24-meric Hemocyanin Research Articles

Crystallization and Preliminary Analysis of Crystals of the 24-Meric Hemocyanin of the Emperor Scorpion (Pandinus imperator)

Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (MW = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.

Bohr-effect and buffering capacity of hemocyanin from the tarantula E. californicum

A previous report showed that binding of oxygen to the 24-meric hemocyanin from E. californicum does not correlate linearly with the release of protons as known from hemoglobin. However, this unusual complex phenomenological observation could not be explained at that time. Here, I present a full analysis of the thermodynamic coupling between protons and oxygen for the 24-meric tarantula hemocyanin in Ringer-solution based on the Nested-MWC-model. A strategy is presented which allows to reduce the number of free parameters when fitting the model to the data by including explicitly the equilibrium constants for binding of protons to the different conformations. The results show that the Nested-MWC-model presents a good description of the cooperative and allosteric properties of spider hemocyanin also under physiological conditions and additional constraints imposed by proton-binding data. The analysis allowed to estimate the average number of allosteric proton-binding sites per subunit and the corresponding p K for each conformation. Furthermore, an estimate of the number and affinity of proton buffering binding sites could be given. Approximately 80% of all proton-binding sites are non-allosteric buffering binding sites. The results obtained in this study allow to predict the relative contribution of the four different conformations under conditions found in vivo . The conformational distribution indicates that the conformation with the highest proton affinity ( tR) might be important for the proton transport in the hemolymph.

Two-photon excitation microscopy of tryptophan-containing proteins.

We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.

On the stability of the 24-meric hemocyanin from Eurypelma californicum

The stability of the 24-meric hemocyanin from Eurypelma californicum towards various denaturants (GdnHCl, urea, urea derivatives and salts of the Hofmeister series) indicates that the quaternary structure is stabilized by hydrophilic and polar forces. Thus, the interaction between the seven different subunit types of this cheliceratan hemocyanin is comparable with that of the closely related crustacean hemocyanins. In contrast, no significant influence of divalent ions such as Ca 2+ and Mg 2+ on the stability is observed at pH 8.0 and pH 8.5 but not at pH 7.0. Studies, both in the presence of urea and GdnHCl indicate that the denaturation process consists of a dissociation of the oligomeric structure into intact subunits at lower concentrations of denaturants followed by denaturation of the subunits at higher concentrations of denaturants. No intermediates such as hexamers or dodecamers were detected after 24 h of incubation. This study also reveals that oligomerization has a stabilizing effect on the heterogeneous subunits. In addition, differences in the primary structures result in different stabilities of the seven different subunit types.

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.

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

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 Å). 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.

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.

Hemocyanins in spiders: XXII. Range of allosteric interaction in a four-hexamer hemocyanin. Co-operativity and bohr effect in dissociation intermediates

The range of allosteric interaction in the 24-meric hemocyanin from the tarantula Eurypelma californicum was studied by measuring the oxygen-binding properties of defined oligomeric fragments. Dissociation intermediates comprising 19, 12, 7 or 6 subunits were obtained by incubation of native hemocyanin with 10 m m-cysteine at pH 4.4, with 40 m m-dithiothreitol at pH 7 or 8, by short-term alkaline (pH 9.6) treatment or by treatment with 4 m-urea. These could be stabilized by returning to neutral buffer conditions and, in the case of the dodecamer, by carboxymethylation. Conditions were chosen so that part of the starting material remained intact to serve as control in the oxygen-binding measurements. Oxygen equilibrium curves were obtained by a very sensitive fluorimetric/polarographic method. Oxygen affinity and the magnitude of the Bohr effect remain constant from the hexamer up to the control four-hexamer. Co-operativity, in contrast, increases with aggregate size in two steps: n (hexamer) = n (heptamer) < n (dodecamer) = n (19-mer) < n (4-hexamer). The characteristic pH-dependence of n h is absent in the hexa- and heptamer but is weakly indicated in the dodecamer, and fully established in the four-hexamer. Results from different preparations are highly consistent, if n h is expressed as a percentage of the control values. Full co-operativity is reached only in the four-hexamer, disproving the dodecameric half-molecule (the smallest repeating unit) as the allosteric unit. The stepwise increase in co-operativity appears to be correlated with higher levels of symmetry in the hierarchy of quaternary structure.