Hemocyanin
Hemocyanin is a protein present in the hemolymph of some crustaceans, arachnids, and mollusks that is responsible for transporting oxygen. Its function is equivalent to that of hemoglobin in the blood of other animals and humans, although oxygen transport is not as efficient.
Instead of iron, it has two copper atoms in its active center. For this reason the color of oxygenated hemocyanin is not red but greenish blue, which has given it its name (cyan = blue). Mollusks and crustaceans with this type of oxygen transport system in their hemolymph show that color in their fluids.
The two metallic centers are not in direct contact, but they are very close to each other. The oxygen molecule is inserted between the two copper atoms, which change their oxidation state, from +I to +II, giving up one electron each to the oxygen molecule. As a consequence, it is reduced to peroxide (deprotonated hydrogen peroxide). The transfer of electrons in pairs prevents the formation of the superoxide ion (Cowan, J. A. (1993). Inorganic Biochemistry, An Introduction. ).
History
The history of hemocyanin dates back to 1867, when it was reported that the blood of cephalopods turned blue when it passed through the gills. In the following decade, in 1878, the Belgian physiologist Leon Frederiq showed that the blue color was caused by the oxygenation of the copper that contained the protein, a process that was reversible; calling it hemocyanin. By 1932, the assimilation of oxygen by hemocyanin had already been investigated in 13 animal species and, for each case, it was found that the hemocyanin molecules had copper atoms, likewise when the molecule was fully oxygenated it contained one oxygen molecule per every two copper atoms.
In 1976, Senozan carried out research on hemocyanin, a name that is misused, since it does not have any heme group: copper is directly attached to the protein, this binding being strong, since according to the dissociation constant calculated for the loss of a copper atom is of the order of 10-20. However, the binding site of the copper atom was not well elucidated, even though there was circumstantial evidence that possibly the sulfhydryl groups of the amino acid cysteine and the imidazole rings of histidine were involved in the bond with the metal.
Conformations
Currently, it is known that the hemocyanin molecule can adopt two different conformations, the aggregated and the disintegrated form, each of which has a different affinity for oxygen: deoxy-hemocyanin, being found preferably in the conformation of low affinity towards oxygen (split form), at intermediate saturation both conformational forms are present, and in highly saturated solutions the high affinity conformation is predominant (aggregate form). This is explained on the basis of cooperativity, through which the affinity for oxygen progressively increases as oxygenation proceeds.
Although oxy-hemocyanine contains two Cu(II) ions with incomplete d configuration, it is a diamagnetic molecule. This has been explained by proposing a coupling between the two metallic nuclei that gives rise to an antiferromagnetic electronic arrangement, which means that oxy-hemocyanin does not exhibit paramagnetism. This characteristic had led to the assumption that the two copper nuclei were linked by a binder, in addition to the oxygen molecule, which was responsible for the antiferromagnetic coupling.
On the other hand, each copper ion in deoxy-hemocyanin is coordinated to three imidazole nitrogens of the protein chains, with a distance between each ion of 3.54 Angstrom. The coordination spheres of the two copper ions consist of two short Cu-N bonds (1.95 to 2.104 Angstrom) and one notably longer Cu-N bond (2.66 to 2.77 Angstrom). The four short bonded nitrogens and the two copper ions form a plane and the other two ligands are axial, lying at opposite ends of the plane, ie the configuration of the axial ligands is trans.
Species distribution
Hemocyanin is found only in two phyla of the animal kingdom: in mollusks and in arthropods, within which it has been found in all decapod crustaceans and within mollusks in all cephalopods. However, in these two phyla the distribution of hemocyanin is erratic, for example, in the edible snail it is found in large quantities, but in the equivalent freshwater species Planorbis no hemocyanin has been found; likewise, hemocyanin has been found in certain types of scorpions and spiders.
The amount of hemocyanin in the blood varies considerably in different species, even within members of the same species. In some cases, the hemocyanin disappears completely during the molting season, as in the case of the maia spider crab.
On the other hand, in molluscs there is approximately 0.25% by weight of copper and in crustaceans 0.17%. This difference has led some zoologists to consider, from an evolutionary point of view, that both hemocyanins are not related and that arthropods and mollusks have developed their respiratory pigments independently. Others consider that there is a relationship between both types of hemocyanins and consider that both are made up of the same "fundamental unit".
Immune activity
Hemocyanins are used as carriers to produce antibodies and as a carrier-adjuvant in experimental therapeutic vaccines for cancer. Among its advantages, in addition to its reasonable cost and the absence of side effects, is that they direct the immune response towards Th1-type responses, unlike other adjuvants, such as alumina, which does so towards Th226-type responses. Likewise, these metalloproteins have been used in the production of a myriad of antibodies against small molecules of diagnostic interest, including hormones, drugs, antibiotics, and toxins, which are chemically coupled. In this way, immunized animals produce antibodies against hemocyanin and against the transported substance, which are subsequently purified and applied in various tests based on antigen-antibody reactions.
On the other hand, the hemocyanin inside the crazy mollusk (Concholepas concholepas), has immunological properties comparable to that of the keyhole limpet (Megathura crenulata), being a good alternative as a carrier protein, in experimental vaccines and in the treatment of superficial bladder carcinoma. Even though the keyhole limpet and the keyhole limpet differ in their origin and structure, the fact that both proteins induce similar immunostimulatory responses suggests that they have a conserved pattern that would be recognized by the immune system, stimulating an ancestral immunogenic mechanism present in mammals.