Sequencing of the Lumbricus terrestris genome reveals degeneracy in its erythrocruorin genes.

The erythrocruorin of Lumbricus terrestris (LtEc) is a relatively large macromolecular assembly that consists of at least four different hemoglobin subunits (A, B, C, and D) and four linker subunits (L1, L2, L3, and L4). The complexity and stability of this large structure make LtEc an attractive hemoglobin-based oxygen carrier that could potentially be used as a substitute for donated red blood cells. However, the sequences of the LtEc subunit sequences must be determined before a scalable recombinant expression platform can be developed. The goal of this study was to sequence the L. terrestris genome to identify the complete sequences of the LtEc subunit genes. Our results revealed multiple homologous genes for each subunit (e.g., two homologous A globin genes; A1 and A2), with the exception of the L4 linker. Some of the homologous genes encoded identical peptide sequences (C1 and C2, L1a and L1b), while cDNA and mass spectrometry experiments revealed that some of the homologs are not expressed (e.g., A2). In contrast, multiple sequences for the B, D, L2, and L4 subunits were detected in LtEc samples. These observations reveal novel degeneracy in LtEc and other annelids, along with some new revisions to its previously published peptide sequences.

Increasing the stability of Lumbricus terrestris erythrocruorin via poly(acrylic acid) conjugation.

Since donated red blood cells must be constantly refrigerated, they are often unavailable in remote areas and battlefields. The goal of this study was to synthesize a highly stable blood substitute that does not require refrigeration. Specifically, the extracellular haemoglobin (a.k.a. erythrocruorin, Ec) of the earthworm Lumbricus terrestris erythrocruororin (LtEc) was cross-linked with poly(acrylic acid) (PAA) and ethylene diamine (EDA). PAGE analysis of the LtEc nanoparticles reveals cross-linking between subunits, while dynamic light scattering and scanning electron microscopy show that cross-linking significantly increases the size of the LtEc nanoparticles (164 ± 13.9 nm). Cross-linking also significantly increased the thermal stability of the LtEc nanoparticles by 10 °C (Tm = 72 ± 0.84 °C) relative to native LtEc (Tm = 62 ± 0.6 °C). In addition, while native LtEc rapidly dissociates at pH 9, the LtEc nanoparticles resist subunit dissociation up to pH 10. The oxygen affinity of the LtEc nanoparticles (P50 = 6.85 ± 0.13 mm Hg) is much higher than native LtEc (P50 = 26.67 ± 0.4 mm Hg), but the cooperativity (n = 2.43 ± 0.12) is not affected. Altogether, these results show that cross-linking LtEc with PAA and EDA provides a potential blood substitute with increased stability and oxygen affinity.

Dynamic properties of monomeric insect erythrocruorin III from Chironomus thummi-thummi: relationships between structural flexibility and functional complexity.

We have investigated the kinetics of geminate carbon monoxide binding to the monomeric component III of Chironomus thummi-thummi erythrocruorin, a protein that undergoes pH-induced conformational changes linked to a pronounced Bohr effect. Measurements were performed from cryogenic temperatures to room temperature in 75% glycerol and either 0.1 M potassium phosphate (pH 7) or 0.1 potassium borate (pH 9) after nanosecond laser photolysis. The distributions of the low temperature activation enthalpy g(H) for geminate ligand binding derived from the kinetic traces are quite narrow and are influenced by temperature both below and above approximately 170 K, the glass transition temperature. The thermal evolution of the CO binding kinetics between approximately 50 K and approximately 170 K indicates the presence of some degree of structural relaxation, even in this temperature range. Above approximately 220 K the width of the g(H) progressively decreases, and at 280 K geminate CO binding becomes exponential in time. Based on a comparison with analogous investigations of the homodimeric hemoglobin from Scapharca inaequivalvis, we propose a link between dynamic properties and functional complexity.

A cooperative model for ligand binding to biological macromolecules as applied to oxygen carriers.

This paper presents a model describing the thermodynamics of cooperative ligand binding to multimeric biological macromolecules and integrating some of the features of the two-state and induced-fit models. The protein is taken to be partitioned into a number of noninteracting functional constellations, each one existing in two possible quaternary conformations. Furthermore, the model postulates that a functional constellation is organized in several subsets of sites (called cooperons), in which subunits interact according to an induced-fit mechanism. In the present version the number of subunits forming a cooperon has been limited to two and the total number of parameters used for fitting experimental data is four, all having a precise physical meaning. Although the present application is limited to oxygen-carrying proteins (hemoglobins, hemocyanins, erythrocruorins), the model appears suitable to describe other biological macromolecules with functional interactions.

Principles in the assembly of annelid erythrocruorins.

Erythrocruorins are giant extracellular respiratory proteins found freely dissolved in the blood of annelids. We present here results from our ultracentrifugation, electron microscopy, spectroscopy, and diffraction experiments on these erythrocruorins. These data are rationalized in terms of a three-dimensional model of the quaternary structure. The proposed structure is arranged in a hierarchy of symmetry. The implications of this structure for the assembly process are considered with special attention to uniqueness and self-limitation. The hypothesis is consistent with observations not used in its construction and it serves as a working hypothesis to focus further experimentation.

The structure of invertebrate extracellular hemoglobins (erythrocruorins and chlorocruorins).

The knowledge accumulated over the last 30 years concerning the subunit structures of the invertebrate extracellular hemoglobins permits us to classify them into four distinct groups. Single-domain, single-subunit hemoglobins consisting of single, heme-binding polypeptide chains which have a molecular mass of ca. 16 KDa. These molecules are found in multicellular parasitic organisms such as the trematodes Dicrocoelium and Fasciolopsis and in a few insects, namely in the adult Anisops and in the larvae of Chironomus and of Buenoa. Two-domain, multi-subunit hemoglobins consisting of 30-37 KDa polypeptide chains each containing two, linearly connected heme-binding domains, which form polymeric aggregates with molecular masses ranging from 250 to 800 KDa. These hemoglobins are found extensively among the carapaced branchiopod crustaceans: Caenestheria, Daphnia and Lepidurus hemoglobins have been found to consist of 10, 16 and 24 two-domain chains, respectively. Judging from their electron microscopic appearances, some of the hemoglobins may possess different molecular symmetries. Multi-domain, multi-subunit hemoglobins consisting of two or more polypeptide chains, each comprising many heme-binding domains of ca. 15-20 KDa each. Examples of this class are found among the carapaceless branchiopod crustaceans, the planorbid snails and the clams from the families Astartidae and Carditidae. Artemia hemoglobin consists of two chains of ca. 125 KDa, each containing 8 heme-binding domains. Planorbis and Helisoma hemoglobins possess a molecular mass of ca. 1700 KDa and consist of 10 chains of 170-200 KDa. Astarte and Cardita hemoglobins appear in electron micrographs as rod-like polymers of variable dimensions, 20-30 nm in diameter and 20-100 nm in length and consist of polypeptide chains of ca. 300 KDa. The crustacean and gastropod hemoglobins vary in their electron microscopic appearance and may possess different molecular symmetries. Single-domain, multi-subunit hemoglobins consisting of aggregates of several small subunits, some of which are disulfide-bonded and not all of which contain heme. These molecules are widely distributed among the annelids and possibly also among the pogonophores. They are characterized by a two-tiered, hexagonal electron microscopic appearance, with a vertex-to-vertex diameter of 30 nm and a height of 20 nm, an acidic isoelectric point, a sedimentation coefficient of 50-60 S and a low iron content of 0.24 +/- 0.03%.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxygen binding to Octolasium complanatum erythrocruorin. Modulation of homo- and heterotropic interactions by cations.

The functional properties of erythrocruorin from Octolasium complanatum (a common earthworm of Central Italy) have been characterized in great detail. Special attention has been given to the reciprocal effects of the various ligands, namely oxygen, cations and protons. The data obtained under a variety of experimental conditions bring out the dominant role played by cations in the modulation of both homotropic and heterotropic interactions. In this respect, the most interesting observation concerns the unusual interplay between protons and cations that occurs in this erythrocruorin, the first respiratory pigment in which the Bohr effect is due totally to the O2-linked binding of an allosteric effector. The oxygen binding data collected under the various experimental conditions have been analyzed in terms of a modified two-state model, which takes into account the fact that allosteric effectors may also influence the ligand binding properties of the state that they stabilize. The analysis shows that the number of interacting sites necessary for the observed co-operativity in O2 binding is much smaller than the number of heme groups carried by the whole molecule, in accordance with previous findings on hemocyanins, the other class of giant respiratory pigments. Moreover, the analysis indicates that the dimensions of these "functional constellations" are under the control of allosteric effectors.

The erythrocruorin of Eisenia fetida. II. Properties of the principal subunit.

The erythrocruorin of Eisenia fetida can be dissociated partially into its principal subunits, the putative one-twelfths of the molecule and smaller subunits, by three different methods: freezing and thawing (Af), exposure to alkaline pH (Aa) and aging (Ao). The isolated subunits possess a relative molecular mass of 310,000 +/- 20,000 by gel filtration and their SDS-polyacrylamide gel electrophoretic patterns are identical to (Af) or slightly different from (Aa, Ao) that of the erythrocruorin. The absorption spectra and Hill constant h of the principal subunit Af correspond to the values of the whole erythrocruorin in the state of low cooperativity, which results from freezing and thawing, exposure to alkaline pH, and aging. Electron microscopic studies of the principal subunits and of the 'treated' erythrocruorin showed that their dimensions had increased relative to the native erythrocruorin: a diameter of 9 nm vs. 8.5 nm and (26.4 +/- 0.4) X (18.3 +/- 0.4) nm vs. 25.0 X 16.5 nm, respectively. Erythrocruorin reconstituted from the Af subunits possessed the dimensions (26.6 +/- 0.4) X (18.6 +/- 0.4) nm. Based on the subunit model of Eisenia erythrocruorin proposed previously it is suggested that there can exist an association-dissociation equilibrium between the principal subunits, smaller subunits, and the erythrocruorin when it is in the state of low cooperativity, but not when it is in its native, high-cooperativity state.

Amphitrite ornata erythrocruorin. II. Molecular controls of function.

In the marine terebellid worm Amphitrite ornata the vascular fluid contains a high molecular weight erythrocruorin, while cells of the coelom contain a monomeric hemoglobin. The structural integrity of the erythrocruorin molecule is known to be dependent on the presence of a minimal concentration of divalent cations (1-3 mM) in the medium. The functional properties of Amphitrite erythrocruorin are also affected by cations. The oxygen affinity tends to increase with increasing cation concentration and the degree of cooperative interactions, expressed in the kinetics and equilibria of ligand binding, goes through a maximum. Maximal Hill coefficients of 3-4 are observed with 50 mM CaCl2, 50 mM MgCl2 or 1 M NaCl in measurements at the physiological pH of 7.75. Only 2 mM CaCl2 is required for maximal cooperativity at pH 8.5. This suggests partial deprotonation of the cation binding site at high pH. It is somewhat unusual that pH effects on cooperativity are reversible, since this is not a common feature of the giant erythrocruorin molecules. The oxygen binding experiments revealed a marked effect of divalent cations of Amphitrite erythrocruorin at high pH and cation concentration. Above pH 8.5, at 50 mM CaCl2 and 12 degrees C, the erythrocruorin will form a polymer upon deoxygenation. This polymerization is readily reversible by bringing the temperature for 12 to 20 degrees C or by oxygenation. Under physiological conditions of pH and cation concentration and at 12 degrees C, the erythrocruorin and the monomeric coelomic hemoglobin require a similar oxygen pressure for half saturation. However, the allosteric regulation of function is absent for the coelomic protein.

Isomeric incorporation of the haem into monomeric haemoglobins of Chironomus thummi thummi 3. Comparative study of components, I, III and IV.

13C-NMR studies on 13CO complexes of the components III and IV of the monomeric haemoglobins from the subspecies Chironomus thummi thummi have been carried out in order to confirm the existence of two conformational isomers differing by the isomeric incorporation of the haem group and the extent of the Bohr effect. In addition, the allosteric linkage between the ligand binding site and the Bohr proton binding site in the component IV is described from investigations of various pH-dependent proton resonances including the C-2 proton resonances of the titratable histidines. In comparison to the data obtained for the component III it is assumed that in both conformational isomers of the component IV the allosteric linkage between the distal site of the haem and the main Bohr proton donating group is present. From corresponding NMR investigations of the component I an isomeric incorporation of the haem group into this monomeric haemoglobin seems unlikely. Also, any correlation between the ligand binding site and a titratable group of the protein had not been found in this haemoglobin, in agreement with previous results of other laboratories that a Bohr effect in the component I cannot be detected.

Isomeric incorporation of the haem into monomeric haemoglobins of Chironomus thummi thummi. 1. Isolation of chemically homogeneous haemoglobins. Evidence for the isomerism of the haem in the component III.

With high-resolution NMR spectroscopy it has been possible to detect various types of isomerisms in the monomeric component III of the haemoglobins of Chironomus thummi. In the component III prepared from commercially available larvae of Ch. thummi a chemical heterogeneity has been found to occur at the position E6 (Ile or Thr). The imidazole C-2 proton resonance of the adjacent His-E7 is split because of this alternative occupancy. From larvae of an inbreeding of the single subspecies Chironomus thummi thummi a component III material has been isolated in which the position E6 is occupied by one single amino acid. An additional isomerism of this haemoglobin III is produced by an isomeric incorporation of the haem group into the haem pocket. This isomerism has been established from the complex signal pattern of the mesoproton resonances of the diamagnetic ligated haemoglobin III. Other evidences of this isomerism have been obtained from an analysis of th high-field proton resonances.

Isomeric incorporation of the Haem into monomeric haemoglobins of Chironomus thummi thummi. 2. The Bohr effect of the component III explained on a molecular basis and functional differences between the two isomeric structures.

The Bohr effect in the monomeric haemoglobin component III of Chironomus thummi thummi is described on a molecular basis using various pH-dependent proton resonances. One of the three titratable histidines changes its pK from 7.9 in the unligated state to 7.7 upon ligation with CO and up to 7.0 upon ligation with O2. It can be concluded from an analysis of the C-2 proton resonance intensities that these shifts in pK occur in only one of the two conformational isomers of the component III, whereas in the second isomer these pK shifts are not observed. The differences between these functional properties are also derived from pH-dependent changes of the mesoproton resonances as well as of the methyl resonances of Ile-E11. The conformational change of the component III, which is connected with the Bohr effect, influences a variety of other resonances which could not be assigned. The pK values derived from the pH dependence of these changes agree exactly with the pK value of the Bohr proton donating group, which is supposed to be His-G2. Also the corresponding signal intensities indicate the presence of two conformational isomers in the haemoglobin component III. Although the change in chemical shift of the His-G2 C-2 proton resonance is only small upon deprotonation of the imidazole ring, it has been shown with potentiometric and calorimetric methods that this histidine is deprotonated in this pH range. Considering the tertiary structure obtained from an X-ray study of Steigemann and Weber we can readily explain the conformational changes in the Bohr active isomer of the component III. With binding of ligands dislocations of side chains at the proximal site produce a change in the interactions of His-G2 with the carboxyl group of the C-terminal methionine-H22.

Physicochemical and functional properties of Perinereis cultrifera (Grübe) erythrocruorin.

Perinereis erythrocruorin has the following physicochemical properties: So20,w = 55S, corresponding to a molecular weight around 2.7-10(6); minimum molecular weight (on the basis of the heme content) 23 700 +/- 500; isoelectric point 5.1; alpha-helix content approximately 40%. At alkaline pH values in the oxygenated form the 55-S molecules dissociate into subunits with a weight average sedimentation coefficient of 3S, corresponding to a molecular weight approximately 35 000. Deoxygenation of partially dissociated samples promotes association of the 3-S subunits into a 9S component. The functional properties of Perinereis erythrocruorin are characterized by a low cooperativity in oxygen binding (n 1/2 = 1.5) at neutral pH. Cooperativity increases reversibly towards both the acid and alkaline pH range, irrespective of changes in molecular weight. This finding, taken together with the ultracentrifuge results, suggests that a subunit may represent the functional unit of the protein. The pH dependence of the oxygen affinity can be accounted for in terms of a single oxygen linked group with a pK of 8.