Presence of functional domains in Limulus polyphemus hemocyanin

In an attempt to isolate structural domains of arthropod hemocyanins and possibly to investigate their functional properties, we have undertaken proteolytic digestion experiments of isolated subunits from Panulirus interruptus and Limulus polyphemus oxy-hemocyanin. Satisfactory results have been obtained using trypsin at high concentration and short digestion times. Results show that, in the case of Panulirus hemocyanin, only subunit alpha is susceptible to trypsin digestion, but that proteolytic cleavage is associated with the loss of the copper-oxygen band; on the other hand, in the case of Limulus hemocyanin, four subunits (I, II, III and IV) show a significant susceptibility to trypsin, and their fragmentation takes place with preservation of the oxygen-binding capacity. A more detailed study of the digestion products of subunit IV from Limulus hemocyanin reveals that the proteolytic fragments keep together in a single non-covalent complex. Attempts to separate the native fragments result in the precipitation of the digestion products. Subunit IV of Limulus with proteolytic cuts binds O2 and CO with the same affinity as the native subunit, suggesting that the copper site is still preserved structurally and is functionally active in a 37 kDa trypsin-resistant domain.     

Dissociation and reassembly of Limulus polyphemus hemocyanin.

Limulus polyphemus hemocyanin is a 3.3 x 10(6)-Mr protein containing 48 subunits in an assemblage of eight hexamers. The molecule can be dissociated into monomers and dimers at pH 8.9 in the presence of 0.01 M EDTA. These subunits are heterogeneous and can be separated into five zones (I--V) by DEAE-Sephadex chromatography. Reassembly experiments were carried out with varied subunit mixtures, based on different combinations of the five chromatographic zones, in order to study the structural role of the diverse subunits in the eight-hexamer molecule. The reassembly products were analysed by electron microscopy and ultracentrifugation. No structural role for zone I could be found. Zone V and possibly zone II are needed to form structures larger than hexamers. Absence of zone III causes irregular aggregation of hexamers. Zone IV and perhaps zone II are needed to make eight-hexamer molecules from four-hexamer molecules. From these results we conclude that there is a high degree of subunit specificity in the inter-subunit contacts in the native Limulus hemocyanin molecule.     

Subunit association and heterogeneity of Limulus polyphemus hemocyanin

The molecular weights of the 6S, 24S, 36S, and 60S components of Limulus polyphemus hemocyanin were determined by high speed sedimentation equilibrium to be 69 400, 856 000, 1 690 000, and 3 160 000. The behavior of this hemocyanin appears to be similar to that of other arthropod hemocyanins where the first aggregation step is the formation of a hexamer of the 6S monomer. Here the larger aggregated states (24S, 36S, and 60S) are successive dimers of an unobserved hexamer (16S). The 24S-36S-60S association was found to be heterogeneous, suggesting that 24S components of different composition may be present.     

Mass spectrometric characterization of Limulus polyphemus hemocyanin.

Electrospray ionization-mass spectrometry of ion-exchange and reverse-phase purified hemocyanin from Limulus polyphemus revealed six predominant isoforms with molecular weights ranging from 71,730 to 72,695 Da. The heaviest of these agreed closely with the molecular weight calculated for the previously determined Edman sequence with substitution of isoleucine for valine at position 9 and inclusion of three internal disulfide bonds and one copper atom. Proposed structures for the other isoforms were made on the basis of the molecular weight measurements. Reverse-phase chromatography can be used in addition to the traditional ion-exchange step to produce hemocyanin preparations of greater purity that might be valuable for further detailed investigations of the physicochemical properties of these important proteins. The results reflect yet again the value of mass spectrometry for recognizing molecular microheterogeneity in biological macromolecules and for following protein purification.     

An oxygenation-linked dye binding to Limulus polyphemus hemocyanin

The reaction of Limulus polyphemus hemocyanin with a dye, bromthymol blue, was examined by equilibrium dialysis, spectrophotometric titration and stopped-flow methods. Oxy-hemocyanin contained one binding site per hexamer unit. The dye binding was linked to oxygenation, and the affinity of the dye for the oxy form was about 10 times as high as that for the deoxy form. Conversely, the dye increased the O2 affinity of hemocyanin. Hemocyanin showed a simple hyperbolic binding curve in the bromthymol blue titration, whereas the time course of the reaction was generally biphasic. It was inferred from the kinetic analyses that the reaction proceeds in two steps. The first bimolecular step is characterized by an increase in the apparent pKa of the bound dye, while the second unimolecular step by a red shift of the absorption band of the unionized dye. The dye binding to partially oxygenated hemocyanin was examined spectrophotometrically; the fractional change in the binding was found to be ahead of the increase in the average degree of O2 saturation. It was concluded that the structural changes in hemocyanin which lead to the increased dye affinity take place at an early stage of the ligand binding sequence.     

Crystals of a functional 70,000 molecular weight subunit of hemocyanin from Limulus polyphemus

Crystals have been obtained of a subunit of Limulus polyphemus hemocyanin. The blue crystals have the symmetry of the space group R32 with hexagonal lattice parameters $\text{a = 115}\text{a}\text{?}$, $\text{c = 285}\text{A}\text{?}$. There is one 70,000 molecular weight subunit per asymmetric unit. Each subunit contains two non-heme copper atoms and can reversibly bind one oxygen molecule.     

Limulus polyphemus hemocyanin: 10 A cryo-EM structure, sequence analysis, molecular modelling and rigid-body fitting reveal the interfaces between the eight hexamers

The blue copper protein hemocyanin from the horseshoe crab Limulus polyphemus is among the largest respiratory proteins found in nature (3.5 MDa) and exhibits a highly cooperative oxygen binding. Its 48 subunits are arranged as eight hexamers (1x6mers) that form the native 8x6mer in a nested hierarchy of 2x6mers and 4x6mers. This quaternary structure is established by eight subunit types (termed I, IIA, II, IIIA, IIIB, IV, V, and VI), of which only type II has been sequenced. Crystal structures of the 1x6mer are available, but for the 8x6mer only a 40 A 3D reconstruction exists. Consequently, the structural parameters of the 8x6mer are not firmly established, and the molecular interfaces between the eight hexamers are still to be defined. This, however, is crucial for understanding how allosteric transitions are mediated between the different levels of hierarchy. Here, we show the 10 A structure (FSC(1/2-bit) criterion) of the oxygenated 8x6mer from cryo-electron microscopy (cryo-EM) and single-particle analysis. Moreover, we show its molecular model as obtained by DNA sequencing of subunits II, IIIA, IV and VI, and molecular modelling and rigid-body fitting of all subunit types. Remarkably, the latter enabled us to improve the resolution of the cryo-EM structure from 11 A to the final 10 A. The 10 A structure allows firm assessment of various structural parameters of the 8x6mer, the 4x6mer and the 2x6mer, and reveals a total of 46 inter-hexamer bridges. These group as 11 types of interface: four at the 2x6mer level (II-II, II-IV, V-VI, IV-VI), three form the 4x6mer (V-V, V-VI, VI-IIIB/IV/V), and four are required to assemble the 8x6mer (IIIA-IIIA, IIIA-IIIB, II-IV, IV-IV). The molecular model shows the amino acid residues involved, and reveals that several of the interfaces are intriguingly histidine-rich and likely to transfer allosteric signals between the different levels of the nested hierarchy.     

Phenoloxidase activity and thermostability of Cancer pagurus and Limulus polyphemus hemocyanin

The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, l-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and l-Dopa (K_m value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for l-Dopa), while k_cat is highest for catechol (2.44 min^? 1 versus 0.67 min^? 1 for l-Dopa and 0.71 min^? 1 for dopamine). On treatment with 4 mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with l-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (T_m ~ 80 °C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (T_m ~ 92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.     

Preliminary crystallographic studies of Limulus polyphemus hemocyanin subunits IIIa,IIIb and IV

Crystals of Limulus hemocyanin subunits IIIa, IIIb and IV are suitable for X-ray diffraction analysis. The three-dimensional structure of subunit IV is determined by molecular replacement and non-crystallographic symmetry averaging methods. A tentative model of subunit IIIa is obtained from a partial data set. Both structures, similar to subunit II, could provide primary structure segments suitable for oligonucleotide probe synthesis.     

Dissociation of Limulus polyphemus (horseshoe crab) hemocyanin. I. Solution X-ray scattering study in equilibrium

A solution X-ray scattering study has been performed on Limulus polyphemus (horseshoe crab) hemocyanin and its dissociated fragments at various pH values in the presence and absence of Ca2+. The scattering patterns of native hemocyanin (48-mer), the half molecule (24-mer), quarter molecule (12-mer) and monomer fraction were measured. The radii of gyration for the four molecular species were calculated from the Guinier plots to be 110.7, 91.3, 77.3, and 36.5 A, respectively. Models which yield good fits to the experimental data are presented. The models were constructed using eight, four and two spheres with a radius of 58 A, assuming the sphere to be the submultiple composed of six subunits. The radii of gyration were calculated on the basis of the model and the values found to be 106, 94 and 73 A, respectively, in good agreement with the experimental results.     

Nervous control of respiration: oxygen-sensitive elements in the prosoma of Limulus polyphemus.

1.Responses of oxygen-sensitive units in the prosomal haemal nerve of Limulus polyphemus were examined while varying the oxygen content of sea water bathing the intercoxal cuticle. 2. When exposed to high oxygen levels these units units maintained a continuous background discharge of spikes. Unit activity was inhibited when oxygen content decreased. Upon reintroduction of oxygen tonic spike discharge resumed. 3. Mechanosensitive units with receptive fields on the prosomal shield or intercoxal cuticle were also present in the haemal nerve. Neither the mechanosensitivity nor the background discharge of these units was affected by changes in oxygen content. 4. It is proposed that the oxygen-sensitive respiratory reflexes of Limulus are an adaption to existence in the tertidal zone. Published observations of the respiratory stress responses of many intertidal animals support this hypothesis.