Structure Of Hemocyanin Research Articles

Subunit structure of hemocyanin from the gastropod Levantina hierosolima

Subunit structure of hemocyanin from the gastropod Levantina hierosolima

Structure of arthropod hemocyanin

Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. The amino acid sequence of subunit a of Panulirus interruptus hemocyanin (657 residues) has been completed and fitted to the electron-density map (3.2 Å resolution). Comparison of amino acid sequence data for several different hemocyanin subunits of arthropod species indicated that the general features of the polypeptide architecture as found in spiny lobster hemocyanin occur in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, 540–600 million years ago.

The structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. The amino acid sequence of the second largest cyanogen bromide fragment.

Fourteen fragments have been isolated from hemocyanin component II of Limulus polyphemus by cleavage with CNBr. The amino acid sequence of the largest fragment, CNBr Ia has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequence of the 12 smaller fragments is reported in an accompanying paper (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1985) J. Biol. Chem. 261, 10511-10519). We have determined the amino acid sequence of the second largest fragment, CNBr Ib. The fragment contains 142 residues and has a molecular weight of 16,095.

Structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. Sequences of the overlapping peptides, ordering the CNBr fragments, and the complete amino acid sequence.

The amino acid sequence of the largest fragment, CNBr Ia (203 residues) has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequences of the second largest fragment, CNBr Ib (142 residues), and of the 12 smaller fragments are reported in accompanying papers (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10511-10519; Behrens, P. Q., Nakashima, H., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10520-10525). The complete amino acid sequence of hemocyanin component II has been established by isolation and analysis of 13 methionine-containing peptides from either a tryptic digest or a Staphylococcus aureus strain V8 protease digest of whole carboxamidomethylated hemocyanin II. Hemocyanin II is composed of 628 residues and has a molecular weight with two copper atoms of 72,946.

The structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. The amino acid sequences of the smaller cyanogen bromide fragments.

Fourteen fragments have been isolated from hemocyanin component II of Limulus polyphemus by cleavage with CNBr. The amino acid sequences of the two largest fragments, CNBr Ia and Ib, have been determined (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749; Behrens, P. Q., Nakashima, H., Yokota, E., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10520-10525). We have determined the amino acid sequence of the remaining 12 smaller fragments.

The structure of arthropod hemocyanins.

Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. Comparison of the amino acid sequence data for seven different subunits of arthropod hemocyanins from crustaceans and chelicerates shows many highly conserved residues and extensive regions of near identity. This correspondence can be matched closely with the three domain structure established by x-ray crystallography for spiny lobster hemocyanin. The degree of identity is particularly striking in the second domain of the subunit that contains the six histidines which ligate the two oxygen-binding copper atoms. The polypeptide architecture of spiny lobster hemocyanin appears to be the same in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, about 540 to 600 million years ago.

The role of copper and quaternary structure on the conformational properties of Octopus vulgaris hemocyanin

Some structural properties of Octopus vulgaris hemocyanin have been investigated by fluorescence spectroscopy. The three-dimensional structure of Octopus hemocyanin is remarkably tight, resulting in a deep burial of almost all the tryptophyl residues of the protein. The hemocyanin conformation has been studied in the two main aggregation states (11 S, 50 S) of the protein, and with respect to the presence or absence of copper in the active site. Upon changing the pH of the solution, Octopus hemocyanin in the 50 S aggregation state can assume at least three different conformations. During the transition between each conformation the fluorescence quantum yield changes, but the environment of tryptophans does not change. Dissociation of the protein from 50 S to 11 S strongly enhances its susceptibility toward denaturating agents such as pH or temperature, and modifies the effects of fluorescence quenchers such as acrylamide. Moreover, these effects are more pronounced when copper is removed from the active site. A comparative analysis of the results shows that the subunit-subunit interactions exerted within the 50 S species are more important in the maintenance of the conformational stability than the copper ions present in the active sites. This behavior can be accounted for by the large amount of Ca(II) ions linked to 50 S hemocyanin.

Allosteric modulation of Callinectes sapidus hemocyanin by binding of L-lactate.

Hemocyanin of the blue crab Callinectes sapidus has the typical structure of crustacean hemocyanins in that its smallest in vivo structure is a hexamer of subunits each having a molecular mass of approximately 75 000. As found in the blood, Callinectes hemocyanin consists of a mixture of hexamers and dodecamers (typically 1:4). As in other crustacean hemocyanins, the affinity with which oxygen binds to the binuclear copper site has been reported to be very sensitive to pH and to a variety of inorganic allosteric effectors. We report here the interaction of L-lactate, a natural metabolite,with the native hemocyanin and with chromatographically purified hexamers and dodecamers. Under ionic conditions that approximate those found physiologically, the addition of 10 mM L-lactate to native Callinectes hemocyanin substantially increases its oxygen affinity (Δ log P(50) = -0.28). The data from lactate titrations were fit to a theoretical equation,and the best fit was obtained with a lactate dissociation constant of 1.8 mM for the oxy state and 2.2 lactate binding sites for every 6 oxygen binding sites. Independent measurements by ultrafiltration techniques indicated a dissociation constant of 3.2 mM with 2.8 lactate binding sites per 6 oxygen binding sites. The two sets of data clearly indicate that there is less than one lactate binding site per oxygen binding site. The fit to the titration was not improved with the assumption of more than one class of lactate binding site. The hexamers and dodecamers of native Callinectes hemocyanin are not in equilibrium and are stable after separation by gel-filtration chromatography. Polyacrylamide gel electrophoresis of the subunits of the dissociated dodecamers shows five major bands.Two of these bands, which constitute one-sixth of the total dodecameric hemocyanin, do not appear upon gel electrophoresis of dissociated hexamers. The oxygen affinities of the hexameric and dodecameric hemocyanin forms are similar to one another but show differences in their sensitivity to L-lactate.The oxygen affinity of native Callinectes hemocyanin was increased appreciably more by L-lactate than by glycolate,D-lactate, and pyruvate (listed in decreasing order of effectiveness).Propionate, acetate, succinate, D-alanine, and L-alanine were without effect, thus illustrating the selectivity of the L-lactate effect on the hemocyanin. We found the magnitude of the Bohr effect to be unchanged by the addition of L-lactate over the pH range 7.5-8.0. Moreover, there is no significant effect of L-lactate on the aggregation state of the hemocyanin or on its 340-nm copper-oxygen absorption band.The foregoing results are consistent with the role of L-lactate as a specific allosteric effector of Callinectes hemocyanin that acts by preferential binding to a stereospecific site of the oxy hemocyanin.

Quaternary structure of arthropod hemocyanins

The models for the quaternary structure of 16S arthropod hemocyanin molecules based on the arrangement of six identical spheres in a trigonal antiprism and in a regular octahedron are shown to be identical. The effect of replacing the spheres with non-identical, non-symmetrical units is discussed.

TheLimulusII hemocyanin structure at 5.5 Å resolution

The<i>Limulus</i>II hemocyanin structure at 5.5 Å resolution

Structural basis of subunit heterogeneity in arthropod hemocyanins

1. 1. Higher than 16 S arthropod hemocyanin structures are described in terms of a model of the 16 S unit made up of six identical spherical subunits occupying the corners of a regular octahedron. 2. 2. The well-established subunit heterogeneity of arthropod hemocyanins is traced to the fact that the positions allotted for the subunits in the models of higher than 16 S hemocyanins are not all equivalent. 3. 3. The analysis presented provides a basis for the rationalization of the number and relative abundance of subunit types established in 24 S hemocyanin from the crayfish Chrax destructor and 36 S hemocyanin from the scorpion Androctonus australis.

Oxygen binding properties of stripped (calcium ion and magnesium ion free) hemocyanin from the scorpion Leirus quinquestriatus.

The binding of oxygen to stripped Ca2+,-Mg2+-free) hemocyanin from the scorpion Leirus quinquestriatus was studied over a wide range of pH and temperature. Oxygen binding was cooperative in the entire range of pH where the native hemocyanin structure is preserved. Probably 12, or even the totality of the 24 oxygen binding sites of the molecule, act as a cooperative unit. The oxygen binding affinity and the degree of cooperativity were both affected by pH. The dependence on pH of the half-saturation pressure-Bohr effect-was interpreted in terms of two oxygen-linked protons, one opposing and the other promoting oxygen binding. The effect of pH on the maximal slope of the Hill plot indicates that proton dissociation and cooperativity are linked phenomena. Analysis of the binding data using linkage theory shows that an efficient coupling of the homotropic (oxygen-oxygen) and the heterotropic (proton-oxygen) free energies of interaction can provide a satisfactory interpretation of the cooperative behavior. Our findings suggest that in arthropod hemocyanin, as in molluskan hemocyanin, cooperativity of oxygen binding can be attributed to ligand-ligand linkage. In molluskan hemocyanin, however, cooperativity is generated solely by alkaline earth ion-oxygen linkage. In arthropod hemocyanin, both alkaline earth ion- and proton-oxygen linkage can generate cooperative behavior.

Conformational changes of Carcinus maenas hemocyanin induced by urea

The effect of urea up to 9 m on the structure of hemocyanin from Carcinus maenas has been studied by different spectroscopic techniques. The renaturation of the protein before and after reduction of the disulphide bonds has also been investigated. Dissociation of the quaternary structure and drastic conformational modifications occur while the concentration of the denaturant increases. Some ordered structure, however, is still present in 8 m urea. In the absence of urea, evidence for tyrosine to tryptophan energy transfer is found ( e = 0.6). No transfer occurs in 9 m urea. Different classes of tryptophan residues are evidenced by the shape of the emission spectra and by the fluorescence quenching with acrylamide. The renaturation of both apohemocyanin and reduced and methylated apohemocyanin does not reconstitute the original conformation. The unique -S-S- bridge seems to have some influence on the protein structure; however, also the reduced protein shows a refractory core to the denaturation.

Arthropod hemocyanin structure: Isolation of eight subunits in the scorpion

Eight immunologically pure subunits were isolated from Androctonus australis hemocyanin. Antisera specific against each of these were prepared. Two subunits associate to form a heteroöligomer which is probably one of the bridges visible in electron microscopy of the native molecule. There is no cross-reactivity between native subunits. When denatured by 7 m urea, the specific antigenic determinants disappear and a broader specificity appears. This is probably due to the unfolding of the molecule which unmasks deeply buried antigenic sites.

Structure and function of hemocyanin from thalassinid shrimp

The subunit structure, association equilibria, and oxygen binding of the hemocyanins of three thalassinid shrimp have been investigated. The organismsCallianassa californiensis, Callianassa gigas andUpogebia pugettensis are found in similar but distinct habitats in a limited region. All of the hemocyanins exhibit a common pattern of subunit structure, but differ in details of the response of that structure to variations in ionic environment. All three are heterogeneous in polypeptide chain composition, but have quite different distributions of components. The oxygen binding of the twoCallianassa hemocyanins is virtually identical under conditions approximating physiological; that ofUpogebia, which shows lower tolerance to anoxia, is significantly different. These similarities and differences are discussed in terms of the physiological requirements of the species.

The structure of arthropod and mollusc hemocyanins.

The hemocyanins from molluscs and from arthropods differ in the size of their polypeptide chains. A variety of physical techniques including sodium dodecyl sulfate polyacrylamide gel electrophoresis and column chromatography in sodium dodecyl sulfate and guanidine HCl indicate that the polypeptide chain of mollusc hemocyanin has a molecular weight of 290,000. These results were corroborated by quantitative end group analyses. Several experiments designed to count the number of tryptophan and methionine-containing peptides in the hemocyanin from the whelk Busycon canaliculatum indicate that sequence homology within the polypeptide chain of the mollusc hemocyanins accounts for their large size. Digestion of the native protein with subtilisin produces a 50,000-dalton fragment in high yield which corresponds to one binding site for oxygen. On the other hand, the polypeptide chain molecular weight of lobster hemocyanin is 76,000 to 78,000 and this seems to be a general property of all arthropod hemocyanins. The pigment from lobster consists of two very similar polypeptide chains which are not present in equal amount. Analysis of the cysteine-containing and of the tryptophan-containing tryptic peptides confirms the value of the molecular weight. However, separation of fragments which contain methionine indicates that there is sequence homology withing the polypeptide chain of this protein. It is concluded that the mollusc and arthropod hemocyanins have little structural similarity.

Structure and properties of hemocyanins: XII. Electron microscopy of dissociation products of Helix pomatia α-hemocyanin: Quaternary structure

Electron microscopy of the dissociation products of α-hemocyanin of the snail Helix pomatia reveals two distinctly different conformations of 1 10 -size molecules: a C(compact)-form at high ionic strength and pH near 8, and an L(loose)-form at low ionic strength and/or higher pH. The size and shape of the C- 1 10 -size molecules indicate a dissociation of the 1 2 -size molecules along the (10,9) helical grooves, which have been shown to be boundaries between the morphological units of the cylinder wall (Mellema & Klug, 1972). The 5-fold collar is distributed evenly among the C- 1 10 -size molecules. The C → L conformational change is characterized by a drastic loosening of the 1 10 -size molecules to a flexible cluster of globules, with a concomitant decrease of sedimentation coefficient and increase of intrinsic viscosity and frictional ratio. The 1 20 -size molecule appears as a flexible, linear chain of seven or eight globules with a diameter of 55 to 60 Å. These globules are inferred to be the minimal oxygen binding units with a molecular weight of about 50,000, linked together by short stretches of the polypeptide chain. Stable intermediate dissociation products, 2 10 , 3 10 and 4 10 -size molecules, were obtained by intramolecular crosslinking of 1 2 -size molecules with dimethyl-suberimidate prior to dissociation.

Oxygen-linked association-dissociation of Helix pomatia hemocyanin.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTStructure and properties of hemocyanins. XV. Oxygen-linked association-dissociation of Helix pomatia hemocyaninRoel Van Driel and Ernst F. J. Van BruggenCite this: Biochemistry 1974, 13, 20, 4079–4083Publication Date (Print):September 1, 1974Publication History Published online1 May 2002Published inissue 1 September 1974https://doi.org/10.1021/bi00717a003RIGHTS & PERMISSIONSArticle Views26Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (565 KB) Get e-Alerts Get e-Alerts

Oxygen binding and subunit interactions in Helix pomatia hemocyanin.

Oxygen binding and subunit interactions in Helix pomatia hemocyanin.

Structure and properties of hemocyanins: VIII. Microheterogeneity of α-hemocyanin of Helix pomatia

The degree of dissociation of Helix pomatia α-hemocyanin is independent of its concentration. The dissociation products can be separated by ultracentrifugation or gel filtration. The isolated fractions do not reequilibrate. Subfractions of α-hemocyanin differ from total α-hemocyanin is a pH-dependent dissociation behaviour. It is suggested that α-hemocyanin is a population of closely similar molecular species differing in pH of dissociation. Independent evidence for microheterogeneity is obtained from reversible boundary spreading of α-hemocyanin and some of its dissociation products by electrophoresis on cellulose acetate.