Immunogenic activity of hemocyanin from the hemolymph of Helix pomatia and Octopus vulgaris

The immunogenic activity of Helix Pomatia Haemocyanin (HPH) and Octopus vulgaris Haemocyanin (OVH) has been tested in rabbits. Primary and secondary antibody response has been evaluated in sera by agar double immuno-diffusion test (Ouchterlony), counter immunoelectrophoresis and complement fixation. Bath haemocyanins proved highly antigenic: antibody titers rising up to 1/3.200 after the second antigenic challenge. The Ouchterlony test showed a partial identity between H.P.H. and O.V.H. No adverse effect was evident in injected animals.     

The oxidation of Octopus vulgaris hemocyanin by nitrogen oxides

The reaction of Octopus vulgaris hemocyanin with nitrite was studied under a variety of conditions in which the green half-met derivative is formed. Analytical evidence shows that the amount of chemically detectable nitrite in various samples of the derivative is not proportional to the cupric copper detected by EPR. The kinetics of oxidation of hemocyanin as a function of protein concentration and pH, in the presence of nitrite and ascorbate, is consistent with a scheme in which NO2 is the reactive oxidant. We suggest that the green half-methemocyanin contains a metal center with one cuprous and one cupric copper without an exogenous nitrogen oxide ligand.     

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 α-helical domain that directly binds the copper ions of the reaction center and a β-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.     

Association-dissociation equilibria of Octopus hemocyanin

The equilibria between the native (decameric) Octopus hemocyanin and its subunits were studied by analytical sedimentation. Equilibrium is obtained slowly, but the reaction is thermodynamically reversible. The mass action law for a monomer-decamer reaction is obeyed. The reassociated hemocyanin is virtually identical in its sedimentation behavior and oxygen binding with the native protein. The association-dissociation equilibria are mediated by cations; Mg2+, Ca2+, Na+, and H+ are all effective in stabilizing the decameric form at appropriate concentrations. About three to four cations per monomer must be bound for association to occur. Under some conditions, dimers of the subunits can be observed, but formation of this dimer does not depend on cation concentration, and it does not appear to be an obligate intermediate in the association to decamer.     

Nucleoside phosphorylase activity of Octopus vulgaris hepatopancreas

From a research on partially purified extracts of Octopus vulgaris hepatopancreas a number of properties of the nucleosidase present in such extracts have been defined, such as enzyme affinity towards different purine and pyrimidine ribo- and deoxyribo-nucleosides. The phosphorolytic mechanism of action of the Octopus enzyme system is similar to the mechanism already known for many nucleosidases from animal tissues. The possibility is envisaged of two different nucleosidases being present, one specific for ribonucleosides, with an acid optimum pH, and the other one for deoxyribonucleosides, with an alkaline pH optimum. Adenosine deaminase also appears to be a component of Octopus hepatopancreas.     

Oxygen equilibria of Octopus dofleini hemocyanin

Oxygen binding by Octopus dofleini hemocyanin was examined under very nearly physiological conditions. The effects of pH, ionic composition, temperature, and aggregation were controlled so that the role each plays in modulating oxygen binding can be isolated. There is a very large effect of pH on affinity, the Bohr effect (delta log P50/delta pH = -1.7), which is the same at 10 and 20 degrees C. However, cooperativity is substantially altered over the same range of pHs at the two temperatures. The allosteric properties were examined by comparing the experimental data points to curves generated by use of the Monod-Wyman-Changeux model. A computer-fitting process was developed which allowed the individual allosteric parameters to be varied independently until the best fit could be determined. The relationship between kR and kT is responsible for the effect of pH on cooperativity. A change in the allosteric properties of the T form is primarily responsible for the differences due to temperature. Changing cation concentrations when the molecule is in the fully aggregated 51S form alters affinity without influencing cooperativity. The effect of Mg2+ is much greater than that of Na+. If the 51S decamer is dissociated to 11S monomers by removing divalent cations, oxygen binding is noncooperative. There is evidence for negative cooperativity, indicating heterogeneity of function within the subunit which contains seven oxygen binding domains. Association into decamers generates conformational change which results in a much wider range of allosteric function.     

The binding of 1-anilino-8-naphthalene sulfonate to the hemocyanin of Octopus vulgaris.

The binding of 1-anilino-8-naphthalene sulfonate (ansyl) to native and copper-free hemocyanin of Octopus vulgaris has been studied in different conditions by measuring the fluorescence properties of the probe in the presence of hemocyanin. Native hemocyanin, either in the oxygenated or in the deoxygenated state, does not bind ansyl. The binding of ansyl with apohemocyanin induces a strong increase (from 0.004 to 0.6 -- 0.7) of the quantum yield and a blue shift from 520 nm to 460 nm of the emission maximum indicating the presence of ansyl binding sites in the protein. Experimental evidence is reported that the binding occurs at the copper-binding site of the protein. The dissociation constants of the ansyl-hemocyanin complexes are equal to about 10(-4) M, i.e. they are of the same order of those obtained with other proteins. The number of binding sites (n) of apohemocyanin for ansyl depends on the conformational state of the protein and ranges from 0.15 -- 0.80 mol/mol protein (Mr 50,000), depending on pH, ionic strength, and urea concentration. A negative interaction between the ansyl binding sites has been suggested.     

Monoamine oxidase activity in hepatopancreas of Octopus vulgaris

1. Monoamine oxidase activity has been studied in hepatopancreas of Octopus vulgaris using 5-HT and PEA as substrates.2. Time courses of MAO activity against 5-HT and PEA show that the enzyme has higher affinity for PEA than for 5-HT.3. MAO activity against 5-HT appears more sensitive than MAO activity against PEA, to variations of the temperature (range 17–67°C).4. The inhibition curves obtained with clorgyline and deprenyl indicate that MAO activity is due to a single form of the enzyme, not corresponding to type A and type B MAO.5. Semicarbazide 10⁻⁴ M does not affect the deamination of 5-HT and PEA, demonstrating that a semicarbazide-sensitive amine oxidase is not involved in this process.     

Crystals of the carboxyl-terminal functional unit from Octopus dofleini hemocyanin

The carboxyl-terminal oxygen-binding unit of the polypeptide from Octopus dofleini hemocyanin has been crystallized in a form suitable for three-dimensional X-ray analysis. This proteolytic fragment has a molecular weight of 47 kDa and reversibly binds O2 while exhibiting a slight Bohr effect. Two types of crystals have been grown. Type I crystals, currently under analysis, belong to the orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of 92.6 A x 167.4 A x 59.2 A. A composition of two protein molecules per asymmetric unit and 50% solvent content is consistent with a self-rotation function that identifies a non-crystallographic 2-fold axis of symmetry relating these molecules. Diffraction extending beyond 1.9 A Bragg spacings can be detected with synchrotron X-radiation.     

The mandibular movements of Octopus vulgaris

The movements of the isolated buccal mass of Octopus vulgaris have been investigated. The beaks undergo rhythmic cycles of activity in the absence of applied stimulation and after electrical stimulation of the inter-buccal connective. Initial opening, closing, retraction and re-opening phases of movement are described. This cycle of movements is taken to resemble those in the intact animal. Anatomical and electrical evidence identifies the superior mandibular muscle as being partly responsible for the closing and retraction phases of movement. The inferior buccal ganglion determines the sequence of these buccal movements, but modification by sensory feed-back from the musculature is also implied. The preparation will allow a closer comparison of the control of movement in cephalopods and gastropods.