Oxygen binding by the hemocyanin of Busycon canaliculatum

• 1.1. This study examined the effect of the monoamines dopamine and octopamine, as well as tyrosine on the oxygen affinity and cooperativity of oxygen binding by the hemocyanin of the marine gastropod Busycon canaliculatum. The effect of temperature on hemocyanin oxygen affinity was also examined.
• 2.2. Freezing Busycon hemocyanin did not affect the binding of oxygen.
• 3.3. Dopamine, octopamine and tyrosine had no significant effect on the oxygen affinity or cooperativity of oxygen binding by the hemocyanin of B. canaliculatum.
• 4.4. It was concluded that Busycon hemocyanin either has no binding sites for the two monoamines or for tyrosine, or that binding of the molecules has no functional significance.
• 5.5. Both temperature sensitivity and affinity of hemocyanin-oxygen binding were similar to values previously reported for hemocyanin of Busycon from other localities.

     

Distribution of copper atoms and binding of carbon monoxide in partially copper-depleted hemocyanin

Binding of CO to single cuprous (half-apo) or cupric (met-apo) copper of hemocyanin is investigated by a new method which allows estimation of the total amount of CO bound to hemocyanin. Pure half-apo preparations could not be obtained with the molluscan hemocyanins from Helix pomatia and Octopus vulgaris, and a residual fraction of sites with coupled copper is always present. However, the determination of CO bound to the protein before and after addition of H2O2, used to oxidize selectively single copper sites, reveals that CO binds to half-apo Cu(I), and is released upon oxidation of copper to met-apo Cu(II). Binding of CO to half-apo is not associated to luminescence, proving that luminescence of native carboxyhemocyanin demands the presence of a second cuprous copper in the site. In addition, analysis of data indicates that the residual amount of coupled copper sites in partially copper-depleted hemocyanin is underestimated by the residual O2-copper band measured at 340 nm in air, while faithfully quantitated by the residual luminescence in the presence of CO. A distribution of the copper left in the site of three partially copper-depleted hemocyanins is depicted.     

Lipid binding capacity of spider hemocyanin.

The spider hemocyanin capacity to bind different lipid classes was evaluated by measuring some binding kinetic parameters. A very high lipoprotein (VHDL) which contains hemocyanin, was isolated from Polybetes pythagoricus hemolymph plasma and delipidated. Hemocyanin was bound separately to labelled palmitic acid, phosphatidylcholine, cholesterol, and triolein resulting in several artificial lipoprotein structures. It was possible to corroborate in vitro the lipid-hemocyanin interactions which had been previously observed and, consequently, the apolipoprotein role played by the respiratory pigment of spiders. Lipoproteins were analysed by gel filtration chromatography, and three subfractions with different hemocyanin structures were obtained. The four lipid classes were only bound to the hexameric structure (420 Kda), possibly to low polarity sites. Upon radioactivity measurements of the protein-associated lipids, maximal binding ratios (Mr), dissociation constants (Kd), and the maximal binding effectiveness at low lipid concentrations (Eo) were calculated. Lipid/protein ratios were increased proportionally to each available lipid concentration, following a hyperbolic binding model. Values of saturation, affinity, and maximal binding efficiency to hemocyanin were found to be different for each lipid class assayed. The highest lipid/protein ratio (41.5) was obtained with the free fatty acid and the lowest (7.2) with triolein. Phosphatidylcholine and cholesterol showed the highest relative affinities for hemocyanin (Kd = 63 x 10(-5) M and 74 x 10(-5) M, respectively). Phosphatidylcholine at low concentrations, similar to the physiological ones, presented the highest Eo value. Maximal lipid/protein ratios reached in vitro, were greater than those in P. pythagoricus VHDL, pointing out that hemocyanin could play the apolipoprotein role even under physiological conditions with a very high plasma lipid concentration. J. Exp. Zool. 284:368-373, 1999.     

Properties of the binding of copper by bleomycin

The glycopeptide, bleomycin, binds metal ions including Cu²⁺. It is the copper complex of this material that is isolated from Streptomyces verticillus. Both free ligand and copper complex are excellent antitumor agents in animals. The biochemical and pharmacological relationship between these compounds has not been established. The present study begins an analysis of the chemistry and biochemistry of copper-bleomycin with structural and equilibrium properties of the complex. Potentiometric and fluorometric titrations of bleomycin confirm three acidic groups with pK_a values of 7.50, 4.93, and 2.72. The conjugate nitrogen bases of these groups, comprise three of the binding sites for Cu²⁺ according to similar titrations of copper-bleomycin. The fourth is a conjugate base of an acid with a very large pK_a that cannot be measured by these techniques. The participation of a fourth such group is inferred from both proton release studies of the binding of metal and ligand above pH 8 and from several studies of the thermodynamic stability of copper bleomycin. At low pH binding of copper to bleomycin occurs in two steps, as observed by several independent techniques which monitor either the metal or the ligand. Log stability constants for the reactions Cu²⁺ + H_kBlm ? CuH_k-nBlm + nH⁺ and CuH_k-nBlm ? CuH_k-n-rBlm + rH⁺ are 1.32 and ?4.31, respectively, with n of 2.21 in the first equation and r of 2.07 in the second equation. The derived logarithm of the pH independent stability constant for copper bleomycin multiplied by the protonation constant for the unknown fourth ligand in the binding site is 12.16. This agrees closely with values obtained from measurements of conditional formation constants. One of the groups which binds in the second reaction is the substituted pyrimidine.     

An oxygenation-linked dye binding to Limulus polyphemus hemocyanin

The reaction of Limulus polyphemus hemocyanin with a dye, bromthymol blue, was examined by equilibrium dialysis, spectrophotometric titration and stopped-flow methods. Oxy-hemocyanin contained one binding site per hexamer unit. The dye binding was linked to oxygenation, and the affinity of the dye for the oxy form was about 10 times as high as that for the deoxy form. Conversely, the dye increased the O2 affinity of hemocyanin. Hemocyanin showed a simple hyperbolic binding curve in the bromthymol blue titration, whereas the time course of the reaction was generally biphasic. It was inferred from the kinetic analyses that the reaction proceeds in two steps. The first bimolecular step is characterized by an increase in the apparent pKa of the bound dye, while the second unimolecular step by a red shift of the absorption band of the unionized dye. The dye binding to partially oxygenated hemocyanin was examined spectrophotometrically; the fractional change in the binding was found to be ahead of the increase in the average degree of O2 saturation. It was concluded that the structural changes in hemocyanin which lead to the increased dye affinity take place at an early stage of the ligand binding sequence.     

Positive cooperativity in binding carbon monoxide to hemocyanin

The binding of carbon monoxide to hemocyanin from the crab $\text{Scylla serrata}$ has been studied by thin layer optical absorption and front face fluorescence techniques. The binding to the monomeric form is completely noncooperative whereas the binding to the native oligomeric form is found to be weakly but definitely cooperative. An analysis based on the MWC model of the oxygen and carbon monoxide binding curves indicates that the allosteric constant, L, describing the equilibrium between the 2 unligated forms is different for each ligand. This implies that at least 3 allosteric forms are needed to characterize the binding of oxygen and carbon monoxide to this hemocyanin.     

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.     

Is the oxygen atom of carbon monoxide coordinated to the copper of hemocyanin?

The carbon monoxide binding site of hemocyanins was studied by comparing the isotope shift of the CO-stretching frequencies in CO-hemocyanins with that of carbon monoxide diethylenetriaminecopper(I)tetraphenylboron in which the carbon atom of CO is coordinated to the copper. Coordination by the carbon atom of CO in CO-hemocyanin is suggested.     

A model for L-lactate binding to Cancer magister hemocyanin

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. These data suggest that L-lactate binds to the hemocyanin at all four positions around the chiral carbon. The carboxyl and hydroxyl groups are required for activity. The protein can only partially distinguish between the methyl group and hydrogen atom.     

On the interaction of hemocyanin with lipid bilayer membranes

Osmotic jump experiments were used to measure the ionic permeability induced in lipid vesicles by Megathura crenulata hemocyanin. It was found that this protein strongly increases the conductance of K⁺ and Cl^- through these membranes but not that of SO ₄ ⁼ . These effects were attributed to the formation of ionic channels in the vesicles. We have found that a simple first-order binding model can explain the dependence of the number of pore-containing vesicles both on the time after exposure to hemocyanin and on the protein concentration. Milder effects were attributed to a non-specific adhesion of the protein to the membrane surface. Consistent with the hypothesis of reversible association, vesicles which retained hemocyanin after step sucrose density gradient centrifugation at low ionic strength, lost most of the protein upon recentrifugation at high ionic strength. Consistent with the hypothesis of channel formation bot the above vesicle preparations transferred voltage-dependent hemocyanin channels into planar bilayers when they were made to fuse with them. It is concluded that hemocyanin can interact both specifically, by forming pores within the hydrophobic core of lipid membranes, and non-specifically, probably by means of electrostatic interaction with the surface of the same membrane.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - DOC sodium deoxycholate     

On the structure of the hemocyanin channel in lipid bilayers

Keyhole limpet hemocyanin has been shown by others (Alvarez, O., Diaz, E. and Latorre, R. (1975) Biochim. Biophys. Acta 389, 444-448) to form single conductance channels in black lipid membranes. In an attempt to visualize how the large (300 A) water-soluble hemocyanin molecule interacts with lipid bilayers, we have examined hemocyanin in the presence of lipids with the electron microscope. We find that incubation of lipids with keyhole limpet hemocyanin produces a characteristic 70 A in diameter, ring-shaped particle or annulus associated with the bilayer. This annulus, which appears to be quite distinct from previously observed aggregated and dissociated forms of hemocyanin, may be responsible for the channel formation in black lipid membranes.     

Unusual oxygen binding behavior of a 24-meric crustacean hemocyanin

Hemocyanins from Crustacea usually are found as 1 × 6 or 2 × 6-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.