Panulirus Interruptus Hemocyanin Research Articles

Fluorescence spectroscopy of the tryptophan microenvironment in Carcinus aestuarii hemocyanin.

The steady-state and time-resolved fluorescence properties of the multitryptophan minimal subunit CaeSS2 from Carcinus aestuarii hemocyanin have been studied with the aim of probing the environment of the fluorophores within the protein matrix. Subunit a of Panulirus interruptus hemocyanin, whose X-ray structure is known, has been also studied. The results are compared with those collected with other two monomeric fractions (CaeSS1, CaeSS3) produced by dissociation of the native, oligomeric protein as well as with those of the hexameric aggregate. Three classes of tryptophan residues can be singled out by a combination of fluorescence quenching and lifetime measurements on the holo-Hc (the copper containing, oxygen binding form) and the apo-Hc (the copper-free derivative). One class of tryptophans is exposed to the protein surface. Some of these residues are proposed to be involved in the intersubunit interactions in CaeSS1 and CaeSS3 fractions whereas in CaeSS2 the protein matrix masks them. This suggests the occurrence of conformational rearrangements after detachment of the subunit from the native aggregate, which could explain the inability of CaeSS2 to reassociate. A second class of tryptophan has been correlatively assigned, by comparison with the results obtained with Panulirus interruptus hemocyanin, to residues in close proximity to the active site. The third class includes buried, active site-distant, residues.

Influence of antibody binding on oxygen binding behavior of Panulirus interruptus hemocyanin

Oxygen binding behavior of monomeric subunit a and the hexameric form of this subunit of hemocyanin of Panulirus interruptus is influenced by the binding of various monoclonal antibodies. These antibodies react with other surface parts of the subunit than its second domain in which the oxygen binding site is located. The influence of three monoclonal antibodies and their antigen binding fragments (F ab) has been investigated. Two antibodies increase the oxygen affinity of monomeric hemocyanin from that observed in its low affinity T-state, while the third has little influence on this property. F ab fragments abolish almost completely the cooperativity of oxygen binding by the hexameric hemocyanin molecule. The two antibodies which increase the oxygen affinity of the monomeric molecule stabilize high-affinity states of the hexameric molecule, while the third stabilizes the low-affinity state.

Dimerization of an antigenic peptide leads to strong interaction with its antibody

A sequential epitope reacting with a monoclonal antibody against Panulirus interruptus hemocyanin was localized in the C-terminal CNBr peptide. As the antibody reacted with about equal affinity with different subunits of this and with hemocyanin from another spiny lobster, Palinurus vulgaris, the epitope was assigned to a conserved sequence region. The CNBr peptide, which was linked to another peptide via a disulfide bridge, was reduced and reoxidized. As a result, not the heterodimer but only the two disulfide-linked homodimers were formed. The dimeric C-terminal peptide had a much higher affinity for the monoclonal antibody than the monomeric peptide. This may be explained by the presence of two independent mobile interaction sites in each of the two reacting molecules.

Primary structure of hemocyanin from Palinurus vulgaris

The primary structure of hemocyanin from the spiny lobster Palinurus vulgaris was determined using a mixture of at least four slightly different subunits. Heterogeneities were observed in 32 (5%) of the positions. The amino acid sequence differs at about 20% of the positions from that of subunit a of Panulirus interruptus hemocyanin.

Comparison of three methods for competitive binding of monoclonal antibodies The localization of antigenic sites for monoclonal antibodies on Panulirus interruptus hemocyanin

The competitive binding of a panel of monoclonal antibodies against hemocyanin of Panulirus interruptus hemocyanin was investigated with three different methods. A competitive-binding immunoassay method was more successful in the determination of ternary complexes than gel electrophoresis and gel filtration. The latter two methods could only be applied with antibodies possessing a higher affinity. Eleven monoclonal antibodies were assigned to groups on the basis of their interactions with five antigenic regions.

Three-Dimensional Reconstruction of Androctonus australis Hemocyanin Labeled with a Monoclonal Fab Fragment

An immunocomplex formed by the 4 × 6 meric hemocyanin of the scorpion Androctonus australis with the monoclonal antibody L104 was studied by cryoelectron microscopy and subjected to three-dimensional (3D) reconstruction. The reconstructed particle reflects the structure of the immunocomplex in its hydrated state and is devoid of the flattening that was previously observed with a negatively stained preparation. A 3D fitting of the X-ray data of the Panulirus interruptus hemocyanin and of the Fab R19.9 to the reconstruction volume allowed the first quantitative measurement of the structural parameters of the antigen, and the localization of the epitope at the surface of subunit Aa6. The independent alignment of the Fobs and the hexamers provides a direct verification for the accuracy of the fit and allows the building of the most detailed model of a cheliceratan 4 × 6meric complex and its attached monoclonal antibodies.

Identification of two antibody-interaction sites on the surface of Panulirus interruptus hemocyanin.

Negatively stained complexes of Panulirus interruptus (spiny lobster) hemocyanin with two different monoclonal antibodies, named E and J, were studied by electron microscopy and image processing. The attachment site of the antibodies to the hexameric hemocyanin molecule was deduced from two perpendicular views of hemocyanin/antibody complexes, in which either the threefold axis or one of the twofold axes was oriented perpendicular to the supporting film. Images of complexes in these orientations were searched with reference images simulated from the known X-ray structure of P. interruptus hemocyanin. The two sites were further characterized by combining our results from electron microscopy with structural data obtained by X-ray diffraction and other methods. These two antibodies recognize different non-overlapping epitopes. The epitope for clone E is located on domain 3 at the surface of the beta barrel and consists of certain loops, which form connections between beta-strand structures. The epitope for clone J is situated on domain 1 at the surface of an alpha-helical region and consists mainly of certain alpha-helices connecting loops. The orientation of the hemocyanin hexamers in the two complexes is very different, as is demonstrated most clearly when they form chains. Clone E forms complexes with the threefold axes perpendicular to the chain direction, while for clone J the threefold axes seem to be parallel to the main direction. The angle between the Fab part of an IgG molecule and the threefold axis of the hexamer is 60 +/- 5 degrees for clone E and 35 +/- 7 degrees for clone J. This observation is clearly related to the difference in orientation of the hexamers for the two complexes.

Kinetic studies on the reactions of separated a, b and c subunits of Panulirus interruptus deoxy-hemocyanin with hydrogen peroxide.

The kinetics of the H2O2 oxidation of separated a, b and c subunits (75 kDa, 657 amino acids) of the arthropod Panulirus interruptus hemocyanin (Hc), in the deoxy Cu(I)2 state, have been studied at 25 degrees C, I = 0.100 M (NaCl). Solutions of oxyHc provide small equilibrium amounts of the deoxy reactant deoxyHc + O2<-->oxyHc (K approx. 10(5) M-1). The reaction is of interest because H2O2 is one of the few molecules which is able to access the active site and oxidise deoxyHc to the metHc Cu(II)2 state. The reaction was studied at pH values in the range 6.8-9.6. Traces of Ca2+ (and other 2+ ions) are controlled by addition of EDTA (5 mM). With or without EDTA hexamer forms are present at the lower pH values. At pH > 8.3 and with EDTA added, a and b (3% sequence difference) give monomer forms. The hexamers are however retained at the higher pH values if Ca2+ (10 mM) is added. As in the corresponding studies of deoxyHc with O2, rate constants for subunit c (42% sequence differences) show no variation with pH and the hexamer is retained over the whole pH-range explored. Rate constants for the reactions of H2O2 with the different monomer and hexamer deoxyHc forms of a, b, and c are in the range 1-75 M-1 s-1. A mild pseudo-catalase activity of metHc leading to reformation of oxyHc also contributes to the reaction.

Kinetics of the equilibration of 0 2 with Panulirus interruptus hemocyanin subunits a, b and c

The kinetics of the equilibration (25°C) of O 2 with the separated a, b and c subunits of interruptus hemocyanin (Hc) as monomer and hexamer forms, as well as the native unfractionated abc mix, have been studied at pH values in the range 6.8–9.6, I = 0.100 M (NaCl). Rate constants k on and k off defined by deoxyHc + O2 if oxyHc have been determined by temperature-jump and stopped-flow techniques, respectively. The aggregation of monomer forms of different subunits to give hexamer is favoured by low pH and the availability of Ca 2+, traces of which (or any other 2 + metals) are effectively removed by complexing with EDTA (5 mM). With or without EDTA, the hexamer form is present at the lower pH values. At the higher pH values with EDTA present a and b but hot c give monomer forms. The hexamers are however retained at the higher pH values on addition of Ca 2+ (10 mM). Cooperativity is observed for the hexamer forms at pH > 8, where the existence of relaxed (R) and tense(T) forms gives rise to sigmoidal kinetic plots in the determination of k off. Subunit c is different in that it retains its hexamer structure over the whole pH range, and does not display a Bohr effect. Native unfractionated protein is present as a hexamer mix of a, b and c in non-stoichiometric amounts, which has an enhanced Bohr effect as compared to the separated subunits.

Studies relating to the formation of semi-met Cu I Cu II panulirus interruptus haemocyanin

Reduction of Panulirus interruptus haemocyanin in the methaemocyanin CuII2 state with N2H4 and S2O42– occurs in two stages. The product of the first stage is semi-met CuICuII which has a UV/VIS spectrum approximately midway between those of met- and deoxy-haemocyanin peaks λ/nm (ε/M–1 cm–1 per subunit) at 680 (≈120) and 337 (≈2900). At 25 °C (pH 8.7) with N2H4 as reduction rate constants k1= 0.0268 M–1 s–1 and k2 < 5 × 10–4 M–1 s–1 have been obtained. Formation of semi-met (k1) and deoxy-haemoyanin (k2) at the intermediate and final stages is confirmed by reaction with O2 to yield UV/VIS spectra of met- and oxy-haemocyanin respectively. Reduction of methaemocyanin with the strong reductants eaq– and CO2.– generated by pulse radiolysis occurs at some other sites on the protein, and no reduction of the CuII2 site was observed. With the less strong reductant MV.+ derived from methyl viologen (1,1′-dimethyl-4,4′-bipyridinium dichloride) and with methaemocyanin in excess, formation of semi-methaemocyanin was observed presumably by electron transfer from the protein surface. The same behaviour is noted with the photochemically generated 5-deazaflavin radical dfl.– which does not further reduce the protein to the deoxyhaemocyanin state. With pulse-radiolysis-generated oxidising radicals N3. and (NCS)2.– deoxyhaemocyanin gives spectroscopically identifiable protein Tyr.(410 nm) and Trp.(510 nm) radicals, but no evidence for oxidation of the CuI2 site. Mechanistic features are discussed including a consideration of the extent to which a redox partner can penetrate the protein to access the active site.

Sulfhydryl groups and disulfide bridges in subunit c of Panulirus interruptus hemocyanin

Subunit c of Panulirus interruptus hemocyanin contains, after reduction, three cysteine residues. Determination of sulfhydryls in the native subunit revealed one free SH group. The cysteine-containing peptides were obtained from a pepsin digest. A disulfide bridge was established between Cys 3 and Cys 557 , while Cys 99 bears a free sulfhydryl group. These results are in agreement with expectations based on the three-dimensional structure of subunit a . The position of the disulfide bridge in subunit c is unique among arthropodan hemocyanins, and is possible responsible for the absence of cooperativity in subunit c homo-hexamers.

Crystallization properties and structure of Panulirus interruptus haemocyanin.

Electron-microscopic studies revealed that two types of subunits of Panulirus interruptus haemocyanin crystallize in different ways. Homohexamers of subunit a give close-packed two-dimensional crystals whereas homohexamers of subunit c form open two-dimensional arrays. We applied computer-image analysis to these arrays and studied the differences in crystallization properties by combining the electron-microscopic data with amino acid sequence information and the X-ray diffraction model of subunit a.

Binding of oxygen and carbon monoxide to the hemocyanin from the spiny lobster

A high precision, two-dimensional study of oxygen and carbon monoxide binding to Panulirus interruptus hemocyanin has been carried out. Global data analysis of three types of experiments, probing the molecule in its various states of CO and O 2ligation, revealed the entire hexamer to be the basic allosteric unit involved in a two-state mechnism. The co-operativity and linkage of the two ligands are presented in terms of derivative Hill plot surfaces extended along co-ordinates of CO and O 2 activities giving a detailed and comprehensive view of the binding behavior. Among the findings is an apparent high co-operativity of carbon monoxide binding at high oxygen activity. The results are discussed in view of a general mechanism for co-operative behavior found in larger hemocyanin aggregates concerning “nested” allosteric interactions.

Spectroscopic investigations of Panulirus interruptus hemocyanin in the crystalline state

Single-crystal ultraviolet spectroscopy, X-ray absorption spectroscopy and EPR measurements have been used to examine the oxidation and oxygenation state of the dinuclear copper site of several types of hemocyanin crystals. The crystals contain Panulirus interruptus hemocyanin which forms hexameric molecules with a molecular mass of approximately 470 kDa. Three types of crystals have been investigated. Type-I monoclinic crystals, which have been used for the X-ray structure determination, contain virtually only deoxyhemocyanin. Type-II monoclinic crystals, which are less well ordered than the type-I crystals, contain a mixture of deoxy, oxy and met forms. Older crystals contain relatively more methemocyanin. A third, hexagonal, crystal form is also partially oxygenated, and, like the type-II monoclinic form, subject to gradual conversion to methemocyanin.

Pseudo 2-fold symmetry in the copper-binding domain of arthropodan haemocyanins: Possible implications for the evolution of oxygen transport proteins

Investigation of the copper-binding centre of Panulirus interruptus haemocyanin led to the discovery of a pseudo 2-fold axis relating two helical pairs surrounding and co-ordinating the two copper ions. The pseudo 2-fold symmetry relating one helical pair, co-ordinating Cu-A, to the second helical pair co-ordinating Cu-B is quite precise with 31 equivalent C α atoms having a root-mean-square deviation of only 1.47 Å. The 2-fold consists of a rotation of 174.6 ° and a translation parallel to the rotation axis of 0.7 Å. After superposition of the helical pairs, the two copper ions are within 1.1 Å and the three C α atoms of the histidine ligands of Cu-A are within a root-mean-square deviation of 1.0 Å from the C α atoms of the histidine residues co-ordinating Cu-B. Of the superimposed residues, 26% are identical in sequence. These data suggest that the current oxygen-binding centre of arthropodan haemocyanins is the result of dimerization, gene duplication and gene fusion of an ancestral mono-copper-binding helical pair. This suggestion is supported by the recent discovery that in the sequence of functional domains of molluscan haemocyanins only amino acid sequence homology with the arthropodan Cu-B helical pair has been found and no evidence for similarity with a Cu-A binding helical pair was observed. This provides strong evidence that a mono-copper-binding helical pair has been the ancestor of both the arthropodan and molluscan haemocyanins. Turning to the Fe-binding helical pairs in haemerythrins, it appears that they are less similar to each other than the two Cu-binding helical pairs in arthropodan haemocyanins. Nevertheless, the Fe-B haemerythrin helical pair superimposes well onto the Cu-A helical pair of Panulirus haemocyanin. A root-mean-square deviation of 1.9 Å for 24 equivalent C α carbon atoms is obtained, while Fe-B deviates 1.4 Å from Cu-A after superposition of the helices. Moreover, the three histidine ligands of the Cu-A helical pair are equivalent with three histidine ligands of the Fe-B pair. The structural similarity and correspondence in metal-binding ligands suggests that both haemocyanins and haemerythrins have originated from an ancestral mono-metal-binding helical pair having two ligands provided by the first helix and one ligand by the second helix. The Fe in the haemoglobin family is surrounded by two helices, each helix providing a histidine that is co-ordinating or approaching the metal ion. Although the similarity of the Fe-binding helical pair in the haemoglobins with the metal-binding helical pairs in haemocyanins and haemerythrins is less convincing than within the latter two classes of oxygen-binding proteins, it might be possible that all oxygen transport proteins are descendants from a primordial “metal-binding helical pair”.

Panulirus interruptus hemocyanin. The amino acid sequence of subunit b and anomalous behaviour of subunits a and b on polyacrylamide gel electrophoresis in the presence of SDS.

The primary structure of subunit b of Panulirus interruptus hemocyanin has been derived from two digests (trypsin and CNBr) and, in some cases, with aid from the similarity with the sequence of subunit a. Differences between the amidation states of Asx and Glx residues in subunit b relative to a were investigated more thoroughly. When compared to the sequence of subunit a, 18 differences (2.7%) were found and certain heterogeneities, indicating the presence of a minor subunit b', were observed. Several differences in properties between subunits a and b, including their anomalous behaviour on SDS/polyacrylamide gel electrophoresis, could be explained by amino acid replacements.

Determination of l-lactate binding stoichiometry and differences in allosteric interactions of structurally distinct homohexamers from Panulirus interruptus hemocyanin

The role of structurally distinct subunits from the hemocyanin of Panulirus interruptus was investigated by the analysis of the oxygen-binding properties of reassembled homohexamers. homohexamers reassembled from subunits a and b exhibited cooperative oxygen binding, whereas subunit c did not. The oxygen affinity of homohexamers from subunit b and c was specifically increased by the addition of l-lactate, whereas that of subunit a was not. Both native hexamers and the homohexamers from subunit b have approximately one oxygen-linked lactate binding site per hexamer.

Panulirus interruptus hemocyanin. The elucidation of the complete amino acid sequence of subunit a.

As a final step in the elucidation of the primary structure of subunit a of Panulirus interruptus hemocyanin (657 residues, Mr 75696 excluding two copper ions and carbohydrate), the amino acid sequence of the largest fragment obtained by limited trypsinolysis was determined. The elucidation of the sequence of residues 176-657, comprising domains two and three, was mainly based on two digests, with CNBr and trypsin, respectively, from both of which a complete set of peptides was obtained. Additional sequence information was obtained from a digest with Staphylococcus aureus V8 protease and from one fragment obtained by cleaving subunit a with hydroxylamine. A block during Edman degradations indicated an Asn-Gly sequence at positions 597-598, although only aspartic acid was identified at position 597.

Complete amino-acid sequence of a functional unit from a molluscan hemocyanin (Helix pomatia).

From the beta c-hemocyanin (beta c-Hc) of the vineyard snail, Helix pomatia, the functional unit d (Mr approximately equal to 50,000-55,000) was isolated by limited proteolysis and gel chromatography. A small quantity of functional unit d was obtained intact, but the major part in the form of two peptides (Mr approximately equal to 43,000 and 10,000, respectively) connected by a disulfide bridge. After reduction and carboxymethylation, these were separated from each other and cleaved by conventional methods. The peptides were isolated by gel chromatography and HPLC, and sequenced manually or automatically. The complete sequence of Helix beta c-Hc d comprises 410 residues plus 3 residues at the N-terminus seemingly resulting from incomplete cleavage. There is apparently only one carbohydrate side-chain. Comparison of this gastropodan hemocyanin sequence to the partial sequence of a cephalopodan Hc C-terminal unit revealed sufficient identities to state that the functional units of molluscan hemocyanins have arisen by a series of gene duplications. On the other hand, there is practically no homology with arthropodan hemocyanins except for one section of 42 residues which is clearly homologous. This section corresponds to the "Copper B" site of Panulirus interruptus hemocyanin. It is also found in tyrosinases from Neurospora crassa, Streptomyces glaucescens, and mouse. In the N-terminal half of Helix beta c-Hc d there are other sections clearly homologous to the tyrosinases, but overall homology is limited. The second copper-binding site was not identified but must be completely distinct from the "Copper A" binding site of arthropodan hemocyanins. It is suggested that molluscan and arthropodan hemocyanins have evolved independently from a common ancestral mononuclear copper protein.

Primary structure of the neutral carbohydrate chains of hemocyanin from Panulirus interruptus.

The N-glycosidic carbohydrate chains of Panulirus interruptus hemocyanin, consisting of only mannose and N-acetylglucosamine residues, were liberated by hydrazinolysis of a pronase digest of the purified glycoprotein. The carbohydrate chains were fractionated by high-voltage paper electrophoresis, yielding mainly neutral oligosaccharide-alditols. These were further separated by high-performance liquid chromatography and characterized by 500-MHz 1H-NMR spectroscopy as (formula; see text).