adaptations to low metabolic rates and low general activity habits.     

Changes in blood respiratory properties and cardiovascular function during acute exposure to hypoxic water in an air-breathing teleost, Channa maculata

Changes in blood respiratory properties and cardiovascular function of Channa maculata are described in fish acutely exposed to water of different oxygen tensions. The oxygen dissociation curve of the blood was hyperbolic, and the blood had a high oxygen affinity ( P ₅₀= 7.6 mm Hg at pH = 7.6) and a large Bohr coefficient. The persistently higher blood oxygen level in the dorsal aorta compared with that in the bulbus arteriosus regardless of the ambient oxygen tensions tends to support Ishimatsu and Itazawa's (1983) suggestion that oxygenated and deoxygenated blood could be. at least partially, separated in the heart of Channa . Acute exposure to hypoxic water induced insignificant changes in blood oxygen tension and saturation. However, bradycardia, hypotension, haemodilution and antidiuresis were observed in fish exposed to severely hypoxic (PO₂≤ 30 mmHg) but not to moderately hypoxic (PO₂= 50 mmHg) water. These data suggested that Channa maculala possessed adaptive blood respiratory properties and blood circulation pattern to allow the fish to take up oxygen from air without significant branchial oxygen loss to the surrounding hypoxic water.     

Oxygen transfer characteristics of the blood of reedfish, Erpetoichthys calabaricus

Oxygen transport characteristics and phosphate compounds were measured in the blood of reedfish, Erpetoichthys calabaricus, a bimodal breather. Blood from reedfish possessed the following values (mean +/- SD): hematocrit (21.7 +/- 0.4%), hemoglobin concentration (7.53 +/- 1.75 g%), red blood cell count (0.45 +/- 0.10 X 10(6)/mm3) and oxygen capacity (10.1 +/- 2.3 vol%). Although hematocrit, hemoglobin concentration, red blood cell count and oxygen capacity were all highly intercorrelated (P less than 0.01 in all cases), none of these parameters were significantly correlated with sex, weight or length in our sixteen fish sample. Erythrocyte volumes equalled 480 micrometers3, showed less variation (CV = 10.4%) and did not correlate with any other measured variable. Blood oxygen dissociation curves were sigmoidal and the P50's equalled 17.34 +/- 3.04 at 1% CO2 and 25 degrees C. Mean Bohr shift (delta log P50/delta pH) was -0.274 +/- 0.087. Temperature strongly influenced blood oxygen affinity. At 1% CO2, delta log P50/delta T equalled 0.026 +/- 0.006 (mean +/- SD). These hematological properties indicate that the blood of reedfish is similar to those of other tropical air-breathering species. Concentrations of total phosphate in the erythrocytes and percentage of total phosphate bound as nucleotide triphosphates were high. Surprisingly, 2,3diphosphoglycerate was found which has been reported in the erythrocytes of only two other fish species. Blood characteristics of reedfish exposed to air for 4 hr with one exception (Hill numbers) were not significantly different from water exposed controls. This suggests that the reedfish does not possess blood respiratory mechanisms to facilitate respiration solely by air-breathing.     

Blood oxygen transport in rats under hypothermia combined with modification of the L-Arginine-NO pathway.

Nitric oxide (NO) has high affinity to heme and by interaction with oxyhemoglobin (HbO2) is converted into nitrate to form methemoglobin (MetHb) as a side product. In combining with deoxy-Hb NO yields a stable molecule of nitrosyl-hemoglobin (HbFe(II)NO) that can further be converted into nitrate and hemoglobin (Hb). In addition, Hb was shown to transport NO in a form of S-nitrosohemoglobin (SNO-Hb). These features of the Hb and NO interaction are important for blood oxygen transport including hemoglobin-oxygen affinity (HOA). The present investigation was aimed to study the blood oxygen transport indices (pO2, pCO2, pH, HOA, etc.) in rats under hypothermia combined with a modification of L-arginine-NO pathway. To modify the L-arginine-NO pathway, rats were administered with N(G)-nitro-L-arginine methyl ester (L-NAME), L-arginine, or sodium nitroprusside (SNP) intravenously before cooling. A substantial impairment of oxygen delivery and development of hypoxia, with an important contribution of HOA into the latter accompanied the deep hypothermia in rats. All the experimental groups developed metabolic acidosis, less pronounced in rats treated with L-arginine only. In the experiments with a modification of the L-arginine-NO pathway, an enhanced cold resistance, attenuated oxygen deficiency, and a weaker oxyhemoglobin dissociation curve (ODC) shift leftwards were observed only after the administration of L-arginine. Neither SNP nor L-NAME had not any protective effects. L-Arginine lowered the value of standard P50 (pO2, corresponding to 50% Hb saturation with oxygen at 37 degrees C, pH 7.4, and pCO2 = 40 mmHg). The actual P50 (at actual pH, pCO2 and temperature) decreased by approximately 15 mmHg and was significantly higher than that under hypothermia without the drug treatment (21.03 +/- 0.35 vs 17.45 +/- 0.60 mmHg). NO also can contribute to this system through different mechanisms (HOA modification, vascular tone regulation, peroxynitrite formation, and effects).     

A comparison of myocardial β-adrenoreceptor density and ligand binding affinity among selected teleost fishes

This study quantified the cell surface β-adrenoreceptor density and ligand binding affinity in the ventricular tissue of seven teleost species; skipjack tuna (Katsowonus pelamis), yellowfin tuna (Thunnus albacares), Pacific mackerel (Scomber japonicus), mahimahi (dolphin fish; Coryphaena hippurus), sockeye salmon (Oncorhynchus nerka), rainbow trout (Oncorhynchus mykiss) and an Antarctic nototheniid (Trematomus bernacchii). β-Adrenoreceptor density varied by almost fourfold among these species, being highest for the athletic fish: sockeye salmon among the salmonids and skipjack tuna among the scombrids. β-Adrenoreceptor density was lowest for the Antarctic icefish. β-Adrenoreceptor binding affinity varied by almost threefold. We conclude that there is a significant species-specific variability in myocardial β-adrenoreceptor density and binding affinity and these interspecific differences cannot be attributed to temperature even though intraspecifically cold temperature can stimulate an increase in myocardial β-adrenoreceptor density. Instead, we suggest that interspecifically myocardial β-adrenoreceptor density is highest in fish that inhabit tropical water. Accepted: 12 July 2000     

Temperature sensitivity of calcium binding for parvalbumins from Antarctic and temperate zone teleost fishes

Parvalbumin (PV) is a soluble calcium-binding protein that is especially abundant in fast-twitch muscles of fish and other lower vertebrates. Despite its prevalence in ectothermic taxa, few data address the effects of temperature on PV binding function. In this study, calcium dissociation constants (KD) were measured as a function of temperature (0-25 degrees C) for PV from two Antarctic (Gobionotothen gibberifrons and Chaenocephalus aceratus) and two temperate zone fish species (Cyprinus carpio and Micropterus salmoides). Measurements by fluorometric competitive binding assay show that KD values for PVs from the Antarctic species were significantly higher at all assay temperatures and were less sensitive to temperature relative to carp and bass. However, estimates of KD are fundamentally similar for PVs from the Antarctic and temperate zone species when examined at their native physiological temperature. Variation in pH and ionic strength within a physiologically relevant range had only modest effects on KD. Thermodynamics of calcium binding to PV from G. gibberifrons and C. carpio was measured by isothermal microcalorimetry. When measured at 15 degrees C, the Gibbs free energy change (deltaG) was significantly greater for calcium binding to PV from G. gibberifrons than from carp (-43.4+/-1.5 kJ mol(-1) and -46.6+/-3.0 kJ mol(-1), respectively), and the relative contribution of entropy to deltaG for calcium binding to PV from the Antarctic species was about twice that of carp (deltaS=16.0+/-0.8 J degrees C(-1) mol(-1) for G. gibberifrons; deltaS=7.5+/-0.8 J degrees C(-1) mol(-1) for C. carpio).     

Oxygen-Linked S-Nitrosation in Fish Myoglobins: A Cysteine-Specific Tertiary Allosteric Effect

The discovery that cysteine (Cys) S-nitrosation of trout myoglobin (Mb) increases heme O₂ affinity has revealed a novel allosteric effect that may promote hypoxia-induced nitric oxide (NO) delivery in the trout heart and improve myocardial efficiency. To better understand this allosteric effect, we investigated the functional effects and structural origin of S-nitrosation in selected fish Mbs differing by content and position of reactive cysteine (Cys) residues. The Mbs from the Atlantic salmon and the yellowfin tuna, containing two and one reactive Cys, respectively, were S-nitrosated in vitro by reaction with Cys-NO to generate Mb-SNO to a similar yield (∼0.50 SH/heme), suggesting reaction at a specific Cys residue. As found for trout, salmon Mb showed a low O₂ affinity (P ₅₀ = 2.7 torr) that was increased by S-nitrosation (P ₅₀ = 1.7 torr), whereas in tuna Mb, O₂ affinity (P ₅₀ = 0.9 torr) was independent of S-nitrosation. O₂ dissociation rates (k _off) of trout and salmon Mbs were not altered when Cys were in the SNO or N-ethylmaleimide (NEM) forms, suggesting that S-nitrosation should affect O₂ affinity by raising the O₂ association rate (k _on). Taken together, these results indicate that O₂-linked S-nitrosation may occur specifically at Cys107, present in salmon and trout Mb but not in tuna Mb, and that it may relieve protein constraints that limit O₂ entry to the heme pocket of the unmodified Mb by a yet unknown mechanism. UV-Vis and resonance Raman spectra of the NEM-derivative of trout Mb (functionally equivalent to Mb-SNO and not photolabile) were identical to those of the unmodified Mb, indicating that S-nitrosation does not affect the extent or nature of heme-ligand stabilization of the fully ligated protein. The importance of S-nitrosation of Mb in vivo is confirmed by the observation that Mb-SNO is present in trout hearts and that its level can be significantly reduced by anoxic conditions.     

Resonance Raman and ligand binding studies of the oxygen-sensing signal transducer protein HemAT from Bacillus subtilis

HemAT-Bs is a heme-containing signal transducer protein responsible for aerotaxis of Bacillus subtilis. The recombinant HemAT-Bs expressed in Escherichia coli was purified as the oxy form in which oxygen was bound to the ferrous heme. Oxygen binding and dissociation rate constants were determined to be k(on) = 32 microm(-1) s(-1) and k(off) = 23 s(-1), respectively, revealing that HemAT-Bs has a moderate oxygen affinity similar to that of sperm whale myoglobin (Mb). The rate constant for autoxidation at 37 degrees C was 0.06 h(-1), which is also close to that of Mb. Although the electronic absorption spectra of HemAT-Bs were similar to those of Mb, HemAT-Bs showed some unique characteristics in its resonance Raman spectra. Oxygen-bound HemAT-Bs gave the nu(Fe-O(2)) band at a noticeably low frequency (560 cm(-1)), which suggests a unique hydrogen bonding between a distal amino acid residue and the proximal atom of the bound oxygen molecule. Deoxy HemAT-Bs gave the nu(Fe-His) band at a higher frequency (225 cm(-1)) than those of ordinary His-coordinated deoxy heme proteins. CO-bound HemAT-Bs gave the nu(Fe-CO) and nu(C-O) bands at 494 and 1964 cm(-1), respectively, which fall on the same nu(C-O) versus nu(Fe-CO) correlation line as that of Mb. Based on these results, the structural and functional properties of HemAT-Bs are discussed.     

Oxygen-related processes in red blood cells exposed to tert-butyl hydroperoxide

The correlation between the oxidative processes in tert-butyl hydroperoxide (tBHP)-exposed red blood cells and the reactions of oxygen consumption and release were investigated. Red blood cell exposure to tBHP resulted in transient oxygen release followed by oxygen consumption. The oxygen release in red blood cells was associated with intracellular oxyhaemoglobin oxidation. The oxygen consumption proceeded in parallel with free radical generation, as registered by chemiluminescence, but not to membrane lipid peroxidation. The oxygen consumption was also observed in membrane-free haemolyzates. The order of the organic hydroperoxide-induced reaction of oxygen release with respect to the oxidant (tBHP) was estimated to be 0.9 +/- 0.1 and that of the oxygen consumption reaction was determined to be 2.4 +/- 0.2. The apparent activation energy values of the oxygen release and oxygen consumption were found to be 107.5 +/- 18.5 kJ/mol and 71.0 +/- 12.5 kJ/mol, respectively. The apparent pKa value for the functional group(s) regulating the cellular oxyHb interaction with the oxidant in tBHP-treated red blood cells was estimated to be 6.7 +/- 0.2 and corresponded to that of distal histidine protonation in haemoprotein. A strong dependence of tBHP-induced lipid peroxidation on the oxygen concentration in a red blood cell suspension was observed (P50 = 32 +/- 3 mmHg). This dependence correlated with the oxygen dissociation curve of cellular haemoglobin. The order of the membrane lipid peroxidation reaction with respect to oxygen was found to be 0.5 +/- 0.1. We can conclude that the intensity of the biochemical process of membrane lipid peroxidation in tBHP-exposed erythrocytes is controlled by small changes in such physiological parameters as the oxygen pressure and oxygen affinity of cellular haemoglobin. Neither GSH nor oxyhaemoglobin oxidation depended on oxygen pressure.