Hc-O2 Affinity Research Articles

Molecular and functional characterization of hemocyanin of the giant African millipede,Archispirostreptus gigas

In contrast to other terrestrial arthropods, where gaseous O2 that fuels aerobic metabolism diffuses to the tissues in tracheal tubes, and most other metazoans, where O2 is transported to tissues by circulating respiratory proteins, the myriapods (millipedes and centipedes) strikingly have tracheal systems as well as circulating hemocyanin (Hc). In order to elucidate the evolutionary origin and biological significance of millipede Hc, we report the molecular structure (subunit composition and amino acid sequence) of multimeric (36-mer) Hc from the forest floor-dwelling giant African millipede Archispirostreptus gigas and its allosteric oxygen-binding properties under various physico-chemical conditions. Archispirostreptus gigas Hc consists of only a single subunit type with differential glycosylation. Phylogenic analysis revealed that millipede Hc is a sister group to centipede HcA, which supports an early divergence of distinct Hc subunits in myriapods and an ancient origin of multimeric Hcs. Archispirostreptus gigas Hc binds O2 with a high affinity and shows a strong Bohr effect. O2 binding is, moreover, modulated by Ca(2+) ions, which increase the O2 affinity of the Hc in the tense (T; deoxygenated) as well as the relaxed (R; oxygenated) states, and by (l)-lactate, which modulates Hc-O2 affinity by changing the allosteric equilibrium constant, L. Cooperativity in O2 binding at half O2 saturation (n50) is pH dependent and maximal at ~pH 7.4, and the number of interacting O2-binding sites (q) is markedly increased by binding Ca(2+). The data are discussed in the light of the mutually supplementary roles of Hc and the tracheal system for tissue O2 supply.

Diel vertical migration and the haemocyanin of krill Meganyctiphanes norvegica

Changes in environmental factors (PO2, temperature, salinity and food availability) influence the concentration of the respiratory pigment haemocyanin ((Hc)) present in many crus- taceans. We investigated the possibility that (Hc) and HcO2 affinity altered during the diel vertical migration (DVM) of Nordic krill Meganyctiphanes norvegica in the Gullmarsfjord, Sweden. The (Hc) decreased with increasing depth, for individuals both trawled and caged at different depths. Labora- tory experiments indicated that this pattern could not be explained by differences in PO2, tempera- ture or salinity. Alternatively, starvation had a significant effect on (Hc) over the course of a few h (≤10), i.e. a much shorter time scale than found for other crustacean species. Starved individuals showed a dramatic decrease in (Hc) compared with fed individuals. This decrease was exacerbated by an increase in temperature. We suggest that when M. norvegica migrates into deep water during the day, for whatever reason (predator avoidance or reduced prey abundance, or reduced ability to locate and capture prey), they cannot secure enough energy to meet routine metabolic demands; they therefore break down Hc and use it as an energy source. We conclude that there is likely to be a trade-off between the respiratory function of Hc and its importance in nutrition when krill migrate into deeper, nutritionally poorer waters during their DVM.

Hemocyanin oxygen-binding properties of a deep-sea hydrothermal vent shrimp: Evidence for a novel cofactor

Rimicaris exoculata is a caridean shrimp from the family Alvinocarididae which forms the dominant species around deep-sea hydrothermal vents from the Mid-Atlantic Ridge (MAR). Seeking respiratory adaptations to the hydrothermal environment, we have analysed the oxygen-binding properties of Rimicaris hemocyanin (Hc) in relation with temperature, pH, and lactate variations. Rimicaris native Hc is mostly composed of hexamers. It showed a high oxygen affinity (P50 approximately 3 Torr at pH 7.5, 15°C), a large Bohr effect (ΔlogP50/ΔpH = −1.87 ± 0.25, n = 6), a moderate lactate effect (ΔP50/Δlog[lac] = −0.12) and almost no temperature effect (ΔH = −1.23 kJ.mol−1 15–35°C). Most surprisingly, dialysis of native hemolymph elicited a large increase of HC-O2 affinity, an effect opposite to the usual trend observed for crustacean Hcs. Moreover, this increase in affinity could be reversed by adding an ultrafiltrate of native hemolymph to a dialysed sample, thus unveiling the existence of a dialysable yet unknown cofactor which decreases Hc-oxygen affinity. J. Exp. Zool. 277:357–364, 1997. © 1997 Wiley-Liss, Inc.

Respiratory, blood-gas transport, and acid-base response of Leptograpsus variegatus to long-term immersion and hyposaline exposure.

The respiratory and acid-base responses of the supratidal crab Leptograpsus variegatus to immersion in either 100% or 50% seawater were measured over a 3-wk period. Leptograpsus maintained haemolymph PO2 and CO2 during immersion, owing partly to elevated cardiac output and without recourse to hyperventilation. Both haemolymph PCO2 and CCO2 were reduced following 3 wk of immersion, and L-lactate remained low, but nonetheless Leptograpsus exhibited a marked metabolic acidosis. Specific responses to 50% seawater exposure were observed in Leptograpsus when compared with control crabs immersed in 100% seawater. Improved O2 delivery concomitant with elevated perfusion and ventilation maintained haemolymph oxygenation. In hyposaline-exposed Leptograpsus, both PCO2 and CCO2 were reduced after 24 h, while pH was elevated after 15 min and remained so during the 3-wk immersion. The marked acidosis exhibited in Leptograpsus exposed to 100% seawater was partially reduced in those in 50% seawater, presumably because of net acid excretion associated with elevated Na+ uptake. Immersion of Leptograpsus reduced Hc-O2 affinity to a greater extent in hyposaline-immersed individuals, while the cooperativity of O2 binding by Leptograpsus Hc decreased at low pH in both salinities. When immersed, the respiratory status of Leptograpsus resembles that of water-breathing crabs. Exposure to 50% seawater causes a twofold increase in energetic cost but does not perturb the immersed condition. The metabolic acidosis induced by chronic immersion is partially compensated at the lower salinity. Leptograpsus thus has the ability to take advantage of habitats ranging from supratidal through brackish to fully marine.

ROLE OF LUNGS AND GILLS IN AN AFRICAN FRESH-WATER CRAB, POTAMONAUTES WARRENI (DECAPODA: POTAMOIDEA), IN GAS EXCHANGE WITH WATER, WITH AIR, AND DURING EXERCISE

ABSTRACT Respiratory gas transport and acid-base state were investigated in the crab Potamonautes warreni breathing air or water for at least 24 h to assess the role of the lungs and gills. The efferent pulmonary and arterial samples had similar O2 content and thus either the lungs received most of the hemolymph flow or the lungs and gills were of equal importance during air-breathing. The gills were more important in immersed crabs. The O2 partial pressure in arterial hemolymph decreased from 8.6 kPa in air-breathing crabs to 4.5 kPa in water-breathing crabs. Despite an increase in O2 diffusion limitation during water-breathing (Ldiff = 0.78) compared to air-breathing crabs (Ldiff = 0.57) the arterial-venous O2 content difference did not change. In immersed crabs, the CO, content of the hemolymph (6.1 mmol·L–1) was half that in air-breathing crabs, but the pH remained unchanged at pH 7.4. Potamonautes warreni showed no specific adaptations to the >1400-m altitude of its habitat other than apparently relatively improved O2 diffusion. Polamonauteswarreni performed well during exercise in air. Crabs exercised at slow speed (1.8 m·min–1) experienced smaller hemolymph acid-base perturbations (ApH = 0.18) than crabs exercised at fast speed (ApH = 0.36; 3 m·min–1). While the partial pressure of 0, in the arterial and efferent pulmonary hemolymph during 20 min of fast exercise decreased from near 9 to 6 kPa or less, the hemocyanin 0, saturation was maintained near 80% and the arterial-venous O2 difference doubled. An increase in Hc-O2 affinity in exercising animals partially offset the Bohr effect and assisted in 0, uptake at the gas-exchange surfaces. The relative importance of lungs and gills in gas exchange after exercise in air was apparently similar to that in resting crabs. There was little requirement for anaerobiosis during submaximal exercise. Hemolymph L-lactate levels peaked at 3.6 mmol·L–1 after 20 min of fast exercise and were similar to L-lactate levels in the muscle.

Respiratory, Acid-Base, and Metabolic Responses of the Christmas Island Blue Crab, Cardisoma hirtipes (Dana), during Simulated Environmental Conditions

The dependency of the Christmas Island blue crab, Cardisoma hirtipes, on fresh water for respiratory gas exchange and transport was investigated in laboratory simulations. The gas exchange rates of air-breathing C. hirtipes were similar to those of other land crabs but decreased to 20% in submerged crabs. Crabs with access to air maintained arterial and pulmonary O2 content (CO2) and partial pressure (PO2), while in submerged crabs the PO2 and CO2 rapidly decreased (by 50%). There was no anaerobiosis, but haemolymph glucose concentration and cardiac output decreased when crabs were submersed, which suggests a hypometabolic state. Submersion induced a metabolic rather than a respiratory alkalosis, and since respiratory gas exchange was low, CO2 excretion to water was unimportant. Cardisoma hirtipes haemocyanin (Hc) has high O2 affinity but low pH sensitivity, which facilitates O2 uptake from hypoxic environments. The high Hc-O2 affinity supports O2 loading but may prevent access to a venous O2 reserve. Calcium, magnesium, and urate, but not L-lactate, were effectors of Hc-O2 affinity. In submerged crabs increased circulating urate maintained haemolymph O2 content. The CO2 capacitance and nonbicarbonate buffering of the haemolymph were relatively low. A significant Haldane effect seemed important for CO2 excretion but would require CO2 and O2 exchange to occur at the same organ (gills or lungs). Submersion interferes with respiration and is not needed for haemolymph acid-base balance; thus; C. hirtipes is an air-breathing crab.

Air Breathing by the Purple Shore Crab,Hemigrapsus nudus(Dana). III. Haemocyanin Function in Respiratory Gas Transport

The functional properties of the haemolymph and haemocyanin (Hc) of Hemigrapsus nudus were investigated with respect to the amphibious behaviour of this crab. The O₂ affinity of the Hc was decreased at higher temperature (∆H = −30 to −40 kJ mol⁻¹). Haemocyanin-O₂ affinity was sensitive to pH (Bohr factor = −0.843 at 15° C) but essentially insensitive to L-lactate (Δ log P50}/Δ log [lactate] = −0.053). The pH sensitivity of Hemigrapsus Hc was dependent on temperature (Bohr factor = −0.44 at 5°C, −0.90 at 25° C). Increasing CO₂ had no specific effect, nor was Hc-O₂ affinity increased by increasing levels of dopamine. Urate markedly increased the O₂ affinity of Hemigrapsus Hc (Δ log P50/Δ log [urate] = −0.203), and together with H+, it would seem to be the major organic effector of Hc-O₂ affinity. Increasing haemolymph Ca both potentiated Hc-O₂ affinity and decreased the magnitude of the Bohr shift. The potentiating effect was more pronounced at lower than normal Ca concentration, while Mg had little effect. The CO₂ capacitance of the haemolymph was more dependent on temperature than could be accounted for by the reduction in CO₂ solubility at higher temperature (CO₂ capacitance = 0.44-0.91 mmol L⁻¹ mmHg⁻¹). Consistent with a relatively low haemolymph protein concentration, the nonbicarbonate buffer was low (Δ[]/ΔpH = − 4.80), and thus changes in H+ concentration would promote large changes in pH and Hc-O₂ affinity. A Haldane effect was detected at 25° C Relating these data to in vivo measurements showed that acid-base balance in Hemigrapsus emerging from 10° C water into 25° C air was likely managed separately from CO₂ excretion. In crabs breathing 10° C water, the Hc transported 50% of the used O₂, while in animals breathing 25° C air the amount of O₂ so transported rose to 85%. Emersion-induced acidosis initially promoted a large decrease in O₂ affinity, reduced oxygenation, and promoted a decrease in the arterial-venous O₂ content difference. However, a subsequent metabolic alkalosis and an elevation in haemolymph urate reestablished the transport role of the Hc. Thus the pH sensitivity, especially at high temperatures, and the effect of urate seem crucial to the amphibious behaviour of Hemigrapsus, whose modulator systems are quite unlike those found in land crabs. Further work is required to determine whether these features represent a transitional stage between water and air breathing or whether they are specific adaptations to the supratidal environment or indeed are peculiar to grapsid crabs.

Ontogeny of respiratory function in crustaceans exhibiting either direct or indirect development

AbstractDialysed haemolymph from the planktonic larvae of the Norwegian lobster Nephrops norvegicus possessed a lower O2 affinity than the benthic adult stage at equivalent pH. This difference could be correlated with the ability to maintain O2 uptake during progressive hypoxia; adult individuals showed better developed regulation than the larvae. The intrinsic O2 affinity of both larval and adult haemocyanin (Hc) could be modified by preexposure to chronic hypoxia. In contrast, there were no significant differences noted in either Hc O2 affinity or the ability to regulate O2 uptake during progressive hypoxia between newly hatched and adult individuals of the direct developing amphipod Echinogammarus pirloti. Unlike the case in N. norvegicus preexposure to chronic hypoxia did not result in any change in respiratory performance at the level of the whole animal or in intrinsic Hc O2 affinity. The changes (or absence of them) in these aspects of the respiratory biology of the two species examined are discussed in the context of their ecological requirements. © 1995 Wiley‐Liss, Inc.

Oxygen binding by amphipod (Crustacea) haemocyanins

Haemocyanin (Hc) occurs in the haemolymph of all gammaroidean amphipods examined to date. Amphipod haemolymph is characterised by its comparatively low protein content, much of which is Hc, resulting in a low O2 carrying capacity. However, He has been shown to function as a respiratory pigment in vivo in at least some of the larger species during sustained activity. Amphipod Hcs seem to exist as hexamer aggregates. The number and types of subunit(s) present has still not satisfactorily been determined. The functional properties of amphipod Hcs are qualitatively similar to those of decapod and isopod crustaceans. They possess a moderate to large Bohr effect. Hc O2 affinity tends to be moderate in gammarid amphipods but higher than found in talitrid amphipods. Temperature affects O2 binding by some Hcs but not others: such effects could not be correlated with the ecology of all of the species examined. The colonisation of land by talitrid amphipods is accompanied by a reduction in both Hc O2 affinity and th...

Specific effects of thiosulphate and L-lactate on hemocyanin-O2 affinity in a brachyuran hydrothermal vent crab

The hydrothermal vent crab Bythograea thermydron Williams (Brachyura: Bythograeidae) is exposed to high environmental concentrations of hydrogen sulphide. Hydrogen sulphide has previously been shown to be oxidized to a non-toxic form, thiosulphate (S2O3 2-), that accumulates in the hemolymph (to concentrations>1 mmoll-1). Hemocyanin-oxygen (Hc-O2) affinity was determined in dialysed, fresh or frozen hemolymph samples from B. thermydron. Although freezing is known to alter the affinity or cooperativity of some decapod crustacean hemocyanins, neither Hc-O2 affinity nor cooperativity was significantly altered in B. thermydron hemolymph samples that had been frozen, consistent with previous findings. Oxygen affinity of B. thermydron hemocyanin was significantly increased by the presence of L-lactate. Likewise, Hc-O2 affinity was significantly increased by the presence of 1.5 mmol S2O3 2- I-1. The magnitude of this effect was the same as for similar concentrations of L-lactate. This specific effect of S2O3 2- does not appear to be a general property of crustacean hemocyanins, as there was no significant effect of S2O3 2- on Hc-O2 affinity or cooperativity in dialysed hemolymph samples from the brachyuran crabs Cancer anthonyi Rathbun and C. antennarius Stimpson, or the thalassinid ghost shrimp Callianassa californiensis Dana. In the context of high environmental sulphide concentrations coupled with low PO 2, and the subsequent accumulation of S2O3 2- in crab hemolymph, the increase in Hc-O2 affinity due to thiosulphate appears to be an adaptive response in B. thermydron.

Adaptations to the Deep-Sea Oxygen Minimum Layer: Oxygen Binding by the Hemocyanin of the Bathypelagic Mysid, Gnathophausia ingens Dohrn.

The bathypelagic mysid, Gnathophausia ingens Dohrn, lives aerobically at oxygen partial pressures as low as 6 torr in the oxygen minimum layer off southern California. This study is concerned with the O2 binding properties of this mysid's hemocyanin and the function of the pigment in O2 uptake at low Po2. The effect of temperature on in vivo hemolymph pH (ΔpH/ΔT = -0.018) was measured from 2.5 to 12.5°C. Hemocyanin concentration was estimated to be 24 mg/1, corresponding to an O2 binding capacity of about 0.3 mmol O2/l. Freezing of hemolymph samples significantly decreased the affinity and cooperativity of HcO2 binding, necessitating the use of fresh hemolymph. The HcO2 affinity was high (P50 of 1.4 torr at 5.5°C, pH 7.87), allowing the loading of O2 even at 6 torr. The cooperativity of HcO2 binding was also high (n50 = 3.5 at 5.5°C, pH 7.87); presumably allowing the pigment to function effectively as an O2 transporter within the small Po2 difference between the environment and the tissues. Temperature differences within the environmental range (2-10°C) had no significant effect on the oxygen affinity (ΔH = -6.7 kJ/mol, pH 7.7) or on the cooperativity of O2 binding. A large Bohr shift (Δ log P50/ΔpH = -0.80 to -0.81) was present at all temperatures. L-lactate produced moderate increases in HcO2 affinity (Δ log P50/Δ log [lactate] = -0.13 at pH 7.9) and in cooperativity. Regional and ontogenetic comparisons suggest that regional and ontogenetic differences in HcO2 affinity occur in this species. This mysid has a hemocyanin of unusually high O2 affinity and cooperativity of O2 binding for a crustacean living at low temperatures, and this appears to be an adaptation for oxygen loading and transport at the cold, low oxygen conditions in deep-sea oxygen minimum layers. The reduced temperature sensitivity of HcO2 affinity may also be an adaptation to low oxygen.

Haemocyanin oxygen binding and the physiological ecology of a range of talitroidean amphipods (Crustacea) I. pH, temperature, and l-lactate sensitivity

The effect of pH, temperature, and l-lactate on the O2 bindign properties of haemocyanin (Hc) from three talitroidean species i.e., the aquatic Apohyale pugettensis, the semi-terrestrial Megalorchestia californiana, and the semi-/euterrestrial Traskorchestia traskiana was studied. The Hc of A. pugettensis was characterized by a higher O2 affinity (and more pronounced Bohr shift) than the Hc of either M. californiana or T. traskiana. Apohyale was the only species that possessed He that was sensitive to temperature change. Resuspending Hc from each of the three species in a stock Ringer's solution (based on the ionic composition of the haemolymph of T. traskiana) showed that the persistence of the difference in Bohr shift between Apohyale and the other two species was due to differences in the haemocyanins themselves and not attributable to their respective ionic environments. An inverse relationship was found between the cooperativity (n50) and pH of Hc from T. traskiana and A. pugettensis but not for M. californiana. In each case adding l-lactate increased Hc O2 affinity, but this was most pronounced for A. pugettensis.

The role of the blood in the temperature dependence of oxidative metabolism in decapod crustaceans. I. Intraspecific responses to seasonal differences in temperature

AbstractTotal oxygen uptake in the blue crab Callinectes sapidus doubles with a rise in ambient temperature in the range 15–25°C, and increases precipitously (Q10=4.92) in the range 5–15°C. Both systemic and cellular phenomena are responsible for the pattern. At summer temperatures, where motor activities are maximal, the oxygen affinity of blue crab hemocyanin is low, and considerable volumes of oxygen are delivered to the tissues. At winter temperatures, where feeding and locomotion virtually cease, the hemocyanin‐oxygen (HcO2) affinity is so high that little oxygen can be extracted by the tissue and O2 is transported entirely in the free form. At the transitional temperature of 15°C, a seasonal acclimation of HcO2 affinity permits appreciable oxygen delivery to the tissues in the spring, but curtails oxygen delivery in the fall.Both muscle and hepatopancreas utilize the hexose monophosphate shunt (HMS) in carbohydrate metabolism. Specific activities of the enzymes participating in the HMS are greater in hepatopancreas than muscle, and the sensitivity of O2 uptake to the glycolytic inhibitor iodoacetate (10−2 M) is smaller. The temperature dependence of O2 uptake in the range 15–25°C is much smaller in hepatopancreas than in muscle. The ratio of C‐1/C‐6 labeled glucose utilization indicates that, at low temperature, the activity of the HMS in hepatopancreas is five times greater than that of glycolysis. Thus, the low thermal sensitivity of O2 uptake in hepatopancreas appears to be due to a change in the relative contribution of the two pathways to total carbohydrate metabolism.There is no evidence of a compensatory response of intermediary metabolism in muscle, which comprises the major fraction of total biomass. The thermal dependence of O2 uptake by the animal exceeds that of either hepatopancreas or muscle, due to the large increase in HcO2 affinity, and hence a decrease in tissue O2 supply, at low temperature.

The role of the blood in the temperature dependence of oxidative metabolism in decapod crustaceans. II. Interspecific adaptations to latitudinal changes

AbstractThe effects of acclimation temperature on hemocyanin‐oxygen (HcO2) transport were examined in two species of decapod crustaceans representing different geographic and thermal ranges. In the coldwater species Cancer borealis and the warmwater species Menippe mercenaria, the performance of the HcO2 transport system is maximized at 15 °C, a summer temperature in the northern habitat and a winter temperature in the southern habitat. In C. borealis little unloading at the tissues occurs at 5 °C, and in M. mercenaria oxygenation at the gill is incomplete at 25 °C. The temperature coefficients describing rates of ventilation, heartbeat, and total oxidative metabolism fall within a narrow range (Q10 = 1.9−3.6), providing no indication of ventilatory or cardiovascular adjustments that would allow the HcO2 transport system to function at the maximum level at different temperatures. Comparison of the HcO2 equilibrium properties suggests that HcO2 affinity has been selected for a particular thermal range, but not cooperativity or pH dependence. The thermal limits of the O2 transport system are set by both the direct effect of temperature on HcO2 equilibrium and the indirect effect via the Bohr shift that accompanies the change in blood pH with temperature.