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At least 19 recordsLinked to original sources
PubMed2006

Immunotherapeutic effect of Concholepas hemocyanin in the murine bladder cancer model: evidence for conserved antitumor properties among hemocyanins.

PURPOSE: We determined the antitumor properties of a newly available hemocyanin obtained from the Chilean gastropod Concholepas concholepas (Biosonda Corp., Santiago, Chile) in a syngeneic heterotopic mouse bladder carcinoma model. Since keyhole limpet hemocyanin (Pierce, Rockford, Illinois) is used increasingly in biomedicine as a carrier for vaccines and an immunotherapeutic agent for bladder transitional cell carcinoma, there is a growing interest in finding new substances that share its potent immunomodulatory properties. Considering that keyhole limpet hemocyanin and Concholepas concholepas hemocyanin differ significantly, it was not possible to predict a priori the antitumor properties of Concholepas concholepas hemocyanin. MATERIALS AND METHODS: C3H/He mice were primed with Concholepas concholepas hemocyanin before subcutaneous implantation of mouse bladder tumor-2 cells. Treatment consisted of a subcutaneous dose of Concholepas concholepas hemocyanin (1 mg or 100 mug) at different intervals after implantation. Keyhole limpet hemocyanin and phosphate buffered saline served as positive and negative controls, respectively. In addition, experiments were designed to determine which elements of the immune response were involved in its adjuvant immunostimulatory effect. RESULTS: Mice treated with Concholepas concholepas hemocyanin showed a significant antitumor effect, as demonstrated by decreased tumor growth and incidence, prolonged survival and lack of toxic effects. These effects were similar to those achieved with keyhole limpet hemocyanin. We found that each hemocyanin increased natural killer cell activity but the effect of Concholepas concholepas hemocyanin was stronger. Analysis of serum from treated mice showed an increased interferon-gamma and low interleukin-4, which correlated with antibody isotypes, confirming that hemocyanins induce a T helper type 1 cytokine profile. CONCLUSIONS: To our knowledge our results are the first demonstration of the antitumor effect of a hemocyanin other than keyhole limpet hemocyanin. They suggest that this is an ancient conserved immunogenic mechanism shared by those hemocyanins that is able to enhance T helper type 1 immunity and lead to antitumor activity. Therefore, Concholepas concholepas hemocyanin may be an alternative candidate for providing safe and effective immunotherapy for human superficial bladder cancer.

Animals↗
PubMed1988

Bivalve hemocyanins--a comparison with other molluscan hemocyanins.

1. The hemocyanins of the protobranch bivalves Yoldia thraciaeformis, Yoldia limatula and Acila castrensis have absorption spectra similar to other hemocyanins. 2. Hemocyanins from all three bivalves appear as six-tiered cylinders in the electron microscope (30-32 nm in diameter by 34-38 nm in height). Yoldia thraciaeformis and A. castrensis hemocyanins tend to dissociate to three-tiered half molecules with polar images and also to associate into long tubular polymers. 3. Yoldia thraciaeformis and A. castrensis hemocyanins chromatograph on Sepharose 4B gel close to gastropod hemocyanin (Mr = 9 x 10(6] rather than chiton hemocyanin (Mr = 4 x 10(6]. 4. Hemocyanins from all three vivalves have subunits with electrophoretic mobilities similar to gastropod and polyplacophoran hemocyanin subunits and slower than octopodan hemocyanin subunits. 5. These similarities between bivalve and gastropod hemocyanins are consistent with the hypothesis that bivalves and gastropods have shared a common ancestor.

Animals↗
PubMed1984

Immunological correspondence between arthropod hemocyanin subunits. I. Scorpion (Leiurus, Androctonus) and spider (Eurypelma, Cupiennius) hemocyanin.

The hemocyanins of the scorpions Leiurus quinquestriatus and Androctonus australis, the tarantula Eurypelma californicum (all 24-mers), and the lycosid spider Cupiennius salei (dodecamer) were dissociated into subunits, the subunits isolated and studied by two-dimensional immunoelectrophoresis for interspecific cross-reactivities. Androctonus hemocyanin yielded a pattern of 8 subunit types in agreement with data from Lamy et al. (1979, Arch. Biochem. Biophys. 193, 140-149). Leiurus hemocyanin is also composed of 8 immunologically distinct subunits which could be assigned to the pattern of Androctonus in a subunit-to-subunit correlation. The subunit designations 1 to 6 of Lamy et al. could be adopted for both scorpion hemocyanins; however, in the present communication, Lamy's subunits 3A/3B are designated as 3'/3", because we could not unequivocally decide if 3' = 3A and 3" = 3B or vice versa. The 7 subunit types a to g of Eurypelma hemocyanin could be correlated with the scorpion hemocyanin subunits as follows: a = 3', b = 5B, c = 3C, d = 5A, e = 6, f = 2, g = 4. Additional cross-reactivities were detected between e/4, and f/5A, respectively. No subunit of Eurypelma hemocyanin is homologous to scorpion 3", which could not be precipitated by anti-Eurypelma antiserum. Antiserum against Cupiennius hemocyanin precipitated subunit f of Eurypelma and subunits 2 and 5A of scorpion hemocyanin. The published models of quaternary structure and a possible subunit phylogeny of arachnidan hemocyanins are discussed in view of the present results.

Animals↗
PubMed2005

Biochemical and molecular characterisation of hemocyanin from the amphipod Gammarus roeseli: complex pattern of hemocyanin subunit evolution in Crustacea.

Hemocyanin is a copper-containing respiratory protein that is widespread within the arthropod phylum. Among the Crustacea, hemocyanins are apparently restricted to the Malacostraca. While well-studied in Decapoda, no hemocyanin sequence has been known from the 'lower' Malacostraca. The hemocyanin of the amphipod Gammarus roeseli is a hexamer that consists of at least five distinct subunits. The complete cDNA sequence of one subunit and a tentative partial sequence of another subunit have been determined. The complete G. roeseli hemocyanin subunit comprises 2,150 bp, which translates in a protein of 672 amino acids with a molecular mass of 76.3 kDa. Phylogenetic analyses show that, in contrast to previous assumptions, the amphipod hemocyanins do not belong to the alpha-type of crustacean hemocyanin subunits. Rather, amphipod hemocyanins split from the clade leading to alpha and gamma-subunits most likely at the time of separation of peracarid and eucarid Crustacea about 300 million years ago. Molecular clock analyses further suggest that the divergence of beta-type subunits and other crustacean hemocyanins occurred around 315 million years ago (MYA) in the malacostracan stemline, while alpha- and gamma-type subunits separated 258 MYA, and pseudohemocyanins and gamma-subunits 210 million years ago.

Amino Acid Sequence↗
PubMed1980

Hemocyanins in spiders, IX. Homogeneity, subunit composition and the basic oligomeric structure of Eurypelma californicum hemocyanin.

37S hemocyanin isolated from the tarantula Eurypelma californicum was subjected to a variety of fractionation procedures: Electrophoresis in polyacrylamide gels, isoelectrofocusing, and ion exchange chromatography. Single fractions were dissociated at alkaline pH and/or by sodium dodecyl sulfate and the resulting subunits separated by polyacrylamide gel electrophoresis in gradient slab gels followed, in many cases, by quantitative estimation of the subunit bands. In addition, crossed immunoelectrophoresis was employed to analyze the subunit composition. Separation of the native hemocyanin into fractions with different subunit composition was not observed. Also, hemocyanin samples taken from individual spiders showed a strikingly uniform composition. It is concluded that Eurypelma hemocyanin is homogeneous for all practical means. This implies that the seven different polypeptide chains described previously for Eurypelma hemocyanin all take part in the formation of the 24 subunits, 37S oligomeric molecule. By monitoring chromatographic effluents at 280 nm, scanning stained electrophoresis gels and determining peak areas in these experiments and in crossed immunoelectrophoresis, the relative quantities of the seven different polypeptide chains in the whole molecule were determined as follows (number per 37S molecule): 6a, 2b, 2c2, 2c4, 4d, 4e, 4f. The ratio between a and c2 (6:2) is the least well established. Since 7 different polypeptide chains are incorporated, the hexameric molecule cannot represent the basic oligomeric structure in tarantula hemocyanin, but probably the dodekamer. If Eurypelma hemocyanin is incubated with sucrose, partial dissociation occurs, yielding a fragment sedimenting with ca. 28S. This process is reversible. The 28S fragment still contains all subunit species; it is thought to represent a (3 x 6) or closely related structure. The implications of this finding with respect to the symmetry of the native hemocyanin molecule are discussed.

Animals↗
PubMed1998

Ontogeny of hemocyanin function in the dungeness crab cancer magister: hemolymph modulation of hemocyanin oxygen-binding

The in vivo oxygen-binding characteristics of Cancer magister whole hemolymph were compared across developmental stages with those of purified hemocyanin. When the 25S hemocyanins from first-instar juvenile and adult C. magister were dialyzed against first-instar juvenile saline, the P50 values at pH 7.8 differed by 54 %: 2.16 kPa for the adult and 4.68 kPa for the first-instar juvenile. Since both purified proteins were examined under identical conditions, this represents an intrinsic stage-specific difference in hemocyanin O2-affinity. When the two types of hemocyanin were dialyzed against their respective stage-specific salines, the oxygen affinities differed by only 28 %: 3.39 kPa for the adult and still 4.68 kPa for the first-instar juvenile. Thus, the intrinsic difference in hemocyanin O2-affinity was reduced by the stage-specific differences in hemolymph ion concentrations. Even more significant is the fact that the whole-hemolymph P50 values of the juvenile and adult were indistinguishable at in vivo pH and divalent cation levels specific for each stage. Thus, despite significant differences in the intrinsic oxygen affinity of the purified 25S hemocyanin during development, the whole-hemolymph oxygen-binding properties are conserved. In the juvenile crab, it appears that the low-affinity hemocyanin serves to modulate the effects of a weak renal regulation of [Mg2+]. As ion regulation is enhanced during development and divalent cation levels decrease, the crab synthesizes higher-affinity hemocyanin.

Journal Article↗
PubMed2000

Haliotis tuberculata hemocyanin (HtH): analysis of oligomeric stability of HtH1 and HtH2, and comparison with keyhole limpet hemocyanin KLH1 and KLH2.

The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from keyhole limpet hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31-41; Harris, J.R., Gebauer, W., Söhngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43-56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329-339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100mM concentrations of calcium and magnesium ions in Tris-HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100mM calcium and magnesium ions yielded an almost 100% conversion into didecamers, whereas the HtH2 subunit produced a mixture containing large numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. The association properties of the HtH1 and HtH2 decamers, and the subunit reassociation, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH2 subunits, enables a detailed comparison of the two hemocyanin isoforms from both molluscan species to be made. Biochemical manipulation of the oligomer states and the subunit reassociation of molluscan hemocyanins can usefully be assessed by the study of negatively stained TEM specimens.

Animals↗
PubMed1997

cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gene family.

The complete cDNA sequence and protein reading frame of a developmentally regulated hemocyanin subunit in the Dungeness crab (Cancer magister) is presented. The protein sequence is aligned with 18 potentially homologous hemocyanin-type proteins displaying apparent sequence similarities. Functional domains are identified, and a comparison of predicted hydrophilicities, surface probabilities, and regional backbone flexibilities provides evidence for a remarkable degree of structural conservation among the proteins surveyed. Parsimony analysis of the protein sequence alignment identifies four monophyletic groups on the arthropodan branch of the hemocyanin gene tree: crustacean hemocyanins, insect hexamerins, chelicerate hemocyanins, and arthropodan prophenoloxidases. They form a monophyletic group relative to molluscan hemocyanins and nonarthropodan tyrosinases. Arthropodan prophenoloxidases, although functionally similar to tyrosinases, appear to belong to the arthropodan hexamer-type hemolymph proteins as opposed to molluscan hemocyanins and tyrosinases.

Amino Acid Sequence↗
PubMed1991

Hexamers of subunit II from Limulus hemocyanin (a 48-mer) have the same quaternary structure as whole Panulirus hemocyanin molecules.

Hemocyanins are copper-containing proteins that transport oxygen in a variety of invertebrates. Considerable evidence has accumulated that arthropodan hemocyanins are multimers of a fundamental hexameric unit. X-Ray crystallographic structure determination has revealed that the hemocyanin molecule from the spiny lobster Panulirus interruptus is a single hexamer having 32 point group symmetry. Using crystals of subunit II, one of 8 polypeptide types comprising the octahexameric hemocyanin of the horseshoe crab Limulus polyphemus, and the molecular replacement method for crystallographic phase determination we show that subunit II forms assemblies with the same hexameric quaternary structure as the whole Panulirus hemocyanin molecule. Observation of the same hexameric motif in two widely separated species provides strong additional evidence that this quaternary structural unit is a universal building block of arthropodan hemocyanins.

Amino Acid Sequence↗
PubMed1998

Active-site disruption in native Limulus hemocyanin and its subunits by disulfide-bond reductants: a chemical probe for the study of structure-function relationships in the hemocyanins.

The crystal structure analysis of Subunit II of Limulus hemocyanin has shown that its polypeptide chain is folded into three distinct structural domains. The oxygen-binding, dinuclear copper center is located deep in the core of Domain 2. Two disulfide bonds are located in a bridging domain, Domain 3. These disulfide bonds are remote from the oxygen-binding site, but are positioned so that they could affect its stability. When the disulfide bonds are broken by dithiothreitol or other disulfide-bond reductants, the 340-nm absorption band, associated with oxygen binding, is lost. Disulfide-bond reductants also cause the loss of the oxygen-binding capacity of all seven of the other subunits of Limulus hemocyanin. Thus, disulfide bonding is a general feature of the Limulus hemocyanin subunits that is important to the maintenance of the physiologically effective geometry of the oxygen-binding site. The rate of loss of oxygen-binding capacity, however, is highly dependent on subunit type, aggregation state, and protein conformation. Evidence that protein conformation markedly affects the rate of disruption of the oxygen-binding site comes from the finding that the addition of dithiothreitol to fully oxygenated samples results in a slow initial loss of oxygen-binding capacity followed by an appreciably faster reaction rate. In contrast, in the deoxygenated conformation, the reaction rate is monophasic and never attains the faster rates observed for oxygenated samples. When the disulfide bonds are broken and oxygen-binding capacity is lost, there is subunit-specific variability in the extent of polypeptide-chain unfolding, subunit aggregation, and loss of active-site copper ions. When the disulfide-bond reductant is removed by dialysis so that disulfide bonds can re-form, there is also subunit-specific variability in the extent of restoration of oxygen-binding capacity. Complete restoration of structure and function as the disulfide bonds re-form occurs only for the 48-subunit native molecule, whose architecture is stabilized by bound Ca2+ and extensive intersubunit contacts. We have found a similar loss of oxygen-binding capacity upon breaking disulfide bonds in a number of other arthropod and mollusc hemocyanins, suggesting that the active site of Limulus hemocyanin is not unique in its dependence upon intact disulfides. The results presented in this paper suggest that disulfide-bond reduction may provide a simple, but powerful, chemical tool with which to probe internal and environmental factors that govern physiologically important structure-function relationships in the hemocyanins.

Animals↗
PubMed2002

Hemocyanin and the branchial heart complex of Sepia officinalis: are the hemocytes involved in hemocyanin metabolism of coleoid cephalopods?

Cytobiological experiments using isotopic- and cytochemical-labeled Sepia hemocyanin as well as immunocytochemical localization of the respiratory pigment were carried out to investigate the function of the hemocytes in hemocyanin metabolism of the common cuttlefish Sepia officinalis. For comparison, the rhogocytes (ovoid cells) of the branchial heart complex were included in this study. Hemocyanin molecules were immunocytochemically detected in the lysosomal compartment of the rhogocytes and, at lower levels, in adhesive and circulating hemocytes. (125)I-labeled Sepia hemocyanin was taken up by the rhogocytes only, whereas gold- and/or fluorescein-labeled Sepia hemocyanin was solely taken up by the adhesive and the circulating hemocytes, even though the level of uptake is different. There are also differences in the uptake of pure gold particles and/or fluorescein between rhogocytes and hemocytes. These findings give evidence that circulating and adhesive hemocytes of the branchial heart complex are not involved in hemocyanin turnover, but are a component of the cellular defense and detoxification system of adult coleoid cephalopods.

Animals↗
PubMed1975

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.

Animals↗
PubMed2002

Complete hemocyanin subunit sequences of the hunting spider Cupiennius salei: recent hemocyanin remodeling in entelegyne spiders.

Hemocyanins are large copper-containing respiratory proteins found in many arthropod species. Scorpions and orthognath spiders possess a highly conserved 4 x 6-mer hemocyanin that consists of at least seven distinct subunit types (termed a to g). However, many "modern" entelegyne spiders such as Cupiennius salei differ from the standard arachnid scheme and have 2 x 6-mer hemocyanins. Here we report the complete primary structure of the 2 x 6-mer hemocyanin of C. salei as deduced from cDNA sequencing, gel electrophoresis, and matrix-assisted laser desorption spectroscopy. Six distinct subunit types (1 through 6) and three additional allelic sequences were identified. Each 1 x 6-mer half-molecule most likely is composed of subunits 1-6, with subunit 1 linking the two hexamers via a disulfide bridge located in a C-terminal extension. The C. salei hemocyanin subunits all belong to the arachnid g-type, whereas the other six types (a-f) have been lost in evolution. The reconstruction of a complex hemocyanin from a single g-type subunit, which commenced about 190 million years ago and was completed about 90 million years ago, might be explained by physiological and behavioral changes that occurred during the evolution of the entelegyne spiders.

Amino Acid Sequence↗
PubMed1980

Hemocyanins in spiders, XII. Dissociation and reassociation of Eurypelma hemocyanin.

The dissociation and reassembly of Eurypelma californicum hemocyanin was studied under various conditions of pH, ionic strength and protein, calcium and magnesium concentrations. The hemolymph concentrations of calcium and magnesium were determined to be ca. 4 and 0.9mM, respectively. Eurypelma hemocyanin does not dissociate upon dilution down to 0.04 mg/ml. At physiological pH, phosphate causes partial dissociation. Dissociation at alkaline pH requires two days for completion and is dependent on hemocyanin concentration. Reassociation, starting with the total dissociated subunits, yielded 37 S, 16 S and unreacted 5 S material. The yield of 37 S particles after one day was 75%, but rose slowly to 84% after two weeks. Raising the calcium concentration above 1mM resulted in an increase of 16 S particles (hexamers). High magnesium concentrations interfered with ordered reassembly. However, 37 S, native-like particles are readily formed in the absence of both calcium and magnesium. Reassociation is strongly favoured if ionic strength is increased. The significance of the 16 S reassociation product was studied by repeated dissociation and reassociation and analysis of subunit composition. It was found that the 37 S reassociate contained the whole set of subunits, their quantitative proportions being identical compared to native 37 S hemocyanin. In contrast, the 16 S material was lacking the dimeric subunit bc4 (= 4D). Evidently, this hexameric product arises by incorrect reassociation of monomeric subunits. P50 and nH values were recorded for the reassociated hemocyanins. The 37 S component had a higher affinity and lower cooperativity than native hemocyanin, possibly due to ageing of the subunits.

Animals↗
PubMed1981

Hemocyanins in spiders, XIV. Subunit composition of dissociation intermediates and its bearing on quaternary structure of Eurypelma hemocyanin.

The 37 S hemocyanin (24 subunits of 7 types) isolated from the tarantula, Eurypelma californicum, was dissociated partially by various agents and the dissociation intermediates analyzed for their subunit composition by crossed immunoelectrophoresis. The subunit composition of the native hemocyanin was reexamined and the pending problem of the ratio between subunits a and g (= c2) clarified. The subunits are present in the ratio of a:b:c:d:e:f:g = 4:2:2:4:4:4:4. Breakdown products were shown to contain 19 (20?), 16, 12, 8, 7 and 6 polypeptide chains (possibly, there is also a 15-mer). The 19-mer is formed by removal of 5 monomeric subunits from one of the constituent hexamers of the native (4 x 6) hemocyanin, the 16-mer by losing one further copy each of e, f and g. The dodecamer is formed by cleavage with 2-mercaptoethanol and represents a half-molecule. The octamer could not be clearly analyzed but probably contains one copy of chain d more compared to the heptamer. The heptamer represents a one-quarter hemocyanin with one additional polypeptide chain sticking out of the hexameric structure. This can be either subunit b or c of the heterodimer bc. Only one type of hexamer was obtained after cleavage of the 37 S hemocyanin with 4M urea, containing one copy each of chains a, b, d, e, f and g. It is concluded that the 37 S hemocyanin is composed of two identical dodecameric halves linked by dimerization of subunit f, and that each half-molecule is constituted by two non-identical though similar hexamers, both encompassing a complete set of subunits a, d, e, f and g, but differing in their share of the rather stable, the 'c'-hexamer unstable. The relative positions of some of the subunits within the native oligomer are discussed.

Hemocyanins↗
PubMed1995

The oxidation of hemocyanin. Kinetics, reaction mechanism and characterization of met-hemocyanin product.

The reaction that gives met-hemocyanin from Octopus vulgaris oxy-hemocyanin has been reinvestigated under several experimental conditions. Various anions including azide, fluoride and acetate have been found to promote this reaction. Kinetic data indicate that the reaction mechanism is different from that currently accepted involving a peroxide displacement of bound dioxygen through an associative chemistry on an open axial position of the copper ions [Hepp, A. F., Himmelwright, R. S., Eickman, N. C. & Solomon, E. I. (1979) Biochem. Biophys. Res. Commun. 89, 1050-1057; Solomon, E. I. in Copper proteins (Spiro, T. G., ed.) pp. 43-108, J. Wiley, New York]. Our study suggests that the protonated form of the anion is likely to be the species reacting with the oxygenated form of the protein. Furthermore, it is also proposed that protonation of bound dioxygen generates an intermediate hydroperoxo-dicopper(II) complex to which the exogenous anion is also bound. This intermediate in not accumulated and preceds the release of hydrogen peroxide by reaction with water. Upon dialysis it leads to the met-hemocyanin form. The structure of this dinuclear copper(II) derivative contains a di-mu-hydroxo bridge but there is evidence from optical and circular dichroism spectra for partial protonation of these bridges at low pH. As a consequence, while one azide molecule binds in the bridging mode to met-hemocyanin with low affinity (K = 30 M-1) at pH 7.0, it binds with much higher affinity at pH 5.5 (K = 1500 M-1), where a second azide ligand also binds in the terminal mode (K = 20 M-1). The coordination mode of the azide ligands is deduced from the optical and circular dichroism spectra of the protein complexes.

Animals↗
PubMed1982

Hemocyanins in spiders, XVII. A presumptive active-site sequence of arthropod hemocyanins.

Peptides containing the sequence -His-His-Trp-His-Trp-His- have been isolated from the subunits e and a of tarantula (Eurypelma californicum) hemocyanin and III B of Limulus polyphemus hemocyanin. In view of the published spectroscopic and titration data on the copper-polypeptide complex and the strong quenching effect of oxygenation on hemocyanin fluorescence, it is concluded that this sequence represents the major part of the active site of Eurypelma, Limulus, and possibly other arthropod hemocyanins.

Amino Acid Sequence↗
PubMed1985

Immunological correspondence between arthropod hemocyanin subunits. II. Xiphosuran (Limulus) and spider (Eurypelma, Cupiennius) hemocyanin.

The hemocyanins of the horseshoe crab Limulus polyphemus (48-mer), the tarantula Eurypelma californicum (24-mer), and the lycosid spider Cupiennius salei (dodecamer, hexamer) were dissociated into subunits, the subunits isolated and studied by two-dimensional immunoelectrophoresis for interspecific cross-reactivities. Among the subunits a to g of Eurypelma on the one side, and I to VI of Limulus on the other, a number of cross-reactions were obtained which agree with the topologic subunit positions in the published models of quaternary structure: a = II, b-c = V-VI, d = IV, e = I, f = IIIb, g = IIIa (IIa). However, cross-reactivity was only strong in the following combinations: a/II, d/IV, b-c/V-VI (the monomers of the two heterodimers could not be correlated individually). A rather weak cross-reaction was obtained in the case of e/I and g/IIIa (IIa); a cross-reaction between f and IIIb was almost undetectable. On the other hand, f/IV clearly cross-reacted, and so did e/IIIa (IIa), which apparently is not in agreement with the two models of quaternary structure. These unexpected relationships, however, indicate the possible phylogeny of the subunits. Antiserum against Cupiennius hemocyanin precipitated subunit f of Eurypelma and subunit IV of Limulus and, moreover, revealed common antigen determinants present on these subunits. Denaturation of hemocyanin subunits of the three species with 8M urea yielded a completely different immunological behavior in that in all intra- and interspecific combinations the reaction of immunological identity was obtained. The published models of quaternary structure and a possible subunit phylogeny of cheliceratan hemocyanins is discussed in view of the present results and the results of the preceding paper. [Markl, J. et al. (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 619-631.]

Animals↗