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Short (10- to 11-mer) hydrophilic peptides based on the structure of the 55-kDa bactericidal protein (BP55, B/PI, and CAP57) from human neutrophil granules were identified from the hydropathy plot of the 456-amino-acid sequence predicted from the nucleotide sequences of cDNA clones for BP55 and B/PI. Peptides corresponding to amino acid residues 90 to 99 (peptide #90-99), 86 to 99, or 90 to 102 of BP55 were bactericidal toward 5 x 10(6) Pseudomonas aeruginosa cells at 0.6 x 10(-5) to 1.5 x 10(-5) M and killed an Escherichia coli rough strain at 3 x 10(-5) M. The #90-99 peptide with a cysteine added at the amino terminus (C#90-99) was approximately 10 times more active than #90-99, killing P. aeruginosa at 1.5 x 10(-6) M. Peptides representing amino acid residues 27 to 37, 118 to 127, and 160 to 170 and the first 10 amino acids of the signal sequence for BP55 were not bactericidal. When coupled to either keyhole limpet hemocyanin or ovalbumin protein carriers through the thiol group, the C#90-99 peptide was not diminished on a molar basis in its capacity for killing of P. aeruginosa. Two other relatively hydrophilic peptides with an added amino-terminal cysteine, peptides C#227-236 and C#418-427, were not bactericidal at 1.2 x 10(-4) M or at 100 times the effective bactericidal concentration of C#90-99. The C#90-99 peptide killed E. coli at 1.5 x 10(-5) M, or at 10 times the concentration required to kill an equal number of P. aeruginosa cells. Although Pseudomonas cepacia and Staphylococcus aureus were resistent to killing by the parent BP55 molecule, they were susceptible to the C#90-99 and #90-99 peptides in the same concentration range as was E. coli. When all peptides were compared for the ability to neutralize E. coli O55:B5 endotoxin in a Limulus amoebocyte lysate assay, the C#227-236, C#418-427, and #160-170 peptides completely inhibited gelation at a 10(-4) M concentration. All other synthetic peptides, including bactericidal peptide #90-99 and its congeners, lacked endotoxin-neutralizing activity at the highest concentration tested (4.5 x 10(-4) M). A hybrid of the C#227-236 and #90-99 peptides (CHybrid) was identical to the C#227-236 peptide component in effectiveness for carrying out endotoxin neutralization and was fivefold better than the #90-99 peptide in its capacity for killing P. aeruginosa.

In a search for adjuvants of non-bacterial origin for immunization with ganglioside, we investigated whether chemical coupling to immune stimulatory protein could increase the immunogenicity of sialoglycosphingolipid. A novel method for the linkage of glycosphingolipids, including gangliosides, to protein was established. The procedure includes lysis of the sphingoid double bond by ozone, reduction of the ozonolysis product to the aldehyde, and coupling to amino groups, either directly by reductamination, or by conjugation via a long aliphatic chain dicarboxylic acid linker. Using this method, gangliosides Gfpt1 (IV2-Fuc-, II3NeuAc-Gg4Cer), Glac2 [II3(NeuAc)2-LacCer], and Gtet1 (II3NeuAc-Gg4Cer) were coupled to keyhole limpet hemocyanin (KLH), and the immunogenicity of the conjugates was tested. Immunization of mice with the KLH-ganglioside conjugates led in each case to the formation of IgG- and IgM antibodies that recognized the underivatized gangliosides, respectively. In contrast to this, mixtures of KLH and ganglioside proved ineffective for immunization. KLH-tumor-associated ganglioside conjugates may, therefore, be considered as possible vaccines in immune therapy of cancer.

The Bohr effect describes the usually negative coupling between the binding of oxygen and the binding of protons to respiratory proteins. It was first described for hemoglobin and provides for an optimal oxygen supply of the organism under changing physiological conditions. Our measurements of both oxygen and proton binding to the 24-meric tarantula hemocyanin establish the unusual case where a respiratory protein binds protons at low degrees of oxygenation but releases protons at high degrees of oxygenation. In contrast to what is observed with hemoglobin and other respiratory proteins, this phenomenon amounts to the inversion of the Bohr effect in the course of an oxygen-binding curve at a given pH value. Therefore, protons in spider blood can act either as allosteric activators or as allosteric inhibitors of oxygen binding, depending on the degree of oxygenation of hemocyanin. These functional properties of tarantula hemocyanin, which cannot be explained by classical allosteric models, require at least four different conformational states of the subunits. Inspection of the known x-ray structures of closely related hemocyanins suggests that salt bridges between completely conserved histidine and glutamate residues located at particular intersubunit interfaces are responsible for the observed phenomena.

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.

Crustacean and cheliceratan hemocyanins (oxygen-transport proteins) and insect hexamerins (storage proteins) are homologous gene products, although the latter do not bind oxygen and do not possess the copper-binding histidines present in the hemocyanins. An alignment of 19 amino acid sequences of hemocyanin subunits and insect hexamerins was made, based on the conservation of elements of secondary structure observed in X-ray structures of two hemocyanin subunits. The alignment was analyzed using parsimony and neighbor-joining methods. Results provide strong indications for grouping together the sequences of the 2 crustacean hemocyanin subunits, the 5 cheliceratan hemocyanin subunits, and the 12 insect hexamerins. Within the insect clade, four methionine-rich proteins, four arylphorins, and two juvenile hormone-suppressible proteins from Lepidoptera, as well as two dipteran proteins, form four separate groups. In the absence of an outgroup sequence, it is not possible to present information about the ancestral state from which these proteins are derived. Although this family of proteins clearly consists of homologous gene products, there remain striking differences in gene organization and site of biosynthesis of the proteins within the cell. Because studies on 18S and 12S rRNA sequences indicate a rather close relationship between insects and crustaceans, we propose that hemocyanin is the ancestral arthropod protein and that insect hexamerins lost their copper-binding capability after divergence of the insects from the crustaceans.

A frozen-hydrated sample of Octopus vulgaris hemocyanin was imaged at 0 degree and 40 degrees tilt angle under low dose conditions by transmission electron microscopy. A three-dimensional reconstruction by the method of random conical tilt series produced a three-dimensional volume to which a D5 symmetry was applied. Examination of serial sections in the volume and surface representation at various thresholds allowed the five arches containing functional unit Ovg to be localized at the interdimeric subunit groove. In another set of experiments specific polyclonal antibodies were used to label functional units Ovb and Ove in the cylinder wall. The observation of the negatively stained immunocomplexes showed that Ovb is located in the external tiers of functional units and Ove in the internal tier. These results suggest that the direction of the polypeptide chains in the cylinder wall may be only partially antiparallel. A model of the quaternary structure is proposed with the following features: (1) the external tiers of functional units comprise four units each (Ova-d) coming from a single polypeptide chain; (2) the internal tier comprises two functional units from each polypeptide chain (Ove-f); (3) the interdimeric subunit arches connect the two copies of a single functional unit (Ovg) located in each polypeptide chain.

The cDNA coding for vitellogenin of the mosquito Aedes aegypti was cloned and sequenced. An immunological analysis of expressed deletions from the 5'-end of the vitellogenin cDNA clones using vitellogenin subunit-specific antibodies showed that the small vitellogenin subunit is located at the N terminus and the large one at the carboxy-portion of the pre-provitellogenin. The position of the cleavage between the vitellogenin subunits in the pre-provitellogenin was identified by locating the N terminus of the large subunit. The cleavage site has a consensus RXRR for the subtilisin-processing endoprotease. Mosquito vitellogenin is highly hydrophilic with 17 putative N-linked glycosylation sites and 13 potential tyrosine sulfation sites. In contrast to known invertebrate vitellogenins, mosquito vitellogenin contains three polyserine domains that are similar to those of phosvitins in vertebrate vitellogenins. These polyserine domains, originally presumed to be vertebrate-specific, have several phosphorylation consensus sites in their sequences. Unlike other known vitellogenins, mosquito vitellogenin is rich in aromatic amino acid residues, tyrosine and phenylalanine, and in this respect is similar to insect serum proteins, arylphorins. This similarity suggests that mosquito vitellogenin may supply aromatic amino acids to the cuticle of rapidly developing embryos.

Arthropod hemocyanins (Hcs) are regular assemblies of 1, 2, 4, 6 or 8 hexameric protein molecules. They transport oxygen and can bind it in a cooperative manner. The hexameric X-ray structures of Panulirus interruptus (spiny lobster) and of Limulus polyphemus (horseshoe crab) subunit II Hc were solved recently by the groups of Hol (Groningen, The Netherlands) and Magnus (Cleveland, U.S.A.). They related cooperativity to a rotational movement of a domain within a subunit and of the two trimers mutually inside the hexamer. In our study a model was derived for the structure and related to the function of the four-hexameric Hc from the tarantula Eurypelma californicum by combining data from electron microscopy and image processing, from the X-ray diffraction studies mentioned earlier and from amino acid sequence studies. Interhexameric contacts were determined at the level of secondary structure elements and in some cases of single amino acids. Loops, undefined in the X-ray structures of the hexamers, were often involved in these contacts. In one case the contact was formed between four parallel alpha-helices, two from each hexamer. Based on these findings a mechanism is proposed for the transmission of cooperativity between the hexamers, in which the concept of "helical friction" plays a key role.

A novel photo-activatable derivative of ryanodine, 9-hydroxy-21-(4-azidobenzoyloxy)-9-epiryanodine, has been synthesized and conjugated to keyhole limpet hemocyanin for the production of antibodies with high affinity and specificity to ryanodine. The anti-ryanodine antibodies reacted specifically on immunoblots with the azido-ryanodine compound covalently conjugated to bovine serum albumin. A radioimmunoassay specific for ryanodine was developed using the anti-ryanodine antibodies, and a dissociation constant for ryanodine of 1 nM was determined. Half-maximal inhibition constants (IC50) for various ryanodine derivatives were found to range between 3.2 and 200 nM. These IC50 values correlated very well with the IC50 values obtained for the compounds binding to the skeletal muscle membrane receptor. These antibodies should be useful for the characterization of the ryanodine binding site on the sarcoplasmic reticulum Ca2+ release channel.

Under physiological conditions, the oxygen-transport protein from the gastropod Megalobulimulus ovatus, an extracellular hemocyanin, is composed of 20 identical subunits organized into a cylindrical structure (M(r) 9 x 10(6); 100 S). It dissociates in the pressure range of 0.4-2.5 kbar, as observed by spectroscopic methods (light scattering and intrinsic fluorescence) and gel filtration. In contrast to what is seen with smaller proteins, especially dimers, the pressure-dissociation curves for hemocyanin show little dependence on concentration, suggesting that native hemocyanin exists as a population of molecules with different free energies of association. The pressure-induced dissociation results from an equilibrium in which each aggregate responds to pressure independently of the others and, at any given pressure, is in one of two states, whole or dissociated, which persists for long times when compared with the duration of the experiment. The subunit-subunit affinity of dissociated hemocyanin is much lower than that of associated subunits, suggesting that a conformational drift of monomers occurs. When hemocyanin undergoes dissociation in the absence of calcium and at high pH (> 7.2), a large fraction of the dissociated products changes to a conformation that generates stable intermediate states of assembly, lacking the ability to fully reassemble into decamers and didecamers. These intermediates consist primarily of dimers (M(r) 900,000), and they bind oxygen reversibly with a higher affinity than the native hemocyanin. The binding of calcium or protons changes the conformation back to the "associable" state, which finally generates the assembled structure. The dissociation process is highly reversible at low pH (6.8-6.0) or in the presence of millimolar concentrations of calcium. At pH 5.7, dissociation is negligible at pressures up to 2.5 kbar. A decrease in pH from 7.6 to 6.6 increases the half-dissociation pressure (p 1/2) by 1.3 kbar, corresponding to a stabilization of 1.35 kcal per mole of subunit. The effects of Ca2+ and H+ may mean that, in vivo, special ionic conditions or other factors are required to be present at the assembly sites of oligomeric proteins such as hemocyanin.

High-resolution x-ray absorption near edge structure spectroscopy was used to characterize the metal sites in three different cobalt-substituted derivatives of Carcinus maenas hemocyanin (Hc), including a mononuclear cobalt, a dinuclear cobalt and a copper-cobalt hybrid derivative. Co(II) model complexes with structures exemplifying octahedral, trigonal bipyramidal, pseudo-tetrahedral, and square planar geometries were also studied. The results provide structural information about the metal binding site(s) in the Co-Hcs that extend earlier results from EPR and optical spectroscopy (Bubacco et al. 1992. Biochemistry. 31: 9294-9303). Experimental spectra were compared to those calculated for atomic clusters of idealized geometry, generated using a multiple scattering approach. The energy of the dipole forbidden 1s-->3d transition and of the absorption edge in the spectra for all cobalt Hc derivatives confirmed the cobaltous oxidation state which rules out the presence of an oxygenated site. Comparisons between data and simulations showed that the mononuclear and dinuclear Co(II) derivatives, as well as the hybrid derivative, contain four-coordinate Co(II) in distorted tetrahedral sites. Although the spectra for Co(II) in dinuclear metal sites more closely resemble the simulated spectrum for a tetrahedral complex than do spectra for the mononuclear derivative, the Co(II) sites in all derivatives are very similar. The Cu K-edge high resolution x-ray absorption near edge structure spectrum of the hybrid Cu-Co-Hc resembles that of deoxy-Hc demonstrating the presence of three-coordinate Cu(I).

An immunocomplex of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis with the monoclonal Fab fragment L104 was reconstructed from electron micrographs of a negatively stained specimen, using the double-carbon-layer technique. The resulting structure enables a clear visualization of the Fab fragments bound to the four copies of the Aa6 subunit and directly confirms a previous localization of the L104 epitope deduced from two-dimensional image processing. Despite a strong flattening effect produced by the negative-staining technique the orientations of the Fab fragments are well characterized. Moreover, the observation of a central hole within the elbow bends of the Fab fragments provides information about the disposition of the Fabs around their main axis.

1. The native Rapana thomasiana grosse hemocyanin is dissociated under mild conditions and fractionated into two dissociation products, RHSS1 and RHSS2, with an apparent molecular mass of approximately 250 and approximately 450 kDa, respectively. The two species are present in approximately equivalent amounts. SDS-PAGE analysis reveals that the latter component is a dimer of approximately 250 kDa polypeptide chains. 2. The amino acid compositions, as well as some spectroscopic properties of RHSS1, are very similar to those of RHSS2. After dissociation under mild conditions of the native hemocyanin both species preserve their capability of binding reversibly molecular oxygen. 3. RHSS1 and RHSS2 are sequenced directly from the amino-terminus for 15 and 20 steps, respectively. These parts of the two polypeptide chains are highly homologous but with microheterogeneity associated with some positions. They also exhibit high homology with the N-terminal region of subunits or functional domains of other gastropod Hcs.

The direction of the polypeptide chains and the location of the functional units in Octopus vulgaris hemocyanin were studied by various methods. Monoclonal antibodies specific for the Ovc (clone Ov409) and Ovg (clone Ov315) functional units produced immunocomplex strings which were examined in the electron microscope. In both cases the immunocomplexes contained more than two hemocyanin molecules in their side view, demonstrating that in the whole hemocyanin neighboring polypeptide chains run in antiparallel directions. The interhemocyanin distances in the immunocomplexes also indicated that Ovg is located inside the cylinder, while Ovc is located in the external layers of functional units. In addition, the fact that the binding point of the Fab arm to the hemocyanin molecule was occasionally visible confirmed the external location of functional unit Ovc. Image processing of the whole hemocyanin cross-linked with dimethyl suberimidate showed that the end-on view is not a perfect cylinder but a regular pentahedron and that the five-arch collar is probably composed of five pairs of functional unit Ovg located inside the cylinder. The accessibility of cross-linked hemocyanin to functional unit-specific polyclonal antibodies, studied in immunoelectrophoresis, showed that Ovb and Ove are highly accessible, while Ovd, Ovf, and Ovg are not. The low accessibility of Ovd may be at least partially explained by its high sugar content which could hamper the accessibility of the antibody to the antigen.

Amino-idazoxan coupled to hemocyanine was used to raise anti-idazoxan antibodies in the rabbit. The antibodies were affinity purified with an amino-idazoxan affinity column. Binding studies with [3H]idazoxan showed a dissociation constant of 2.2 +/- 1.4 nM. The specificity spectrum of these antibodies indicates that the imidazoline part of idazoxan is more important for recognition than the benzodioxan ring as imidazoline substances (clonidine, cirazoline) are powerful competitors of [3H]idazoxan binding on the antibodies. Catecholamines or imidazoles were unable to displace [3H]idazoxan from the antibodies. These anti-idazoxan antibodies present specificity similarities with the imidazoline receptor as did our previously obtained anti-clonidine antibodies. Affinity-purified antibodies represent useful tools for studying the imidazoline receptors particularly with an anti-idiotypic approach.

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.

The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 A. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 A between both copper ions in the oxygen-binding site. Each copper has approximate trigonal planar coordination by three histidine N epsilon atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis-peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the N delta atom of the copper B ligand His 324; (2) localization of a chloride-binding site in the interface between the first and second domain; (3) localization of a putative calcium-binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5 degrees with respect to the other two domains, whereas within the hexamer the major movement is a 3.1 degrees rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.

To examine the effect of interactions between the hexameric units in the dodecameric hemocyanin, crayfish (Procambarus clarki) hemocyanin was partially dissociated. Gel-filtration yielded fractions containing the undissociated dodecamer and a mixture of the hexamer and heptamer (referred to as 'half-dodecamer'). Their O2 equilibria were compared and were analyzed by curve fitting of cooperativity models. The partial dissociation of the dodecamer significantly lowered the cooperativity but little affected the O2 affinities. The O2 equilibrium of the half-dodecamer could be described by the two-state allosteric model of Monod-Wyman-Changeux on the assumption that the cooperative unit is composed of six subunits. We tested the applicability of allosteric models for the dodecamer that deal with the interactions between the hexamers. To reduce the arbitrariness of the fitting, we presumed that some parameters are equal to the two-state model parameters for the half-dodecamer. It was found that the 'nesting model', which assumes allosteric equilibria at two different hierarchical levels, could be applied successfully to the dodecamer.

Many metalloenzymes exhibit distinctive spectral features that are now becoming well understood. These reflect active site electronic structures that can make significant contributions to catalysis. Copper proteins provide well-characterized examples in which the unusual electronic structures of their active sites contribute to rapid, long-range electron transfer reactivity, oxygen binding and activation, and the multielectron reduction of dioxygen to water.

The kinetics of the equilibration (25 degrees C) of O2 with the separated a, b and c subunits of Panulirus 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 kon and koff defined by deoxyHc + O2 <=> 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 Ca2+, 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 not c give monomer forms. The hexamers are however retained at the higher pH values on addition of Ca2+ (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 koff. 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.

In this report, we have described methods to improve the efficiency of coupling synthetic peptides to keyhole limpet hemocyanin (KLH) and for the analysis of the composition of the resulting peptide-protein conjugates. KLH was first dissolved in buffers containing 3 M guanidine hydrochloride to maintain solubility and derivatized with either of two water soluble, heterobifunctional crosslinkers, m-maleimido-benzoyl-N-hydroxy-sulfosuccinimide ester (SMBS), or sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC) (300:1 molar excess over KLH). Synthetic peptides containing an amino terminal cysteine were then crosslinked to the modified KLH via sulfhydryl reaction with the crosslinker maleimide groups. Following dialysis to remove free peptide, the amino acid composition of the conjugate was determined. The molar ratio of peptide to protein within the conjugate was obtained by comparing the conjugate composition with that of both the KLH and peptide analyzed separately, and by a multiple regression, least squares analysis of the data. This method is generally applicable to the analysis of the molar ratios of protein-protein conjugates of unknown sequence or composition, and requires only the prior determination of the experimental amino acid composition of each component of the conjugate separately.

1. The hemocyanin of the Californian whelk, Kelletia kelleti, investigated at pH and ionic conditions close to physiological, has a molecular weight close to 9.0 x 10(6) and a sedimentation constant of 114S, characteristic of the di-decameric structure of molluscan hemocyanins. Light-scattering measurements at pH 8.0, 0.05 M Mg2+, 0.01 M Ca2+ gave a molecular weight of 9.0 +/- 0.6 x 10(6), and scanning transmission electron microscopy produced nearly the same particle mass of 9.22 +/- 0.50 x 10(6) daltons (Da). 2. Light-scattering measurements on the fully dissociated monomers in the presence of 8.0 M urea and at pHs 10.6 and 11.0 gave molecular weights of 4.50 x 10(5)-4.91 x 10(5), that are close to one-twentieth of the mass of the parent di-decameric hemocyanin assembly. 3. Changes in pH produced a bell-shaped molecular weight profile, with molecular weights close to 9.0 x 10(6) in the pH region of about 5.5-8.0, and progressive dissociation to 4.5 x 10(5) Da monomers in the region below pH 4.0 and above pH 9.0 or 10, depending on the absence or presence of stabilizing Mg2+ ions (0.01 M). 4. In the absence of divalent ions some aggregation of hemocyanin was found at pHs close to 5.0, with observed molecular weights above 10 x 10(6) (investigated at a hemocyanin concentration of 0.10 g/l). The early studies of Condie and Langer (Science 144, 1138-1140, 1964) had shown that Kelletia kelleti hemocynanin aggregates at acidic pHs close to the isoelectric point, forming linear polymers of the hemocyanin di-decamers.(ABSTRACT TRUNCATED AT 250 WORDS)

A binuclear cobalt derivative of arthropod hemocyanin (Hc) has been prepared by the reaction of apo-Hc with Co(II) in the presence of thiocyanate. The crude product of the reaction contains specifically and adventitiously bound metal, the latter being removable by EDTA treatment. The specifically bound Co(II) constitutes a binuclear metal center that exhibits optical and CD spectra typical in their absorption maxima and extinction coefficients of Co(II) complexes with near-tetrahedral geometry. The EPR spectrum of the binuclear Co(II) derivative contains a resonance at g approximately 13, which is characteristic of integer spin systems and indicates coupled metal ions; the excess Co(II) bound to crude products exhibits an EPR signal at g approximately 4. The time course of derivative formation was followed by EPR, optical and atomic absorption techniques, and by fluorimetry. The intensity of the optical absorption in the visible region due to Co(II) increases with increasing stoichiometry of specifically bound metal [up to 2 Co(II) per protein monomer], but the intensity of the Co(II) EPR signal increases only during the formation of a mononuclear derivative. As the reaction proceeds over approximately 100 h to the formation of the binuclear derivative, the EPR signal intensity decreases to 10% of the value expected for 2 mol of EPR-active Co(II)/mol of protein. The binuclear cobalt derivative cannot be reconstituted to native Hc with Cu(I), indicating the stable loading of Co(II) in the active site. EPR and optical spectroscopic evidence is presented showing that the binuclear derivative does not bind oxygen.

Electron-transfer reactions of myoglobin, hemocyanin and hemerythrin with the inorganic complexes [Fe(CN)6]3- (oxidant) and [Co(sep)]2+ (reductant) are considered. Rate constants kFe (25 degrees C) have been determined for the [Fe(CN)6]3- (410 mV) oxidation of horse deoxyMb, I = 0.100 M (NaCl). From the decrease in kFe over the range pH 5.5 to 9.0 a pKa of less than 6.2 is obtained, most likely due to the involvement of the heme propionate(s). At the higher pH values the rate constant is 1.2 x 10(6) M-1 s-1. Rate constants kCo (25 degrees C) for the [Co(sep)]2+ (-260 mV) reduction of metMb are also pH-dependent, pKa = 8.82, corresponding to acid dissociation of the H2O axially coordinated to the Fe(III). The rate constant for the aqua-met form is 2.8 x 10(3) M-1 s-1 at pH values less than 7.0. In contrast, no reaction is observed for the deoxy and met forms of P. interruptus hemocyanin monomer subunit a with the same two complexes (k less than 10(2) M-1 s-1). Comparisons are made with rate constants for hemerythrin, also as the monomer, which have been determined previously. Rate constants for the reactions of deoxy forms with the neutral small molecules, here O2 and H2O2, are also considered. Whereas the reactions of [Fe(CN)6]3- and [Co(sep)]2+ are at the protein surface, those of O2 and H2O2 are at the active site. Hemocyanin with the more buried (approximately 20 A) active site compared with myoglobin (3.8 A) and hemerythrin (6.3 A), does not readily undergo electron transfer with reagents at the surface. However, with the small molecules O2 and H2O2 penetration of the surrounding peptide occurs, with reaction at the active site. Rate constants for the three proteins are now of similar magnitude, and in the range (2.3-7.8) x 10(7) M-1 s-1 for O2, and 10.9 to 3600 M-1 s-1 for H2O2.

Extended Huckel theory calculations have been carried out on a model of the oxyhemocyanin active site that includes six imidazoles, the two copper cations, and a dioxygen molecule. The results obtained for the very likely mu-eta 2:eta 2 arrangement of the dioxygen molecule show that the most favorable orientation of O2 is such that the two long Cu-N coordination bonds are perpendicular to the plane formed by the two metal atoms and O2. This arrangement leads to pentacoordinated coppers with a distorted square pyramidal geometry. The molecular electrostatic potential maps of the complexes exhibit a potential well located close to the peroxo anion midbond. The dependence of the energy and of the molecular electrostatic maps on the precise orientation and location of the imidazole rings has been investigated. These results, which show the important role played by the third remote imidazole ligand, are discussed in relation with the first step of tyrosinase-mediated phenol oxidation.

The amino acid sequence of the hemocyanin subunit c from the spiny lobster, Panulirus interruptus, has been determined. The elucidation was mainly based on three digests, with CNBr, trypsin and endoproteinase Glu-C, respectively. Additional evidence was obtained by sequencing of peptides from an endoproteinase Lys-C digest. Subunit c is a polypeptide with 661 amino acid residues and with a carbohydrate group attached to residue 476 in the third domain. No heterogeneity was observed. The degree of identity with subunit a is 59%. Some differences with subunit a are an N-terminal extension of six residues, a one-residue C-terminal extension, and a three-residue deletion. Furthermore, carbohydrate attachment is in a different position, as are most half-cystine residues. Limited trypsinolysis resulted in cleavage at the same site as in subunits a and b.

Monoclonal antibodies (mAbs) that react with soluble mercuric ions have been produced by injection of BALB/c mice with a hapten-carrier complex designed to maximize exposure of the metal to the immune system. Three hybridomas producing antibodies that reacted with bovine serum albumin (BSA)-glutathione-HgCl, but not with BSA-glutathione, were isolated from the spleen of a mouse given multiple injections with glutathione-HgCl conjugated to keyhole limpet hemocyanin. Stable subclones were established from two of these antibodies, designated mAb 4A10 and mAb 1F10. The binding of both antibodies to immobilized BSA-glutathione-HgCl was inhibited by soluble HgCl2, and dissociation constants for mercuric chloride binding were 2.3 and 3.7 nM for mAbs 4A10 and 1F10, respectively. Both antibodies bound mercuric acetate with similar affinities, demonstrating that the antibodies were capable of binding to mercuric ions in the presence of a different counterion than the one used in the immunogen. Reactions were not observed with other metal cations by either antibody. These data demonstrate the successful induction of antibodies that react very specifically with mercuric ions in solution regardless of the presence of a carrier.

The binding of various ligand molecules to the binuclear Cu(I) site of deoxy-hemocyanin has been investigated through the changes produced in the aromatic region of the circular dichroism spectrum of the protein, where a cluster of tryptophan residues located in the vicinity of copper site undergo conformational reorientations in the presence of exogenous ligands coordinated to the metal. In agreement with expectations, the binuclear site of arthropod hemocyanin is severely hindered to the access of exogenous ligands except for very small molecules like CO, O2 or CN- while for mollusc proteins ligands such as thiourea and 2-mercaptoethanol bind easily to the Cu(I) sites. However, the access of the ligand becomes progressively hindered and eventually prevented as the size of substituents on the ligand increases.

The Greek key beta-barrel topology is a folding motif observed in many proteins of widespread evolutionary origin. The arthropodan hemocyanins also have such a Greek key beta-barrel, which forms the core of the third domain of this protein. The hemocyanin beta-barrel was found to be structurally very similar to the beta-barrels of the immunoglobulin domains, Cu,Zn-superoxide dismutase and the chromophore carrying antitumor proteins. The structural similarity within this group of protein families is not accompanied by an evolutionary or functional relationship. It is therefore possible to study structure-sequence relations without bias from nonstructural constraints. The present study reports a conserved pattern of features in these Greek key beta-barrels that is strongly suggestive of a folding nucleation site. This proposed nucleation site, which we call a "beta-zipper," shows a pattern of well-conserved, large hydrophobic residues on two sequential beta-strands joined by a short loop. Each beta-zipper strand is near the center of one of the beta-sheets, so that the two strands face each other from opposite sides of the barrel and interact through their hydrophobic side chains, rather than forming a hydrogen-bonded beta-hairpin. Other protein families with Greek key beta-barrels that do not as strongly resemble the immunoglobulin fold--such as the azurins, plastocyanins, crystallins, and prealbumins--also contain the beta-zipper pattern, which might therefore be a universal feature of Greek key beta-barrel proteins.

The combination of scanning transmission electron microscopy (STEM) and parallel-detection energy-loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze-drying in situ, samples were re-cooled to liquid nitrogen temperature and low-dose (about 10(3) e/nm2) digital dark-field images were obtained with single-electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of +/- 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10-nm length of single-stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of +/- 25 atoms. Near single-atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second-difference acquisition technique and multiple least-squares fitting of reference spectra. Moreover, an extremely high electron dose (about 10(10) e/nm2) is required resulting in mass loss that may be attributable to 'knock-on' radiation damage.

Hemocyanin has been used as a protein marker for Crustacea speciation. The specificity of the hemocyanin fractions of two Idotea species has been confirmed by mono and bidirection electrophoresis. Four protein fractions have been found in the two Idotea species. However, the molecular weight of each Idotea chelipes fraction is slightly higher. The two fractions of high molecular weight are strongly associated. We assumed a molecular weight of 240,000 for I. chelipes and 230,000 for I. balthica basteri. The two other protein fractions are well separated and have a weight of 130,000 and 77,000 in I. chelipes; 120,000 and 75,000 for I. b. basteri. In contrast, the subspeciation of I. chelipes has not been clearly demonstrated by electrophoresis of the hemocyanin.

Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1 x 6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10 x 12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a very minor and a third immunologically separated component remains unclear. A novel 2 x 6 arylphorin particle was detected and isolated. It comprises less than 10% of the total arylphorin material and shows a long, narrow interhexamer bridge in the electron microscope. An arylphorin dissociation intermediate identified as a trimer (1/2 x 6) was isolated; its possible quaternary structure is discussed on the basis of electron micrographs. The epitope of monoclonal antibody Ec-7 directed against tarantula (Eurypelma californicum) hemocyanin subunit d and also reactive to Calliphora arylphorin was traced to a highly conserved peptide of 27 amino acids localized in the center of the protein. The primary structure of Calliphora arylphorin as published in our preceding paper (Naumann and Scheller 1991) is compared in detail to the sequences of spider and spiny lobster hemocyanin. This revealed a basic framework of 103 strictly conserved amino acids. Isofunctional exchanges are proposed for another 76 positions. On the basis of these similarities, and the published three-dimensional model of spiny lobster hemocyanin, a detailed model of the quaternary structure of Calliphora arylphorin is presented. A second larval storage protein previously termed protein II was purified from Calliphora hemolymph. It was demonstrated to be a 500 kDa hexamer of 83 kDa subunits. In the electron microscope it shows a cubic view 9 nm in length with a large central hole and a rectangular view (9 x 10 nm) with a large central cavity. A morphologically very similar hemolymph protein was detected in Drosophila melanogaster larvae. From its structural appearance it is uncertain whether protein II belongs to the hemocyanin superfamily or not.

A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.

Haemocyanin from the lobster Palinurus elephas has been shown to change in its subunit composition according to the time of year. In contrast, in Palinurus mauritanicus, a lobster living at greater depth, no seasonal changes in subunit composition have been observed. The results obtained from a set of experiments performed on some Palinurus mauritanicus acclimatized in an aquarium have clearly indicated that modifications of haemocyanin subunit composition may be involved in the adaptation of arthropods to environmental change.

The kinetics of association of Octopus dofleini hemocyanin subunits to form the native decameric molecule have been studied with a combination of sedimentation, light scattering and electron microscopy. The reaction, initiated by addition of magnesium, is relatively slow, requiring hours to reach completion, with monomer and decamer as predominant molecular species throughout. Analysis of the light-scattering data, including stopped-flow studies, reveals an initial lag period in the reaction, followed by a second-order process that is rate limiting. The lag period depends on both protein and magnesium ion concentration. Electron microscope studies reveal intermediates in the process, and support a model of assembly in which nucleation begins at the dimer level. Theoretical models for the process are compared.

The stabilizing influence of Ca2+, Mg2+, Ba2+ and Na+ on the di-decameric structure of the hemocyanin of the bivalve, Yoldia limatula has been investigated by light-scattering molecular weight measurements and by analytical ultracentrifugation. The molecular weight (Mw) data, examined as a function of decreasing divalent ion and sodium ion concentrations at pH 8.0 and at a constant hemocyanin concentration of 0.10 g.l-1, show biphasic transition profiles, with a sharp initial decline in Mw as the concentration of the stabilizing cations is reduced. The analysis of the molecular weight data is best described in terms of the four-species, di-decamer-decamer-dimer-monomer scheme of association-dissociation equilibria. About 25 to 35 bound divalent ions and about 10 bound Na+ ions per half-molecule or decamer are required in order to account for the initial step of the observed transitions. The subsequent transitions representing the decamer to dimer and the dimer to monomer steps of the reaction account for the additional binding of three to four and two to four cations per dimer and per monomer, respectively. The relatively large number of divalent ions per decamer suggests strong ionic stabilization of the decamer to decamer contacts within the parent di-decameric assembly of Yoldia hemocyanin. This is consistent with earlier observations showing relatively few hydrophobic groups at the decamer to decamer contact areas.

1. The hemocyanins of the Muricidae and Fasciolariidae families of marine gastropods: Chicoreus florifer dilectus, Muricanthus fulvescens, Urosalpinx cinerea, Fasciolaria lilium hunteria, and Pleuroploca gigantea were investigated by sedimentation velocity, scanning transmission electron microscopy, light-scattering, and other physical techniques. 2. The hemocyanins of these species are characterized by sedimentation coefficients close to 100 S and molecular weights of 8.2 x 10(6)-9.0 x 10(6). 3. The hemocyanins have di-decameric structures, with tail-to-tail arrangement of the decameric halves of the cylindrical particles. Only the hemocyanin of U. cinerea was found to contain about 30% higher, tri-, and tetra-decameric particles, with one or two decameric units added in a tail-to-head manner to a central di-decameric particle of the Mellema and Klug tail-to-tail arrangement. 4. The influence of pH, and the urea and Hofmeister salt series of reagents on the subunit structure and denaturation of P. gigantea hemocyanin were also investigated.

1. The hemocyanins of the Fissurellidae, Naticidae and Melongenidae families of marine gastropods as well as some other molluscs including some members of the Opistobranchia and Bivalvia groups have hemocyanins which exist in solution as tri-decameric and mixed, multi-decameric aggregates characterized by sedimentation coefficients close to 100 S, 130 S, 150 S, 170 S and 200 S to 230 S. 2. The particle masses of the molluscan hemocyanins appear to be integral multiples close to 4.4 x 10(6) daltons. Thus, particle mass values of 4.47 x 10(6), 8.67 x 10(6) and 13.40 x 10(6) daltons were obtained for representative decameric, di-decameric, and tri-decameric components of Stenoplax conspicua, Fasciolaria tulipa and Euspira (Lunatia) heros hemocyanins. For Busycon contrarium, a gastropod with a mixed multidecameric hemocyanin, scanning transmission electron microscopic (STEM) measurements gave particle masses ranging from 8.89 x 10(6) and 13.20 x 10(6) for the di- and tri-decameric components to 38.87 x 10(6) and 43.40 x 10(6) daltons for highest nano- and deca-decameric aggregates. 3. The electron microscopic images of both uranyl acetate-stained and unstained specimens of hemocyanin aggregates indicate a non-random mode of assembly of the multi-decameric particles. This is most apparent from the electron micrographs of the moon snail hemocyanins. The tri-decameric and tetra-decameric particles seem to be assembled from a single di-decameric unit of the Mellema and Klug arrangement, with the collar ends facing outward, to which decameric units have been added from one or both ends, in a unidirectional tail-to-head to tail-to-collar manner. Consequently, all the aggregates including the higher, Melongenidae polymers have the appearance of closed cylinders terminating with the collar ends. 4. The radial distribution of the end-on views of the hemocyanin of the moon-snail Calinatioina oldroydii, show that the radial mass drops to zero at the center of the cylindrical particles consisting of one, two, or three decamers. This suggests that no caps are present at the ends of the hemocyanin particles which would inhibit or terminate their linear assembly. 5. The light-scattering behavior of B. contrarium and Marisa cornarietis hemocyanins examined as a function of increasing reagent concentration using the hydrophobic urea and Hofmeister salt series of reagents, show distinct aggregation and increase in molecular weights at low concentrations of reagent. Together with the stabilizing influence of Mg2+ and Ca2+ ions, this suggests polar and ionic stabilization of the inter-decameric contacts between the central di-decamers and the added decameric units of the higher aggregates of molluscan hemocyanins.(ABSTRACT TRUNCATED AT 400 WORDS)

In the haemolymph of Oniscidea haemocyanin exists mainly in the form of hexamer 16S (90%); the 5S and 24S components are present in small amounts. Alkaline dissociation of the examined 16S haemocyanins is dependent on pH and divalent cations (Ca2+,Mg2+). The 16S component consists of 23-24.5% acidic amino acids, 15-18% basic amino acids and small quantities of tryptophan and cysteine.

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.

In order to determine the reactivity on hemocyanin from Androctonus australis, the reaction of superoxide anion has been investigated using pulse radiolysis. The kinetics of O2- decays have been studied in aqueous buffered media at various basic pH (8, 8.5 and 9), first in the absence and then in the presence of hemocyanin (in oxygenated solutions containing formate anion 0.16 mol.l-1). We have shown that, in the presence of hemocyanin, O2- decay is a first-order process whose apparent rate constant is proportional to protein concentration (10(-7) to 10(-6) mol.l-1) and pH independent between 8 to 9. A second-order rate constant of 3.5 +/- 0.1.10(7) mol-1.l.s-1, has been deduced for the catalytic rate constant of hemocyanin with O2-. Meanwhile, this activity is smaller than that described for free copper, eukaryotic Cu-Zn-SOD or some copper chelates. We have verified that apohemocyanin--the copper deprived protein--does not exhibit such an activity vs. SOD (superoxide dismutase).

The complete amino-acid sequence of subunit a of the hemocyanin of the tarantula Eurypelma californicum was determined by manual sequencing. By limited chymotrypsinolysis, subunit a is split into two fragments of 25 kDa and 40 kDa, respectively, only one single peptide bond being attacked. The whole chain contains 15 methionine residues, after cyanogen bromide cleavage, 15 peptides were identified indicating that one residue (Met85) was not split by the cyanogen bromide reaction. For subcleavages, trypsin, chymotrypsin, Staphylococcus aureus proteinase, and Astacus fluviatilis proteinase were employed. The total chain length comprises 627 amino-acid residues, carbohydrate side chains were not found.

Image processing of molecule electron microscopic views allows the observation of structural details which are randomly destroyed in a fraction of the images. The computation of average images reinforces significant details and weakens random variations provided that the image set is homogeneous. Before the computation of the average images the selection of homogeneous image population was carried out by the multiple correspondence analysis method. Two biological systems were used to demonstrate how much this method can help to understand protein-protein interactions: the topological localization of epitopes on a subunit of hemocyanin, and the intramolecular localization of proteases within human alpha 2-macroglobulin.

1. The hemocyanins of the Naticidae family, E. heros, N. duplicata, P. draconis, P. lewisii and C. oldroydii were investigated by sedimentation velocity and scanning transmission electron microscopy. 2. At pH 8.0, 0.05 M Mg2+ E. heros hemocyanin is found to be predominantly in the tri-decameric state with a sedimentation coefficient (So20,w) of 131.3 (+/- 0.6) S. While the hemocyanin of N. duplicata is also mainly in the 130 S form, the hemocyanin of C. oldroydii is largely in the di-decameric form with a sedimentation coefficient close to 100 S. Other Naticidae hemocyanins, those of P. lewisii and P. draconis, have mixtures of the 100 S and 130 S di- and tri-decamers, and minor amounts of 150 S and faster sedimenting components. 3. The average particle masses based on STEM measurements are 8.85 x 10(6), 1303 x 10(6), and 17.1 x 10(6) da for the di-, tri-, and tetra-decameric assemblies of hemocyanin. 4. The subunit mol. wts of C. oldroydii hemocyanin and the published values for E. heros hemocyanin at alkaline pHs and in the presence of 8.0 M urea range from 4.2 x 10(5) to 4.8 x 10(5), suggesting the same decameric organization of the sub-assemblies of the Naticidae hemocyanins as for other molluscan hemocyanins. 5. The appearance of the larger hemocyanin particles in the electron micrographs support the hypothesis for their assembly that was based on similar studies of the hemocyanins of the Melongenidae family. According to this scheme the formation of higher aggregates is accomplished by the tail-to-head addition of each decameric unit to a central di-decamer which itself has the tail-to-tail Mellema and Klug arrangement of decamers. In this model all the higher aggregates terminate from either end with the same "collar" ends.

1. The hemocyanin of the freshwater snail, Marisa cornuarietis exists predominantly as a di-decamer with the approximate mol. wt of 8.5 x 10(6) and a sedimentation coefficient of 100 S. Sedimentation and scanning transmission electron microscopy experiments indicate that about 15-20% of the hemocyanin forms tri-decameric and possibly higher aggregates with mol. wts of 12.5 x 10(6) and 130 S. 2. The fully dissociated subunits in 8.0 M urea and 6.0 M GdmCl have mol. wts of 4.1 to 4.7 x 10(5) which is close to one-twentieth of the major di-decameric component of the native hemocyanin. 3. Subunit dissociation by the urea series and the Hofmeister salt series of reagents suggests hydrophobic stabilization of the decamers or half-molecules of the parent hemocyanin. As with the other molluscan hemocyanins the order of effectiveness of the ureas as dissociating agents shows increased efficacy with increasing hydrophobicity or chain-length of the urea substituents. 4. Denaturation of the hemocyanin subunits by the ureas and Hofmeister salt series, investigated by circular dichroism measurements, essentially follow the same trend in effectiveness as observed by changes in subunit dissociation followed by light-scattering mol. wt measurements. 5. The observed denaturation transitions are shifted to much higher ranges of reagent concentration than the concentrations required for the dissociation of the hemocyanin subunits.

Limulus hemocyanin is a 48-subunit complex that is composed of eight immunochemically distinct subunits.Conditions were established that allowed for comparison of the structural and functional properties of the native molecule with those of its 24-subunit and 12-subunit dissociation products by analytical ultracentrifugation, functional analysis,stopped-flow light scattering, and circular dichroism spectroscopy.The 48-subunit complex was found to be specifically stabilized by calcium ions. When 48-subunit molecules at pH7 were rapidly mixed with a pH 9 buffer containing a calcium chelator, the dissociation to monomers was complete within minutes. The rate is dependent upon the concentration and Ca2+ affinity of the chelator, consistent with the formation of a transient chelator-calcium ion-protein complex during the dissociation process. Dissociation to the level of 24-subunit molecules occurs much more slowly than does the dissociation of 24-subunit molecules to monomers. The dissociation to monomers, from 48-, 24-, or 12-subunit aggregates, is a highly cooperative process, and the corresponding reassembly reactions show appreciable hysteresis. In contrast, the reversible dimerization of dodecamers appears to be a rapidly equilibrating system. The varied aggregation states of Limulus hemocyanin are characterized by different circular dichroism spectra. These are, however, not unambiguously associated with the aggregation-state changes in that the CD spectra were found to be sensitive to pH under conditions where the aggregation state was unaltered. The 24-subunit complex of Limulus hemocyanin was found to be similar in its functional properties to the native 48-subunit aggregate. No unique functional properties thus appear to be associated with the 48-subunit ensemble that is found only in horseshoe crabs.Upon dissociation to the level of dodecamers, the reverse Bohr effect, cooperatively, and modulation of oxygen affinity by NaCl are still present although diminished in magnitude.Taken together, these studies clearly indicate that the interactions that stabilize dodecamers are different in pH and ion sensitivity from the interactions that are involved in formation of the 24-subunit complex from dodecamers and that these in turn differ from the interactions that stabilize the 48-subunit complex. These results are consistent with other lines of evidence that indicate that interactions at these different levels are dominated by structurally and functionally distinct types of subunits.