Hemocyanin Molecules Research Articles

Purified haemocyanin in the blood of fresh water amphibious snail,Pila globosa (Swainson) in relation to aestivation

The copper and protein content in the blood and in the purified haemocyanin of active and aestivated snails are determined. There is a marked increase in the copper content and also in the haemocyanin content in the blood upon aestivation which is discussed in the light of the available literature. The per cent copper/protein ratio was 0·255 in the purified haemocyanin of the active and aestivated snails, suggesting similar nature of haemocyanin molecule whose minimal molecular weight was calculated to be 24,910.

High Resolution Scanning Electron Microscopy in Biology: Artefacts Caused By the Nature and Mode of Application of the Coating Material

Results of high resolution secondary electron observations of the haemocyanin molecule from the marine whelk Buccinum undatum are given. The choice of metal used for surface coating of the sample (gold, gold--palladium or carbon/gold--palldium) and the mode of application of this metal (thermal vacuum evaporation or diode sputtering) were both found to be of utmost importance when working at high magnifications in the scanning electron microscope. Sputter coating with gold gives poor results because of a granular and cracked appearance of the surface film. The haemocyanin molecules were difficult to recognize in these preparations. Best results were obtained by a thermal vacuum evaporation of gold--palladium. It is suggested that the inferior results seen after sputter coating may be due to the high temperatures wihch the specimen experiences during the sputtering, even when cooling of the specimen stage is carried out. Other specimens (diatom valves and latex spheres) were also studied at high magnification giving the same results as the haemocyanin molecules.

Labelling of concanavalin A sites on the plasma membrane of soybean protoplasts

The plasma membranes of protoplasts from cultured soybean cells bind haemocyanin after treatment with concanavalin A. On unfixed protoplasts the binding sites were unevenly distributed. In thin section and in Pt/Pd replicas, the haemocyanin molecules were seen in clusters. After 16 hours incubation, the haemocyanin appeared in tighter clusters and newly synthesized cellulose microfibrils were present. In contrast, the protoplasts prefixed before concanavalin A treatment exhibited a very even distribution of haemocyanin. These observations are discussed in relation to membrane structure and the fluid mosaic model based on analogous studies of animal cells.

Proteolytic fragmentation of Helix pomatia alpha-hemocyanin: structural domains in the polypeptide chain.

alpha-Hemocyanin from the Roman snail Helix pomatia is composed of polypeptide chains with a molecular weight of 360000 +/- 30000. The cylindrically shaped hemocyanin molecule contains 20 of these large chains. The polypeptide chain has been split into components with molecular weights of: 210000, 154000, 147000, 112000, 120000, 98000, 55000, and 50000, by gentle proteolysis with enzymes of different specificities. Most of the fragments have molecular weights which are about 50000 or a multiple of 50000. Departure from these values, as found in the 112000 and 120000 fragments, is probably caused by the high carbohydrates content of these components. A mixture of these fragments has the same oxygen binding properties as the nondigested protein. Subtilisin converts the hemocyanin polypeptide chain, under appropriate conditions, almost completely into fragments of 50000 and 55000 daltons with conservation of the oxygen binding properties of the nondigested protein. We conclude from these studies that the polypeptide chain of Helix pomatia alpha-hemocyanin is folded into about seven compact tertiary structures, which are covalently interconnected. This chain of structural domains has been visualized. (Siezen and Van Bruggen (1974), J. Mol. Biol. 90, 77-89) by electron microscopy, which shows 1/20 hemocyanin molecules to be flexible structures consisting of 7-8 apparently spherical units of 55-60 A diameter.

Tubular polymers derived from Helix pomatia beta-hemocyanin.

Upon trypsinolysis Helix pomatia beta-hemocyanin forms long tubular structures, which appear to be linear polymers of hemocyanin molecules from which the collar structure has been removed. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that only few peptide bonds are hydrolyzed by trypsin. The structure of the polymers has been investigated by electron microscopy, combined with optical diffraction. Preliminary X-ray diffraction data are presented. Functional properties of the polymers are similar to those of the native protein. Both show a calciumion-dependent co-operativity of oxygen binding and a Bohr effect. The results suggest that the collar of a hemocyanin molecule has no special function in the process of (co-operative) oxygen binding, different from that of the wall of the molecule.

Immunochemical properties of the haemocyanins from Buccinum undatum (L.) and Neptunea antiqua (L.)

1. 1. Rabbit antiserum to Buccinum undatum haemocyanin cross-reacted with Neptunea antiqua haemocyanin and vice versa, the reaction in each case being one of partial identity. Antiserum to B. undatum haemocyanin also cross-reacted weakly with Colus graeilis haemocyanin, and very weakly with Murex trunculus haemocyanin. 2. 2. Depending upon the pH and the Ca 2+ or Mg 2+ concentrations, the reaction of the haemocyanins with their rabbit antisera resulted in the formation of single or multiple precipitin lines in agar and different equivalence points in the quantitative precipitin assay. 3. 3. Analytical ultracentrifugation and electrophoretic analysis showed that this phenomenon was caused by pH-induced dissociation of the haemocyanins, both whole molecules and their dissociation products being capable of reacting with antisera. 4. 4. An interpretation of the patterns observed in immunoelectrophoresis is given based on the time taken for haemocyanin molecules to dissociate at certain pH values, and the electrophoretic mobilities and diffusion coefficients of the dissociation products.

The haemocyanin of Lymnaea stagnalis L. (Gastropoda: Pulmonata)

1. 1. The haemolymph of Lymnaea stagnalis (L.) was shown to contain a typical gastropod haemocyanin by virtue of its absorption spectrum, copper content, and electromicroscopic appearance. 2. 2. The native haemocyanin molecule ( s 20, w 0 = 99·7S) underwent partial dissociation into one-half molecules ( s 20, w 0 = 63·0S) in the presence of molar NaCl at pH 5·7 indicating that both β- and α-haemocyanins were present in the haemolymph. The resulting mixture of whole and one-half molecules appeared to be in dynamic equilibrium. 3. 3. The native haemocyanin molecule also underwent dissociation as the pH of the solution was raised, to one-half molecules and at high pH values to one-tenth molecules ( s 20, w 0 = 15·5S). The halves-wholes dissociation again appeared to be one involving dynamic equilibrium. Calcium or magnesium ions repressed the pH-induced dissociation. 4. 4. The oxygen-binding curves of the haemocyanin were sigmoidal for whole molecules but hyperbolic for one-tenth molecules. The corresponding Hill plots had maximum slopes of about 4·0 and 1·2 respectively, indicating that whole molecules, but not one-tenth molecules, bind oxygen co-operatively. 5. 5. A small, positive Bohr effect was found over the pH range 7·3–8·5.

Functional properties of chemically modified hemocyanin. Fixation of hemocyanin in the low and the high oxygen affinity state by reaction with a bifunctional imido ester.

Hemocyanin of Helix pomatia is a respiratory protein with a molecular weight of 9 times 10-6; it contains 180 oxygen binding sites. The reaction of hemocyanin with the bifunctional reagent dimethyl suberimido ester, which reacts with amino groups, has been studied. Up to 75 per cent of the amino groups can be modified without inactivation of oxygen binding sites or dissociation of the protein, It appears that hemocyanin can be fixed in a state with low oxygen affinity by modification of the deoxy protein, and in a state with high oxygen affinity by modification of the oxy protein. Using conditions under which native hemocyanin binds oxygen cooperatively (Hill coefficient 2.9), modification of deoxy- and oxyhemocyanin yields derivatives with different oxygen affinities (P50 equals 10 and 2.2 mm, respectively). Both the deoxy and oxy derivatives show strongly reduced cooperativity (Hill coefficients 1.4 and 1.1, respectively). Modification of oxy- and deoxyhemocyanin subunits (molecular weight one-tenth of the native protein), which bind oxygen noncooperatively, results in derivatives with oxygen binding properties identical with those of unmodified subunits. Parallel experiments have been carried out with a unifunctional reagent, methyl acetoimido ester. Modification of partially oxygenated hemocyanin under conditions at which the protein binds oxygen cooperatively yeilds derivatives with redued cooperativity (Hill coefficents 1.1-1.2) and an oxygen affinity depending on the oxygen saturation whivh modification had been carried out. The results are consistent with a simple two-state model for the cooperativity of oxygen binding by these giant hemocyanin molecules.

Functional differences in the multiple hemocyanins of the horseshoe crab, Limulus polyphemus L.

Hemocyanin in the hemolymph of the horseshoe crab, Limulus polyphemus L., is a high-molecular-weight copper protein which binds oxygen cooperatively and shows a higher oxygen affinity at pH 7 than at pH 9. Treatment with EDTA (ethylenediaminetetra-acetate) disaggregates the hemocyanin molecules and abolishes both the reverse Bohr effect and cooperative oxygen binding. Chloride ions interact with the EDTA-treated material and, in the presence of saturating amounts of NaCl, a reverse Bohr effect is restored, but cooperativity is not. The EDTA-treated hemocyanin contains at least five electrophoretically distinct hemocyanins. These hemocyanins have similar molecular weights (about 66,000) but are functionally dissimilar. They have different oxygen affinities and different responses to chloride ions. The effect of chloride ions on unfractionated hemocyanin is due to pH-dependent chloride interactions with only two of the five hemocyanin components. The functional differences between the hemocyanin components may provide Limulus with a valuable respiratory flexibility in its interaction with the environment. The kinetics of oxygen combination and dissociation for the various hemocyanin preparations show that variations in the rate of oxygen dissociation are primarily responsible for the observed differences in oxygen affinity. The rate of oxygen dissociation varies 20-fold under conditions where the apparent rate of oxygen combination shows less than a 2-fold variation. Cooperative interactions in the untreated hemocyanin are most obvious in the "off" reaction, which increases in rate as successive oxygen molecules are released.

Small-angle x-ray-scattering studies on the substructure of Helix pomatia hemocyanin

Hemocyanin of the snail helix pomatia was studied by small-angle X-ray scattering in 0.15 M phosphate buffer pH 6. The outer portion of the scattering curve showed minima, which indicated that the hemocyanin molecule is composed of a large number of identical (or very similar) nearly spherical subunits. From the position of the minima the radius of gyration Ru of the subunit was calculated to be 1.55 nm; this corresponds a diameter of 4 nm assuming a spherical shape. The number of the subunits is discussed, too; a number of 330 can be given as lower limit, a number of 528 as upper limit (most propable value 350 — 400). Various models were put up by arranging the subunits (diameter 4 nm) in different ways to hollow cylinders. The theoretical scattering curves of these models were compared with the experimental one and a model equivalent in scattering is suggested for the hemocyanin Helix pomatia. In this model, which fits very well to the experimental curve, 360 subunits form a hollow cylinder. The model shows also the essential features found by electron microscopy by VAN BRUGGEN. The essential features are an arrangement of the subunits in 6 layers vertical to the cylinder axis and a 5-fold screw axis parallel to it.

Structure and function of hemocyanins VII. The smallest subunit of α- and β-hemocyanin of Helix pomatia: Size, composition, N- and C-terminal amino acids

1. 1. The amino acid and carbohydrate composition of α- and β-hemocyanin of Helix pomatia were determined. The amino acid composition is strikingly similar in both hemocyanins; the carbohydrate composition shows small quantitative differences. The presence of an unidentified sugar in both α- and β-hemocyanin is observed. 2. 2. Both α- and β-hemocyanin have N-terminal arginine; the C-terminal amino acid could not be identified by several methods. By application of a quantitative dinitrophenylation micromethod it was shown that 1 mole of arginine is present per 25 000 g of hemocyanin. 3. 3. Dissociation of hemocyanin molecules beyond the state of twentieth molecules is extremely difficult. Application of 70% formic acid leads to dissociation into subunits with a molecular weight of 25 000, readily dimerizing to 50 000. 4. 4. The obtained subunits are highly heterogeneous, in accordance with the results of dissociation experiments. The cause of this phenomenon is still unknown.

Structure and properties of hemocyanins. V. Binding of oxygen and copper in Helix pomatia hemocyanin

1. 1. Reintroduction of monovalent copper into apo-α-hemocyanin of Helix pomatia leads to the complete return of the properties of native α-hemocyanin. 2. 2. The copper atoms in hemocyanin are required for the reassociation of tenth and twentieth molecules. 3. 3. In the reintroduction process, the copper atoms are randomly distributed among the empty binding sites of apo-α-hemocyanin. All copper-binding sites are equivalent and grouped in pairs; each pair forms a functional group. 4. 4. Upon storage for a few months, part of the copper atoms in every hemocyanin molecule are oxidized to Cu 2+. This affects the oxygenation properties. 5. 5. The oxygenation curves of fresh α- and β-hemocyanin are nonhyperbolic at alkaline pH values in the presence of Ca 2+ and Mg 2+. The influence of the pH on the oxygenation curves of α- and β-hemocyanin shows marked differences.

Electron microscopy of the glio-vascular organization of the brain of octopus

The glio-vascular organization of the octopus brain has been studied by light and electron microscopy. The structure of the walls of the blood vessels has been described. Two types of neuroglia can be recognized, the fibrous and protoplasmic glia; also enigmatic dark cells. Most blood vessels in the neuropil are surrounded by extracellular zones containing collagen. These zones give off glio-vascular tunnels (strands) that penetrate the neuropil in a complex network. The extracellular zones and tunnels contain in addition to collagen, smooth muscle cells and fibrocytes. Glial processes surround the extracellular zones and incompletely partition them from the neuropil. The small neuronal perikarya have no glial folds around them. The medium-size cells have thin glial sheets or finger processes related to their surfaces, which may indent the cells to form small trophospongia. The large neurons of the suboesophageal lobe have complex glial sheaths interspersed with extracellular channels. Both penetrate the neurons to form complex trophospongia. A new form of extracellular material has been observed in these extracellular channels. The occurrence of trophospongia in vertebrate and invertebrate neurons may be correlated with the absence of dendrites. Special problems discussed include the nature of the trophospongial function, the question of fluid-filled extracellular zones and their possible function as lymph channels, and the presence in some of them of haemocyanin molecules identical with those in the blood vessels. Perhaps of special importance is the observation that the lobes of the octopus brain are permeated with extracellular tunnels containing smooth muscle fibres, but it still needs to be determined whether or not the muscle cells in the tunnels of the neuropil actively contract and massage the neuropil to facilitate metabolic and other exchanges.

Oxygen binding characteristics of lobster hemocyanin and its subunits

1. 1. Dissociation of lobster hemocyanin was broguht about by changes in pH. The effect of dissociation and reassociation of subunits on the oxygen equilibrium characteristics of hemocyanin was studied. Dissociation was determioned by ultracentrifugation and electrophoresis. Oxygen dissociation curves were obtained using a tonometer equipped with both a polarographic sensor and a 1 -cm quartz cuvette. 2. 2. The hemocyanin molecule remained aggregated between pH 4·5 and pH 8·5. Subuniting occured between both pH 3·0–4·5 and pH 8·5–10·5. The subunits sedimented with values of 8·5 S and 6·5 S, while the aggretated form of the molecule and an average value of 16·0 S. 3. 4. The reassociation of subunits was not complete, and although the autocatalytic nature of the ligand-binding reaction was not chagned in reassociated hemocyanin, the total amount of oxygen bound per molecule was slightly less. 4. 5. The numner of ligand-binding sites per molecule, as indicated by Hill equation analysis, is at least four.

甲殻類の血清学的研究

Hemocyanin is the major component of the proteins contained in the crawfish serum, and other kinds of protein than hemocyanin are hardly demonstrable by Tiserius' electrophoretic method or ordinary paper electrophoresis. But by the immuno-electrophoretic method in agar-gel and the electrophoresis on cellulose acetate membrane the presence of 4 fractions of protein in the serum are detected with a hemocyanin zone. These 4 fractions are considered as globulin because of their electrophoretic mobility and precipitation by 40 % saturation of ammonium sulphate. The humoral antibody, precipitin, produced in the blood of the crawfish after the injection of heterogenous serum protein (goat serum) seems to be co ntained in the slowest globulin.Sedimentation measurements of hemocyanin which was isolated by centrifugation under 110,800 G, shows 2 resembled and closed peaks with S20 about 24 and 16. These 2 components of hemocyanin are present in almost equal amount. The change of pH of the solution within a range of 6.4-8.3 seems to have no influence to the amounts of these 2 hemocyanins. It is confirmed that these 2 components have different antigenicity in precipitin reaction. The hemocyanin solution dyalised by the borate buffer containing KCN, shows same precipitin reaction as original hemocyanin, so that the copper in the hemocyanin molecule may have no effect on this antigenicity.

Structure and properties of hemocyanins: III. Electron micrographs of hemocyanins from different gastropoda and crustacea

Electron micrographs of negatively stained hemocyanin molecules of some Gastropoda (Helix pomatia, Buccinum undatum and Neptunea antiqua) and some Crustacea (Palinurus vulgaris, Homarus vulgaris, Nephrops norvegicus, Cancer pagurus and Carcinus maenas) were made. With the Gastropoda the molecular structure and dissociation at the alkaline side of the isoelectric point have been studied. Differences are observed between Helix p. and Buccinum u. on one hand and Neptunea a. on the other. The electron micrographs suggest that the first two hemocyanins are built from six equidistant parallel layers of subunits, while the latter consists of three double-layers of subunits. This makes a dissociation via half molecules highly improbable for Neptunea hemocyanin and thus explains the absence of the corresponding component on the sedimentation patterns. The molecular structures of the 16 s and 23 s component of the Crustacea hemocyanins were studied. The observed projections of the 16 s component are compatible with a molecular model consisting of eight subunits lying on the corners of a cube. The 23 s component is a dimer of two of these cubes.

OXYGEN-HEMOCYANIN RELATIONSHIPS IN THE LAND CRAB, CARDISOMA GUANHUMI

1. The hemocyanin of the land crab, Cardisoma guanhumi, under physiological conditions, possesses a high oxygen affinity. 2. The oxygen equilibrium curve is sigmoid, with a fairly high degree of interaction occurring between the oxygen-combining sites of the hemocyanin molecule. 3. As in all other crustacean hemocyanins investigated, a normal Bohr effect is present. 4. Van Slyke analyses of the oxygen content of pre-branchial and post-branchial blood samples indicated that nearly all of the oxygen present was in combination with the hemocyanin; consequently the oxygen partial pressures throughout the circulatory system were very low. The hemocyanin did not become saturated with oxygen during passage through the gills. These results are similar to those previously found for three other species of decapod crustaceans. 5. The carbon dioxide content of the blood was very high, averaging almost 50 volumes per cent. 6. It is shown that the low half-saturation value of the hemocyanin confers a stability of loading pressure in the face of changing environmental temperature. It is speculated that the high oxygen affinity may also represent an adaptation tending to counter the effect of water-conserving measures on the rate at which oxygen diffuses through the respiratory surfaces into the blood. 7. Zuckerkandl's studies on the crab, Maia squinado, are discussed and it is tentatively concluded that, though the hemocyanin of the decapod Crustacea may serve several functions and may not always be essential for respiration, its primary function is that of a blood respiratory pigment.

Structure and properties of hemocyanins: I. Electron micrographs of hemocyanin and apohemocyanin from Helix pomatia at different pH values

Electron micrographs were made from Helix pomatia hemocyanin at different pH values with the negative-staining method. The pH-dependent dissociation on the alkaline side of the isoelectric point was especially studied. Our observations are in agreement with previously reported results, although they are not compatible with the model and mechanism of dissociation previously proposed. Our observations suggest that the Helix pomatia hemocyanin molecule is roughly cylindrical ; the diameter of the cylinder is about 300 Å and the height is about 335 Å. This cylinder has a fivefold axis; perpendicular to this axis it is built up from 6 parallel layers. The first dissociation step occurs perpendicular to the fivefold axis into halves. Further dissociation is supposed to be a splitting of these flat cylinders via submultiples into subunits. Divalent cations in concentrations of about 10−4 molar or higher are required for the reversibility of this dissociation.

The decomposition of hydrogen peroxide by hemocyanin and by its dissociation products

The experiments reported in this paper give evidence for the ability of hemocyanin to decompose hydrogen peroxide catalytically. This property was common to all the hemocyanins studied (Molluscan and Crustacean hemocyanins). Any possibility that this catalytic property could be due to a catalase incidentally present in the blood or linked to the hemocyanin molecule must be excluded. Not only was iron never found in the protein preparations, but the study of the activity showed several striking differences from that of catalase, among them the pH-activity curve and the action of substrate concentration. Whereas a high concentration of H 2O 2 inhibits Fe porphyrin catalase activity, it had no effect at all on the catalase activity of hemocyanin. The decomposition of hydrogen peroxide catalyzed by hemocyanin is due to the presence of copper. Removal of the metal from the protein brings loss of activity. The activity is restored to the same level when hemocyanin is reconstituted. Hemocyanin from different sources does not have the same activity as calculated per unit of copper. Hemocyanin from Helix, e.g., with a molecular weight of 8,000,000 and a copper content of 0.25%, had activity toward H 2O 2 2.5 times less than that of Octopus hemocyanin which has a molecular weight of 2,800,000 and the same copper content. Moreover, equivalent amounts of inorganic copper had no effect upon H 2O 2 as demonstrated manometrically.

THE SIZE, SHAPE AND HYDRATION OF LOBSTER HEMOCYANIN

1. The sedimentation coefficient of lobster hemocyanin corrected to water at 20° and extrapolated to zero concentration has been found to be 24.5 x 10-13 cgs units.2. The intrinsic viscosity has been found to be 4.85 ml./ml.3. From the variation of the sedimentation rate of hemocyanin in sucrose solutions of different densities and from the partial specific volume, the hydration of hemocyanin was calculated to be 0.15 ml. of water per gram of dry protein.4. The specific refractive increment was determined to be 0.00193 ml. per 10-2 g.5. From the above parameters and others taken from the literature, lobster hemocyanin molecules were calculated to have a molecular weight of 825,000, corresponding to oblate ellipsoids of revolution with hydrated diameter of 32.2 mµ and hydrated thickness of 7.8 mµ.6. Electron micrographs are consistent with the assumption that the molecules are plate-like ellipsoids of revolution 33 mµ in diameter and 7.5 mµ in thickness in the dried state.