Heinz Decker

Spider hemocyanin binds ecdysone and 20-OH-ecdysone.

Fluorescence quenching studies and binding experiments with [3H]ecdysone reveal that the respiratory protein, hemocyanin, of the tarantula Eurypelma californicum binds ecdysone. The binding constant for ecdysone ranges between 0.5 and 5 mm, indicating a low affinity binding. However, it is comparable with those found for the ecdysone binding to hexamerins from insects. Based on a comparison of sequences and x-ray structures of arthropodan hemocyanins, we propose an evolutionary conserved hydrophobic pocket in domain 1 of the hemocyanin subunit that may bind ecdysone.

Quaternary and subunit structure of Calliphora arylphorin as deduced from electron microscopy, electrophoresis, and sequence similarities with arthropod hemocyanin.

SummaryArylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1×6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10×12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a very minor and a third immunologically separated component remains unclear. A novel 2×6 arylphorin particle was detected and isolated. It comprises less than 10% of the total arylphorin material and shows a long, narrow interhexamer bridge in the electron microscope. An arylphorin dissociation intermediate identified as a trimer (1/2×6) was isolated; its possible quaternary structure is discussed on the basis of electron micrographs. The epitope of monoclonal antibody Ec-7 directed against tarantula (Eurypelma californicum) hemocyanin subunit d and also reactive to Calliphora arylphorin was traced to a highly conserved peptide of 27 amino acids localized in the center of the protein. The primary structure of Calliphora arylphorin as published in our preceding paper (Naumann and Scheller 1991) is compared in detail to the sequences of spider and spiny lobster hemocyanin. This revealed a basic framework of 103 strictly conserved amino acids. Isofunctional exchanges are proposed for another 76 positions. On the basis of these similarities, and the published three-dimensional model of spiny lobster hemocyanin, a detailed model of the quaternary structure of Calliphora arylphorin is presented. A second larval storage protein previously termed protein II was purified from Calliphora hemolymph. It was demonstrated to be a 500 kDa hexamer of 83 kDa subunits. In the electron microscope it shows a cubic view 9 nm in length with a large central hole and a rectangular view (9×10 nm) with a large central cavity. A morphologically very similar hemolymph protein was detected in Drosophila melanogaster larvae. From its structural appearance it is uncertain whether protein II belongs to the hemocyanin superfamily or not.

Oxygen transport in Crayfish blood: Effect of thermal acclimation, and short-term fluctuations related to ventilation and cardiac performance

SummaryThepO2 in the hemolymph of the decapod crustaceanAstacus leptodactylus was measured continuously with micro O2 electrodes. PostbranchialpO2, heart rate and ventilation rate were simultaneously recorded. PrebranchialpO2 was measured in a separate series of experiments. Crayfish acclimated for six weeks to warm (20 °C) and cold (10 °C) conditions were studied. 1.Postbranchial bloodpO2 was measured in the pericardium. Typical values for warm-acclimated animals fell in the range 23–35 Torr, with a mean of 28.3 Torr, and those for cold-acclimated animals were 17–30 Torr, with a mean of 24.4 Torr. PrebranchialpO2 was measured in the meropodites of the 8th thoracopod. VenouspO2 levels ranged from 7–13 Torr, mean 9.4 Torr (warm-acclimated), and 6–9 Torr, mean 6.8 Torr (cold-acclimated).2.pO2 values of resting, restrained animals were variable and fluctuations occurred in both the pre- and the postbranchial levels. Fluctuations were either small (1–5 Torr) or large (10–70 Torr).3.PostbranchialpO2 correlated with fluctuations in the ventilation and heart rates. ArterialpO2 rose when the ventilation rate was increased, with a delay of about 10 s in warm-acclimated and 20 s in cold-acclimated animals, respectively. Along with increasingpaO2 an increase of the heart rate was established.4.In resting animals, there is no a-v pH-difference. Cell-free hemolymph, taken from the pericardium or the meropodites, had a pH of 7.48 or 7.47, respectively, in warm-acclimated animals and 7.94 or 7.97 in cold-acclimated animals. Under these conditions the hemocyanin was 99% saturated in the artial blood and 45% in venous (warm-acclimated). Corresponding values in cold-acclimated crayfish were 98% and 61%, respectively.5.TheP50 of the hemocyanin in undiluted hemolymph was 10 Torr at 20 °C and pH 7.42, and 6.2 Torr at 10 °C and pH 7.92. The hemocyanin showed a weak Bohr effect (ΔlogP50/ΔpH=−0.19 in warm-acclimated, and −0.20 in cold-acclimated animals). The pH range of the Bohr effect fell into the in vivo pH range in each group and thus differed by 0.5 pH units between the two groups.

Extreme thermostability of tarantula hemocyanin

Biotops with extreme temperatures such as deserts force animals to avoid or escape high temperatures by biochemical, behavioural or morphological adaptation. In this context we tested the resistance to heat of the oxygen carrier hemocyanin from the ancient tarantula Eurypelma californicum, which is found in arid zones of North America. Differential scanning calorimetry, light scattering, crossed immunogelelectrophoresis and oxygen binding experiments show that the 24‐meric hemocyanin is conformationally stable and fully functioning at temperatures up to 90°C. Our results demonstrate that the cation‐mediated state of oligomerization is not only crucial for the high cooperativity of oxygen binding of this hemocyanin, but also for its extreme stability in the physiological temperature and pH range.

Nested allostery in scorpion hemocyanin (Pandinus imperator)

The oxygen-binding behavior of the 24-meric hemocyanin of the scorpion Pandinus imperator and its dependence on allosteric effectors such as protons can be successfully described by the nesting model; the MWC model is not acceptable. The affinities of the four assumed conformations of the allosteric unit, the 12-meric half-molecule, are not dependent on pH whereas the three allosteric equilibrium constants decrease with decreasing proton concentration. Comparison with the oxygen-binding behavior of the 24-meric tarantula hemocyanin (Eurypelma californicum) reveals that the affinity values for the various conformations seem to be conserved for chelicerata hemocyanin.

Temperature adaptation influences the aggregation state of hemocyanin from Astacus leptodactylus

When Astacus leptodactylus were kept at various temperatures for several weeks, different ratios between di-hexameric and hexameric hemocyanins were observed in their hemolymph. The higher the temperature the more hexamers were present. This long-term adaptation to different temperatures or/and to temperature-induced pH-shifts as observed in the hemolymph has different effects on the expression of subunit types building up hexamers and those which covalently link two hexamers within the di-hexamers. The oxygen binding behaviour of di-hexameric hemocyanins from cold and warm adapted animals do not show differences with respect to affinity, Bohr effect and cooperativity.

Tris: an allosteric effector of tarantula haemocyanin

The effect of the chemical buffering component Tris (hydroxy‐methyl‐amino‐methane) and of chloride ions on the oxygen binding of tarantula hemocyanin was studied at constant pH. It revealed that Tris at micromolar concentrations decreases the oxygen pressure at half‐saturation (P50) by a factor of more than two, whereas chloride does not influence oxygen affinity. A thermodynamic analysis in terms of the nested model of allostery [(1987) Proc. Natl. Acad. Sci. 84, 1891‐1895] indicated that Tris acts a an allosteric activator of oxygen binding by influencing the interaction between the 12‐meric half‐molecules of the 24‐meric tarantula haemocyanin.

Fluorescence properties of hemocyanin from tarantula (Eurypelma californicum): A comparison between the whole molecule and isolated subunits

Abstract The tryptophan (Trp) fluorescence properties of the 24-meric hemocyanin (Hc) from the tarantula Eurypelma californicum and its subunits a, d and e were studied and compared with respect to the influence of the two copper atoms at the oxygen-binding site and the status of oxygenation. Fluorescence quenching experiments (by using acrylamide and iodide as external quenchers) and lifetime determinations suggested the occurrence of different classes of Trp responsible for the emission in oxygenated, deoxygenated and copper-depleted protein. On the basis of the different content and distribution of Trp in the isolated subunits and their position in the primary structure, each class of fluorophores was assigned to defined Trp residues. In 24-meric Hc, Trp-292 is mostly responsible for the fluorescence emission in the protein-oxygenated state. After oxygen removal, the major contribution probably arises from Trp-346 while in copper-depleted Hc, about ∼ 70% of fluorescence is due to Trp-195 and Trp-197. All these Trp are conserved in the different subunit types and appear to be located in the interior of the protein matrix, with the exception of Trp-292, which is partially exposed to the solvent. The ratio between the fluorescence quantum yields of oxy-, deoxy- and apo-24-meric Hc is ∼ 1:10:15. The drastic fluorescence decrease in oxy-Hc is mostly due to static quenching by copper ions on that classes of fluorophors (Trp-195, 197 and 346) localized very close to the active site (within 11 A). Each isolated subunit exhibits a peculiar fluorescence behavior, due to the heterogeneity of Trp distribution. In particular, the fluorescence properties of subunit d are dominated by Trp-386 which is present only in this subunit and is fully exposed on the protein surface.

Labeling of tarantula hemocyanin (Eurypelma californicum) with dansyl-type fluorescent tags: Identification of the dye-binding site by fluorescence spectroscopy

Abstract In this paper, we report a study on the interaction of Eurypelma hemocyanin (Hc) with two fluorescent sulphydryl-reactive probes: N -(iodoacetylaminoethyl)-5-naphthylamino-1-sulfonic acid (AEDANS) and 5-dimethylaminona-phthalene-1-sulfonyl aziridin (DNS) which combine the high specificity of reactivity with the spectral properties of the naphthalenesulfonic acids. Both dyes form 1:1 stable complexes with the heterodimer bc , the other Eurypelma subunits being uninvolved in the binding. The labeled sulphydryl group is identified by means of the fluorescence properties of the labeled protein. Both dyes appear to accommodate in the same binding site which is located in the interior of the protein matrix, in close proximity to the copper active centers. An energy transfer process from Trp residues to the bound dyes is observed but only in copper-depleted protein: on this basis, a mean distance of ∼ 15–17 A between AEDANS or DNS groups and Trp-195 and Trp-197 (which are mostly responsible for the emission in apo-derivatives) is calculated. Based on the fluorescence properties of labeled protein, the only candidate which can bind the dye appears to be Cys-228. Based on the particular location of the protein-binding sites for AEDANS and DNS (only a distance of ∼ 10 A from the metal centers) and their peculiar fluorescence properties, both markers appear particularly suitable to monitor conformational transitions of arthropod Hc-active sites during oxygenation.

Nested Allostery of Arthropod Hemocyanins

Arthropod Hcs are multisubunit respiratory proteins (1,2,3). As extracellular proteins, Hcs are more frequently exposed to changing conditions than is human Hb, which is kept in an almost homeostatic environment within the erythrocytes. The O2 binding of Hcs is highly cooperative. The cooperativity and O2 affinity are sensitive to internal and environmental allosteric effectors. These effectors regulate the pO 2 gradient from the hemolymph to the tissue by affecting the 02-binding behavior of the Hcs (Figure 1). It is of biophysical and physiological interest to understand the functional properties of arthropod Hcs on the basis of their structure.