Oxygen Affinity Of Hemocyanin Research Articles

Oxygen Transport and Acid-Base Balance in the Haemolymph of the Lobster, Homarus Gammarus, During Aerial Exposure and Resubmersion

ABSTRACT Submerged lobsters at 15°C were normoxaemic ( at a of 6·53 kPa) and normocapnie (; [HCO3-] = 9·3mequivl-1 and pHa = 7·78). After 3h in air the haemolymph was markedly hypoxic and hypercapnic (; ; and pHa = 7·64). Disturbance after 3h in air caused a greater increase in to 1·28 kPa and a fourfold increase in lactate levels to 3·6 mmol l-1. The combined respiratory and metabolic acidosis reduced pHa to 7·39. After 14 h in air, undisturbed lobsters remained hypoxic and hypercapnic (; ). Lactate levels had increased to 6·2mmoll-1. Despite this clear limit on respiratory gas exchange in air, oxygen transport by the haemolymph was restored. A rise in buffer base ([HCO3-] = 15·8mequivl-1) compensated for the potential respiratory and metabolic acidosis and pH was unchanged at 7·63. The combined effects of the increase in lactate (ΔlogP50/ Δlog[lactate] =–0·175) and calcium (ΔlogP50/Δlog[Ca2+] = — 0·20 at pH7·63) levels contributed to an increase in oxygen affinity of haemocyanin at constant pH. Consequently, mean increased from 0·2 to 0·38 mmol l-1 between 3h and 14 h in air. Resubmergence after 14 h in air resulted in a transient alkalosis due to retention of bicarbonate; oxygen and CO2 were rapidly restored to submerged levels. The lobster possesses the appropriate respiratory adaptations for survival during the relatively long periods of exposure in air encountered during commercial shipment.

Potentiation of Hemocyanin Oxygen Affinity by Catecholamines in the Crab Cancer magister: A Specific Effect of Dopamine

The hemocyanin from Cancer magister was confirmed to be sensitive to L-lactate, which increased oxygen affinity. Additions of various monoamines, reported to be released as neurohormones, from the pericardial organs of crab hearts were found also to potentiate in vitro the oxygen affinity of hemocyanin from C. magister. Of the compounds tested, the catechol dopamine was found to be the most active. In addition to increasing oxygen affinity, high concentrations of dopamine decreased the cooperativity of oxygen binding. Analysis indicates that these effects are due to an increase in the affinity of the deoxygenated hemocyanin. The effect of dopamine could be summated with that of lactate to increase the oxygen affinity of dialyzed hemolymph to that of the original blood sample. Preincubation of the hemocyanin with the dopamine antagonist butaclomol was found to abolish the effect Thephysiological significance of a potential neurohormonal modulator of hemocyanin oxygen affinity is discussed.

Neurohumor effects on crustacean haemocyanin oxygen affinity

AbstractVarious monoamines released as neurohormones from the pericardial organs of crab hearts were found to potentiate the oxygen affinity of haemocyanin from Cancer magister. Of the compounds tested, the catechol dopamine was the most active. Preincubation of the haemocyanin with a dopamine antagonist abolished the effect, indicating a specific site. Haemocyanin may also retard the rapid auto‐oxidation of dopamine that normally occurs at physiological pH.

Adaptations to a Terrestrial Existence by the Robber Crab Birgus Latro: I. An in vitro Investigation of Blood Gas Transport

ABSTRACT The gas-transporting properties of the haemolymph of Birgus latro L. were investigated in vitro. This terrestrial anomuran is restricted in distribution to the tropics, and on Christmas Island inhabits a highly stenothermal environment. The effect of temperature on haemocyanin oxygen-affinity was pronounced (ΔH = –39 kJ mol−1) and was considered to represent the absence of any specific adaptation to environmental temperature. The Bohr effect was large for a terrestrial decapod (ϕ = –0·60), although reduced at low pH. Changes in [Ca] had a significant effect on oxygen affinity of dialysed haemolymph (ΔlogP50/ Δlog[Ca] = –0·39) whereas [Mg] had no effect. Increasing, [L-lactate] had a small effect on the oxygen affinity of dialysed haemolymph (ΔlogP50/ Δlog[lactate] =–0·013) but not whole haemolymph. Dialysis increased oxygen affinity, suggesting the presence of a dialysable component that suppresses affinity. The effect of L-lactate was inhibited in whole haemolymph. The oxygen affinity of Birgus haemolymph was largely insensitive to effector substances, with the possible exception of Ca. In the case of lactate, at least, this was not due to a reduced sensitivity of the haemocyanin, a situation different from that in closely related species. Carbon dioxide transport was also affected by temperature. Birgus haemolymph showed a high nonbicarbonate buffer capacity which could be correlated with a high haemocyanin concentration. It is concluded that terrestrial anomuran decapods depend on mechanical adjustments of ventilation and perfusion, rather than employing direct modulation of haemocyanin function, to optimize oxygen delivery.

Temperature Sensitivity of Molluscan and Arthropod Hemocyanins

The temperature sensitivity of hemocyaninoxygen affinity and cooperativity was measured at 5, 15, 25, and 35°C in a variety of manine molluscs and arthropods from different thermal environments. These environments included a subtidal habitat in which the temperature is generally less than 15°C and the diurnal temperature variation is small, and an intertidal habitat in which the temperature varies more than 30°C. The temperature sensitivity of P50 showed considerable variation (▵H = 0 to ▵H = -67 kJ/mol) depending on species and experimental temperatures. Sensitivity generally decreased as temperature increased. In several species temperature sensitivity was either absent or greatly reduced above 15°C. The horseshoe crab Limulus polyphemus showed a minimum temperature sensitivity between 15 and 25°C but higher sensitivity above and below this range. The hypothesis that a greater interaction between hemocyanin molecules and calcium ions at high temperatures offsets the temperature effect, resulting in a pi...

The haemocyanin of the tarantula Lasiodora erythrocythara—the influence of CO 2, organic cofactors and temperature on oxygen affinity

1. 1. Whole blood oxygen equilibrium curves for the haemocyanin of the Honduran tarantula Lasiodora erythrocythara have for the first time been investigated in vitro. 2. 2. Oxygen affinity was pH dependent with a mean Bohr coefficient of −0.71 ± 0.06 between pH 7.3 and 8.1. At 25°C and pH 7.6 the P 50 value was 23 Torr. 3. 3. The fixed acid Bohr effect was not significantly different from the CO 2 Bohr effect indicating the lack of a specific effect of CO 2 on oxygen affinity. 4. 4. Dialysis had no measurable effect on haemocyanin oxygen affinity, indicating the absence of unidentified dialysable factors. The haemocyanin was also insensitive to changes in the concentration of d and l lactate, succinate, guanosine and urate. 5. 5. Temperature was found to have little influence on oxygen affinity between 20 and 25°C illustrated by the low Δ H value of −5.6 kJ mol −1. At the temperature extremes of 15 and 30°C Δ H values increased to −29kJmol −1. 6. 6. Cooperativity, expressed as n 50, was both temperature and pH dependent.

The Regulation of Haemocyanin Oxygen Affinity During Emersion of the Crayfish Austropotamobius Pallipes : III. The Dependence of Ca2+-Haemocyanin Binding on the Concentration of L-Lactate

ABSTRACT The binding of Ca2+ to the haemocyanin of the crayfish Austropotamobius pallipes was investigated. The amount of bound Ca2+ was determined using an ultrafiltration technique to produce haemocyanin-free solutions, the Ca2+ concentration of which could then be compared with that of the original, unfiltered solution. Any difference between the two values would indicate the amount of calcium bound by haemocyanin. The effect of L-lactate on Ca2+ binding was investigated by determining the amount of bound ion at different concentrations of L-lactate. In addition, oxygen equilibrium curves were constructed for some of the solutions to verify that the haemocyanin oxygen affinity remained sensitive to L-lactate and to determine whether the haemocyanin was functionally similar to that used in previous investigations. With 17 mmol 1−1 total Ca2+ and approximately 1 mmol 1−1 L-lactate the number of Ca2+ binding sites was estimated to be between eight and nine per haemocyanin molecule. Without taking into account the formation of calcium lactate, the observed dependency of Ca2+-haemocyanm binding on L-lactate concentration could best be described by the equation: Ca2+/Hc = 8·64– 0·32[lactate−]. A ‘worst case’ estimate for maximum calcium lactate formation, assuming Ca2+ to be the only counterion available to lactate, altered the relationship slightly to: Ca2+/Hc = 8 ·65– 0·35[lactate-]

The influence of serum lipids on oxygen binding of Callinectes sapidus hemocyanin

1. 1. Addition of the detergent Triton XI00, which removes lipid moieties from lipoproteins, to the serum of Callinectes sapidus did not change hemocyanin-oxygen affinity. 2. 2. Addition of the soluble fraction and/or vesicles of phosphatidyl choline, the major lipid in crustacean blood, to Triton X-treated hemocyanin did not alter oxygen affinity. Addition of lipids prepared from native serum did not influence oxygen affinity. 3. 3. Despite the close correlation between the two in nature, the seasonal changes in serum phospholipids cannot explain the seasonal changes in hemocyanin-oxygen affinity in this species.

The effect of ambient oxygen and temperature on haemolymph l-lactate and urate concentrations in the shore crab Carcinus maenas

1. 1. Shore crabs, Carcinus maenas, were exposed to various oxygen levels at medium (15°C) and high (30°C) ambient temperatures. 2. 2. Elevated l-lactate concentrations were found in haemolymph only in severely hypoxic water (Po 2 about 10 torr) in resting crabs, concomitant with a marked alkalosis and an increase in haemolymph calcium. 3. 3. Haemolymph urate increased at both hypoxic levels tested and at high ambient temperature, but decreased in hyperoxic water. 4. 4. It is concluded that urate may act as a true in vivo modulator of haemocyanin oxygen affinity.

Oxygen Binding by the Hemocyanin of the Terrestrial Hermit Crab Coenobita Clypeatus (Herbst): The Effect of Physiological Parameters in vitro

In situ, the terrestrial hermit crab Coenobita clypeatus may be exposed to extremes of temperature together with the associated problems of dehydration. In the present in vitro study, the effect of temperature on hemocyanin oxygen affinity was small over the range 25-30 C (ΔH = -14.8 kJ · mol⁻¹). Outside this range, however, changes in enthalpy were >39 kJ · mol⁻¹. Changes in calcium and hemocyanin concentration over the range expected in vivo did not appreciably affect oxygen affinity. A dilution of the hemocyanin concentration to 50% of the original value decreased oxygen affinity only slightly. The addition of L-lactate, a known effector of hemocyanin oxygen affinity to nondialyzed and dialyzed hemolymph, did not influence hemocyanin oxygen affinity. The hemolymph of Coenobita contained a large amount of urate (~1 mM) and 1.2 mM L-lactate. The removal of this ion, also an effector of hemocyanin oxygen affinity, resulted in a hemolymph preparation that exhibited the same oxygen-binding properties as whole hemolymph. The hemocyanin-free plasma of Coenobita was, however, capable of increasing the O₂ affinity of crayfish hemocyanin, confirming that Coenobita hemocyanin itself is extremely insensitive to identified effectors of hemocyanin oxygen affinity in other crustaceans.

The potentiating effect of purine bases and some of their derivatives on the oxygen affinity of haemocyanin from the crayfishAustropotamobius pallipes

1. At pH 7.8 theP50 of dialysedAustropotamobius pallipes haemolymph (P50=1.7 Torr) was 2.9 Torr greater than that of nondialysed haemolymph (P50=4.6 Torr). This difference was not attributable to the specific effect ofl-lactate but rather to unidentified factors (U.F.) which increase the affinity of haemocyanin for oxygen. 2. The presence of 0.35 mM urate in the haemolymph of this species was confirmed as was the potentiating effect of this ion on oxygen affinity. In this study 2 mM urate reduced theP50 of dialysed haemolymph by approximately 50% from 4.6 Torr to 2.1 Toor. 3. The specificity of the haemocyanin for a specific purine structure was demonstrated to be low as the urate analogues, caffeine and theobromine, isolated from plants also increased the oxygen affinity of dialysed haemolymph. 4. Metabolites arising from the degradation of ADP/ATP to urate can also increase haemocyanin oxygen affinity to a variable extent but not as effectively as urate. Compounds in which the purine ring was cleaved evoked very little or no specific enhancement of haemocyanin oxygen affinity. 5. A combination of purine bases and derivatives, shown to produce individually an increase in oxygen affinity, did not produce an effect in excess of that due to urate alone. The presence of high concentrations of urate in the haemolymph abolishes the specific effect ofl-lactate. 6. Additive and synergistic effects ofl-lactate and the various purine derivatives may account for 80% of the U.F. effect.

The Regulation of Haemocyanin Oxygen Affinity During Emersion Of the Crayfish Austropotamobius Pallipes: II. An Investigation of In Vivo Changes in Oxygen Affinity

ABSTRACT The oxygen-transporting properties of the haemolymph from the crayfish Austropotamobius pallipes were investigated. The haemolymph concentrations of K+, Mg2+ and Ca2+ ions, together with the concentration of L-lactate were measured in crayfish before, during and after 24 h emersion. The concentrations of K+ and Mg2+ increased during aerial exposure and returned to pre-emersion levels during immersed recovery. Large increases in the level of circulating L-lactate and Ca2+ were correlated with short- and long-term aerial exposure respectively. Changes in the concentrations of these ions could also be correlated with changes in haemocyanin oxygen affinity. Reimmersion and recovery returned all parameters to near control values. The effect, on haemocyanin oxygen affinity, of the Bohr shift alone was calculated and compared with the change in oxygen affinity (ΔP50) actually determined during aerial exposure. These data were also compared with predictions, calculated from in vitro data, for the potentiation of haemocyanin oxygen affinity by Ca2+ and L-lactate ions in aerially exposed crayfish. The physiological significance of the regulation of haemocyanin oxygen affinity by these ions is discussed.

The Regulation of Haemocyanin Oxygen Affinity During Emersion of the Crayfish Austropotamobius Pallipes: I. An In Vitro Investigation of the Interactive Effects of Calcium and L-Lactate On Oxygen Affinity

Abstract The haemolymph of the crayfish Austropotamobius pallipes exhibits a high affinity for oxygen (P50 = 2·7 Torr at pH 7·9 and 15°C) and a modest Bohr effect (φ =−0·455). The affinity of haemolymph dialysed against a crayfish Ringer was lower with a P50 value of 6·4 Torr at the same temperature and pH. The oxygen affinity of the dialysed haemolymph can be increased markedly by increased concentrations of L-lactate and to a greater extent by elevated concentrations of calcium ions. In the dialysed preparation, the potentiating effects of L-lactate and Ca2+ on haemocyanin oxygen affinity were found to be interdependent. Elevating the concentration of one of these two ions reduced the effect of the other. The increase in the oxygen affinity of the haemocyanin brought about by elevated Ca2+ and L-lactate was insufficient to account for the difference in affinity between dialysed and nondialysed haemolymph. The mutually agonistic effects of Ca2+ and L-lactate are described both empirically and graphically.

Modulation of haemocyanin oxygen affinity in the prawn, Palaemon elegans (Rathke) under environmental salinity stress

The effects of salinity acclimation on the oxygen-transporting properties of the blood of the intertidal prawn Palaemon elegans (Rathke) have been examined. Although there was considerable variation in the haemocyanin content of the haemolymph between individual animals, an inverse relationship was obtained between the haemocyanin content of the blood and the salinity to which the animals were acclimated. This resulted in an increase in the oxygen-carrying capacity of the haemocyanin from 1.2 mmol · 1 ∮-1 to 1.48 mmol · 1 −1 in prawns acclimated to salinities of 30 and 10%., respectively. No significant differences were observed in either the oxygen affinity ( P 50 = 31.3–34.0 Torr, pH = 7.8, temperature = 10 °C) or in the size of the Bohr effect (mean value of Φ = −0.74) of the haemocyanin in animals maintained in salinities of 10, 20, and 30%., but blood from animals kept in water having a salinity of 40%. had both a higher oxygen affinity ( P 50 = 11.8 Torr, pH = 7.8) and a higher Bohr value (Φ = −1.26). These increases were primarily due to changes in the concentration of ions (mainly Mg 2+) in the blood of these animals. The co-operativity of the haemocyanin, however, did not differ significantly between the four groups of prawns. Similarly, the effect of l-lactate on the modulation of haemocyanin oxygen affinity was found to be unaffected by changes in the ionic composition of the blood associated with acclimation to differing salinities.

A new role for uric acid: Modulator of haemocyanin oxygen affinity in crustaceans

AbstractUric acid enhances the oxygen affinity of some crustacean haemocyanins and can account for some of the potentiating effects due to unidentified factors in the haemolymph of the freshwater crayfish Austropotamobius pallipes. A similar effect can be elicited by an analogous purine, caffeine, which demonstrates a low specificity of haemocyanin for urate. This suggests that other haemolymph purines might produce a cumulative effect on haemocyanin oxygen affinity.

Evidence for the presence and specificity of an unidentified plasma cofactor increasing haemocyanin oxygen affinity in two natantid crustaceans

AbstractInvestigation of haemocyanin oxygen affinity in two species of Palaemon has demonstrated the presence of an unidentified factor (s); (U.F.), in the plasma, that increases oxygen affinity. Plasma exchange experiments have further demonstrated that this factor is not species‐specific and that the magnitude of the effect is dependent on the particular haemocyanin molecule. It has not been possible to demonstrate conclusively that the effect of this factor(s) can be reduced by dilution of the plasma. The variability in the extent of this cofactor effect, throughout the Crustacea, may be explained by variable sensitivity of haemocyanin to U.F.

Gas Exchange, Acid-Base Balance, and the Oxygen Supply to the Tissues During a Molt of the Blue Crab Callinectes Sapidus

At temperatures compatible with molting the hemocyanin in the blood of an intermolt stage C blue crab transports all but a very small fraction of the oxygen consumed. A number of previous investigations suggest that the respiratory mechanisms permitting the oxygen carrier to function at such a high level during stage C are perturbed during a molt. Many of these suggestions prove to be correct. The uptake of water and the apparent decrease in net hemocyanin synthesis during stages D3 through A result in the virtual loss of the hemocyaninoxygen transport system. A pronounced premolt alkalosis compensates for a concomitant hypercapnia. During exuviation lactate frequently accumulates in the blood, which presumably results from the inadequacy of gas exchange and aerobic metabolism at this time. When the molt is successful, however, the blood becomes only slightly acidotic, if at all, and the acidosis rapidly disappears. The changes in net metabolism of hemocyanin do not entail shifts in the composition of functionally distinct subunits that might permit an adaptation of the oxygen transport system to altered operating conditions in the blood during the early postmolt. There is, however, an increase in oxygen permeability of the general body surface, which becomes a site of oxygen uptake from the medium directly into the metabolically activated epidermis and also into the blood. In animals that survive a molt, including groups exposed to moderately hypoxic as well as normoxic water, there is no sign of tissue hypoxia and total 02 uptake actually increases during the early postmolt. The critical role of these compensatory mechanisms is indicated by severe acidosis and tissue hypoxia prior to mortality in cases of aborted molts and molts that result in morphological aberrations. Later in the postmolt period the decrease in blood calcium that accompanies hardening of the new skeleton lowers hemocyanin-oxygen affinity, permitting the carrier to function at elevated blood PO2. In the decapod crustaceans the hemocyanin (Hc) in the blood is responsible for a very large fraction of the oxygen supplied to metabolizing tissue, especially at the high temperatures compatible with molting in temperate zone species. At least 80% and more commonly 90% of the oxygen consumed is transported to the tissues of a stage C animal in the form of HcO2 (reviewed by Mangum, 1983a). And yet numerous observations suggest that the mechanisms of gas exchange and transport are profoundly perturbed both during and immediately following a molt.

Effects of hypoxia and l-lactate on the haemocyanin-oxygen affinity of the lobster, Homarus vulgaris

1. 1.Dialysis of lobster haemolymph against a physiological saline entailed a decrease of the haemocyanin-oxygen affinity. 2. 2. L-Lactate added to dialysed haemolymph increased the oxygen affinity of the haemocyanin without changing the Bohr effect. The effect of adding D-lactate was much less. 3. 3.In lobsters exposed to ambient hypoxia ( P O 2 = 35 Torr at 15°C), whole haemolymph oxygen affinity increased irrespective of pH. This in vivo increase cannot be accounted for by L-lactate accumulation alone, but must involve another, as yet unidentified dialysable factor.

A model for l-lactate binding to Cancer magister hemocyanin

1. 1. l-Lactate raises the oxygen affinity of Cancer magister hemocyanin. The l-lactate analogs, d-lactate, glycolate and 2-methyl-lactate cause smaller increases in an oxygen binding affinity. Other analogs have no detectable effect. 2. 2. These data suggest that l-lactate binds to the hemocyanin at all four positions around the chiral carbon. 3. 3. The carboxyl and hydroxyl groups are required for activity. The protein can only partially distinguish between the methyl group and hydrogen atom.

The CO2‐specific sensitivity of hemocyanin oxygen affinity in the decapod crustaceans

AbstractThe CO2‐specific sensitivity of hemocyanin oxygen affinity and oxygen binding site cooperativity were investigated in five species of crabs. The hypothesis that CO2‐specific effects on hemocyanin are related to the molecular CO2 concentrations that occur naturally in crab hemolymph was tested in crabs that experience a variety of hemolymph CO2 pressures. Crab species were chosen from subtidal, intertidal, and semiterrestrial habitats. Hemolymph samples from each species were added to physiological salines buffered with 50 mM HEPES. O2 equilibrium curves were determined spectrophotometrically by gassing the hemolymph‐saline mixtures with different P at constant P and temperature. We found no CO2‐specific effects on oxygen equilibrium properties of hemocyanin in any species under the conditions investigated. It is concluded that molecular CO2 and bicarbonate ions are not important in directly modulating the oxygen equilibrium properties of crustacean hemocyanins.