Unusual oxygen binding behavior of a 24-meric crustacean hemocyanin.

Hemocyanins from Crustacea usually are found as 1x6 or 2x6-meric assemblies. An exception is the hemocyanin isolated from thalassinidean shrimps where the main component is a 24-meric structure. Our analysis of oxygen binding data of the thalassinidean shrimp Upogebia pusilla based on a three-state MWC-model revealed that despite the 24-meric structure the functional properties can be described very well based on the hexamer as allosteric unit. In contrast to the hemocyanins from other thalassinidean shrimps the oxygen affinity of hemocyanin from U. pusilla is increased upon addition of l-lactate. A particular feature of this hemocyanin seems to be that l-lactate already enhances oxygen affinity under resting conditions which possibly compensates the rather low intrinsic affinity observed in absence of l-lactate. The fast rate of oxygen dissociation might indicate that in this hemocyanin a higher cooperativity is less important than a fast response of saturation level to changes in oxygen concentration.

Cytosolic carbonic anhydrase in the Gulf toadfish is important for tolerance to hypersalinity.

Carbonic anhydrase (CA) is a ubiquitous enzyme involved in acid-base regulation and osmoregulation. Many studies have demonstrated a role for this enzyme in fish osmoregulation in seawater as well as freshwater. However, to date CA responses of marine fish exposed to salinities exceeding seawater (approximately 35 ppt) have not been examined. Consequently, the aim of the present study was to examine CA expression and activity in osmoregulatory tissues of the Gulf Toadfish, Opsanus beta, following transfer to 60 ppt. A gene coding, for CAc of 1827 bp with an open reading frame of 260 amino acids was cloned and showed high expression in all intestinal segments and gills. CAc showed higher expression in posterior intestine and rectum than in anterior and mid intestine and in gills of fish exposed to 60 ppt for up to 4 days. The enzymatic activity, in contrast, was higher in all examined tissues two weeks following transfer to 60 ppt. Comparing early expression and later activity levels of acclimated fish reveals a very different response to hypersalinity among tissues. Results highlight a key role of CAc in osmoregulation especially in distal regions of the intestine; moreover, CAc play a role in the gill in hypersaline environments possibly supporting elevated branchial acid extrusion seen under such conditions.

Role of the tertiary structure in the diphenol oxidase activity of Octopus vulgaris hemocyanin.

The functional differences between the oxygen transport protein Hemocyanin and the enzymes Tyrosinase and Catechol oxidase are believed to be governed, at least in part, by the tertiary structure, which differs in these molecules and controls the accessibility of their copper containing active site for substrate(s). Accordingly, Octopus vulgaris Hemocyanin catalyses the o-diphenol oxidation to o-quinone at a very low rate. The crystallographic structure of one of the functional units (called Odg) of O. dofleini Hemocyanin shows two domains, a mainly alpha-helical domain that directly binds the copper ions of the reaction center and a beta-strand domain that precludes access to the active site to ligands bigger than molecular oxygen. In this work, we have first cleaved the whole protein and then purified different oxygen binding functional units from O. vulgaris Hemocyanin. These functional units were used in activity assays with l-DOPA, the paradigmatic substrate for Catechol oxidase. All functional units show a negligible enzymatic activity. The procedure to generate the functional units induces in only one of them a proteolytic cleavage. Amino terminal sequencing and mass spectroscopy of the fragments allow to place the cleavage site between the alpha and beta domains of the functional unit homologous to Odd, in the O. dofleini sequence. An increase, up to three orders of magnitude, of Tyrosinase-like activity was observed when the cleaved Odd-like was incubated with the substrate in the presence of trifluoroethanol or hexafluoroisopropanol.

The molecular heterogeneity of hemocyanin: its role in the adaptive plasticity of Crustacea.

Crustacean hemocyanin (Hc) represents a unique case of molecular heterogeneity among oxygen-carrying proteins. The existence of different genes, encoding single polypeptide chains, constitutes the genetic basis for the inter- and intra-specific polymorphism. In addition, the large number of Hc subunits within crustacean species, together with their flexible expression, provides an efficient intrinsic mechanism of modulation of oxygen transport. This review presents a description and classification of the various aspects of crustacean Hc heterogeneity and defines its role in a perspective of crustacean adaptive physiology.

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4x6-meric protein of Upogebia pusilla (Crustacea).

The structural properties of the hemocyanin isolated from the Mediterranean mud shrimp, Upogebia pusilla (Decapoda: Thalassinidea), were investigated. Our intent was to make use of the U. pusilla case to perform a structural comparison between crustacean and chelicerate 4x6-meric hemocyanins. The thalassinidean hemocyanin appears similar in size but different in structural organization compared to the chelicerate 4x6-mer. Ultracentrifuge analyses on the purified protein revealed a sedimentation coefficient of 39S, typical of 4x6 hemocyanins. Electron micrographs are in agreement with a model in which four 2x6-meric building blocks are arranged in a tetrahedron-like quaternary structure and not in the quasi-square-planar orientation characteristic of the chelicerate protein. Size-exclusion chromatography-fast protein chromatography analysis showed elevated instability of the protein in absence of divalent ions or at pH values higher than 8.0. This analysis also shows that the dissociation of the U. pusilla 4x6-meric hemocyanin into hexamers occurs without any intermediate 2x6-meric state, in contrast with the dissociation profile of the chelicerate protein exhibiting several dissociation intermediates. The oxygen-binding properties of U. pusilla hemocyanin were studied to disclose possible effects by the typical allosteric effectors that modulate the functional properties of crustacean hemocyanin. A marked Bohr and lactate effect, but no significant influence of urate, on the oxygen affinity of U. pusilla hemocyanin were found.

The reaction of CN- with the binuclear copper site of Neurospora tyrosinase: its relevance for a comparison between tyrosinase and hemocyanin active sites.

The equilibrium and the kinetics of the reaction between Neurospora crassa tyrosinase and cyanide have been studied. Cyanide reacts with the binuclear copper active site of the protein competitively with respect to dioxygen and displaces the metal ions. This process occurs stepwise and involves transient intermediates containing mononuclear Cu(I) sites. The reaction mechanism proved to be the same as described earlier for molluscan and arthropodan hemocyanins, which share with tyrosinase the same copper active site organization, but perform different physiological functions. A comparison of the kinetic parameters between the different proteins shows that the tyrosinase copper active site has a greater accessibility than that of hemocyanin. The relevance of these data in terms of structure-function relationship and evolution of the binuclear copper proteins is discussed.

Cobalt substitution studies on bovine erythrocyte superoxide dismutase: evidence for a novel cobalt-superoxide dismutase derivative.

Three cobalt derivatives of bovine erythrocyte superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) have been prepared under different pH conditions using a cobalt-thiocyanate complex which has already proved to yield specific substitutions on other copper proteins. The cobalt-protein derivatives have been characterized by optical, circular dichroism and fluorescence spectroscopies. One derivative, referred to as Co2Co2-protein, contains Co(II) ions specifically bound at both Zn(II) and Cu(II) sites. On the basis of their spectroscopic properties, the other two derivatives can be referred as E2Co2- and Co2E2-superoxide dismutase, with cobalt substituting, respectively, at the zinc and the copper sites leaving the contiguous site empty (E). The Co2E2-protein complex represents a novel derivative, since it has never been described in literature. The optical spectrum in the visible region of Co2-Co2-protein well corresponds to the sum of the spectra of the other two derivatives. The circular dichroism spectrum of Co2Co2-derivative, however, is not the sum of individual E2Co2- and Co2E2-proteins, suggesting that the presence of Co(II) in one site strongly affects the geometry of the neighbouring site. Some discrepancies between our spectroscopic data and those reported in literature are discussed. The results obtained from fluorescence experiments indicate that Co(II) ions exert a different quenching effect on the tyrosine emission, depending on whether they are located in the Zn(II) or in the Cu(II) site. The fluorescence quenching can be attributed to a 'heavy atom' and 'paramagnetic ion' effect by Co(II) ions.

Dissociation kinetics of hemocyanin from Octopus vulgaris.

The native form of hemocyanin (Hc) from Octopus vulgaris can be completely dissociated, at alkaline pH and in the presence of EDTA, from 49S decamers to 11S monomers. The kinetics of this process was studied, using a Bio-Logic stopped flow system, by following the time dependence of the 450-nm light intensity, scattered at 90 degrees, in the 7.9-8.8 pH range. All experimental traces were best fitted by a sum of three exponential decay functions. We then tried to best fit these decay functions with a series of kinetic models, the best of them resulting in one whose dissociation of decamers to monomers takes place in three consecutive and irreversible steps, with a highly cooperative step concerning dissociation of octamers to dimers, which appears to be the only intermediate species. This model was preferred over several others, not only for the best norm value but also for the best accordance between each calculated and experimental kinetic parameter (rate constants and amplitudes). Although other more complex models may be considered, our best fit model represents the simplest one, which is able to describe the observed dissociation kinetics.

The aromatic circular dichroism spectrum as a probe for conformational changes in the active site environment of hemocyanins.

The binding of various ligand molecules to the binuclear Cu(I) site of deoxy-hemocyanin has been investigated through the changes produced in the aromatic region of the circular dichroism spectrum of the protein, where a cluster of tryptophan residues located in the vicinity of copper site undergo conformational reorientations in the presence of exogenous ligands coordinated to the metal. In agreement with expectations, the binuclear site of arthropod hemocyanin is severely hindered to the access of exogenous ligands except for very small molecules like CO, O2 or CN- while for mollusc proteins ligands such as thiourea and 2-mercaptoethanol bind easily to the Cu(I) sites. However, the access of the ligand becomes progressively hindered and eventually prevented as the size of substituents on the ligand increases.

Evidence for the molten globule state of human apo-ceruloplasmin.

The conformational features of copper-free ceruloplasmin (CP), as compared to the holo-protein, were evaluated utilizing far- and near-UV circular dichroism and fluorescence spectroscopy. The results obtained indicate that apo-CP maintains the secondary structure of the holo-protein, while the tertiary interactions are much weaker. In addition, the removal of copper from the holo-protein leads to the exposure of hydrophobic patches to solvent, as shown by the fact that apo-CP, at variance from the holo-protein, binds the hydrophobic probe ANS. It is proposed that the CP molecule, upon copper removal, acquires the conformational features typical of a molten globule, which might be the conformational state of CP during its biosynthesis before metal incorporation.

The o-diphenol oxidase activity of arthropod hemocyanin.

Arthropod hemocyanin (isolated from the crab Carcinus maenas and the lobster Homarus americanus) is usually referred to as an oxygen transport-storage protein. The protein, however, also catalyses with low efficiency the oxidation of o-diphenol to quinone, similarly to tyrosinase (monophenol, o-diphenol:oxygen oxidoreductase). The enzymatic parameters of hemocyanin are affected by the aggregation state of the protein; namely V(max) exhibited by a dissociated subunit is one order of magnitude greater than that of aggregated species. The reaction velocity is increased by the presence of perchlorate, an anion of the Hofmeister series. The results are also discussed on the basis of active site accessibility in comparison with tyrosinase.

Evidence for a major role of plasma lipoproteins as hematoporphyrin carriers in vivo.

Hematoporphyrin (5 mg/ml), administered intravenously to tumor-bearing patients, becomes associated with different serum proteins, including lipoproteins (mainly HDL), globulin and albumin. No residual porphyrin is bound to the two latter classes of proteins after 48 h, whereas the complexation with the lipoproteins appears to be particularly stable probably owing to the hydrophobic nature of hematoporphyrin. The late persistence of hematoporphyrin in serum is due to the binding to the VLDL fraction with special regard to its cholesterol moiety. The importance of hematoporphyrin transport by lipoproteins for the photodynamic therapy of tumors is briefly discussed.

Primary structure and spectroscopic studies of Neurospora copper metallothionein.

When Neurospora crassa is grown in the presence of Cu(II) ions, it accumulates the metal with the concomitant synthesis of a low molecular weight copper-binding protein. The molecule binds 6 g-atom of copper per mole protein (Mr = 2200) and shows a striking sequence homology to the zinc- and cadmium-binding vertebrate metallothioneins. Absorption, circular dichroism, and electron paramagnetic resonance spectroscopy of Neurospora metallothionein indicate the copper to be bound to cysteinyl residues as a Cu(I)-thiolate complex of the polymeric mu-thiolate structure [Cu(I)6RS7]-. This metal-binding mode is also in agreement with the unusual luminescence of the protein. Spectral perturbation studies with HgCl2 and p-(chloromercuri)benzoate suggest that the 6 Cu(I)ions are coordinated to the seven cysteinyl residues in the form of a single metal cluster. Neurospora apometallothionein is also capable of binding in vivo group IIB metal ions [Zn(II), Cd(II), and Hg(II)] as well as paramagnetic Co(II) ions with an overall metal-to-protein stoichiometry of 3. The spectroscopic properties of the fully substituted forms are indicative of a distorted tetrahedral coordination. However, metal titration of the apoprotein shows the third metal ion to be differently coordinated than the other two metal ions. This difference can be explained by the presence of only seven cysteine residues in Neurospora metallothionein as opposed to nine cysteine residues in the three-metal cluster of the mammalian metallothioneins.

An oxygenation-sensitive dye binding to Carcinus maenas hemocyanin.

The interaction of 4',6-diamidino-2-phenylindole (DAPI) with Carcinus maenas hemocyanin has been investigated by steady state fluorescence, dynamic fluorescence and circular dichroism measurements. The dye binds to apohemocyanin (without copper) as well as to oxygenated hemocyanin and to deoxygenated hemocyanin with very similar affinities (kd approximately equal to 1 microM ) and number of binding sites (one per subunit). In contrast, the fluorescence quantum yield enhancement of DAPI bound to oxygenated hemocyanin is nearly 60% lower than that observed for deoxygenated and apo forms. The decrease of fluorescence of the dye bound to deoxygenated hemocyanin is a sigmoidal function of the oxygen partial pressure, specular to that observed by following the absorbance of the copper-oxygen charge transfer band at 340 nm. This result provides preliminary evidence that DAPI may be used as a functional probe to monitor the cooperative binding of oxygen to the protein. The higher fluorescence quantum yield of DAPI bound to either apohemocyanin or deoxygenated protein is characterized by a single fluorescence decay with lifetime of about 3 ns, while with the oxygenated protein two components of about 1 ns and 3.0 ns are observed. This result is interpreted assuming the existence of two rotamers of DAPI in solution (Szabo et al. Photochem. Photobiol. 44 (1986) 143-150) both able to interact with oxygenated hemocyanin but only one to deoxygenated and apo forms. We conclude that the different fluorescence behaviour of the dye induced by the presence of oxygen bound to the protein is probably due to a structural change of hemocyanin in cooperative interaction with oxygen. Furthermore, the interaction is confirmed by the induced negative ellipticity of DAPI bound to apohemocyanin and deoxy-hemocyanin and by the increase of fluorescence anisotropy of DAPI bound to all forms of protein investigated.

Copper depletion/repletion of human ceruloplasmin is followed by the changes in its spectral features and functional properties.

Copper ions of different types were gradually eliminated from ceruloplasmin (CP1; ferro-O2-oxidoreductase, EC 1.16.3.1.) by dialyzing the enzyme against KCN. Protein was sampled 2, 4, 6, 22, and 28 h after the dialysis started. Atomic absorption allowed us to estimate the amount of copper atoms per CP molecule. Light absorption in the UV and visible regions along with fluorescence and EPR spectra were also registered. Oxidase and dismutase activities of the enzyme were measured at each step. The combination of the data thus obtained allowed us to trace the sequence of CP depletion of certain copper ions. The same methods were applied in reconstitution studies to detect the return of different types of Cu2+. The experiments were performed on CP samples differing in the amount of copper still bound after CN- treatment. It is shown that the oxidase activity is efficiently brought back to CP if, after the dialysis against cyanide, the catalytic center had preserved its type 3 Cu2+. Dismutase activity of CP did not depend greatly on the presence or absence of type 1 and type 2 copper ions. The results obtained allow a more precise evaluation of the role of different types Cu2+ in the assembly of the complex catalytic center of CP and in the accomplishment by the enzyme of its multiple functions.

Comparative analysis of structural properties of Portunid Crab Hemocyanin.

In order to explore the hemocyanin adaptative potential and evolutive dynamics, we have analyzed the structural properties of this oxygen-carrier protein, in some species of portunid Crabs, (Brachyura, Portunida). We have compared the intra- and interspecific subunits patterns, in native and denaturant conditions, to estimate the phenetic relationships and the different stabilities of the protein.

Structural role of the copper ions in the dinuclear active site of Carcinus aestuarii hemocyanin.

In this work we show, by a combination of biochemical and biophysical approaches, that the copper ions bound in the binuclear active site of Carcinus aestuarii hemocyanin play a stabilizing role on the tertiary structure of the protein. Upon removal of copper, the monomeric hemocyanin, but not the hexameric oligomer, undergoes changes at the level of tertiary structure while the secondary structure is almost unaffected. By Small-Angle X-Ray Scattering, supported by gel chromatography measurements, it can be concluded that the apo-monomer, but not the holo form or the hexameric form, undergoes a slow time-dependent oligomerization process.

Resistance to stress and Hc functional modulation in Liocarcinus sp.

This study is included in a project aimed to study the alterations on the structure of the Northern Adriatic Sea ecosystem produced by fishing activity. The indirect or secondary effects of fishery such as the changes of the structure and trophic relationships of the ecosystem are under investigation and we have particularly considered the effects on species such as Liocarcinus depurator that are captured and then rejected because devoid of commercial value. The objective of this study is the Liocarcinus sp. adaptative resistance to stress and the effects of biochemical parameters (allosteric effectors) on Hc functional modulation.

Structural properties, conformational stability and oxygen binding properties of Penaeus monodon hemocyanin.

Hemocyanin sequences allineament shows the presence of highly invariant regions especially in the active site and in the tight intersubunits interaction sites. Comparing the aminoacids in contact regions between monomers is possible to interpret the stability of hexamers.

Carbohydrate moieties of molluscan Rapana venosa hemocyanin.

Using Zn2+ ions as new method, several FUs have been isolated from molluscan Hc Rapana venosa without formation of non-functional proteolytic side products. N-terminal sequences of these fragments in comparison with FUS from other gastropodan Hcs show a very high degree of structural identity. Four Fus, purified from enzyme-treated structural subunits RvH1 and RvH2 (RvH1-a, RvH1-f, RvH2-a and RvH2-e) show identical N-terminal sequences compared to fragments isolated after treatment with Zn2+ ions. However, in some cases trypsin cleaves RvH chains at different positions if compared to the Zn2+ treatment. To analyze the oligosaccharide composition of two FUS from the first structural subunit of Rapana Hc, RvH1-a and RvH1-f, several techniques were applied: capillary electrophoresis, MALDI-MS, ESI-MS in combination with glycosidase digestions. On basis of these results and the determined amino acid sequence two N-linkage sites were identified in the FU RvH1-a, but only one in the FU RvH1-f.