Ezra Daniel

Subunit Structure of Earthworm Erythrocruorin

Electron microscopy of earthworm erythrocruorin shows two hexagonal rings stacked on one another (1–5). Existing models of the molecule involve 12 identical subunits arranged in dihedral symmetry (6–10). In the present report, the adequacy of this model to account for the dissociation pattern of earthworm erythrocruorin is examined. A new model is proposed.

Oxygen binding properties of haemocyanin from Levantina hierosolima

Abstract The oxygen binding properties of haemocyanin from Levantina hierosolima in the absence and in the presence of calcium ions were studied. A fluorometric method for the determination of saturation curves, based on the difference in emission intensities of the reduced and oxygenated protein, was developed. In divalent ion-free solutions, the binding was found to be of the simple type. In the presence of calcium ions interaction between the binding sites takes place. Thermodynamic quantities for the oxygen binding reaction in the two cases were determined. Corresponding values for the interaction among the oxygen binding sites were also calculated. The binding behaviour of the oxygenated haemocyanin in calcium-containing solutions was found to be identical to that of haemocyanin in the absence of the ion. The findings show that, as is the case in the haemoglobin system, deoxyhaemocyanin has a lowered affinity towards oxygen; oxygenation returns the molecule to the high affinity characteristic of the calcium-free protein.

The binding of calcium ions to hemocyanin from Levantina hierosolima at physiological pH.

Abstract The binding of Ca2+ to Levantina hierosolima hemocyanin at physiological pH, 8.2, was studied. 20 moles of binding sites per unit of 50 000 g protein were found. In parallel, the effect of Ca2+ on the aggregation state of the macromolecules was examined. The correlation between the binding of Ca2+ and the association behavior is explained by the formation of calcium chelates involving carboxylate and imidazole side chains of the proteins.

Structural diversity of arthropod extracellular haemoglobins

Abstract 1. 1. Extracellular haemoglobins from several arthropod species were investigated by sedimentation in the ultracentrifuge and sodium dodecyl sulfate gel electrophoresis. 2. 2. Haemoglobins from Lepidurus apus (19.3S), Daphnia sp. (17.6S) and Cyzicus hierosolymitanus (11.4S) are each composed of many identical polypeptide chains (mol. wt 34,000, 31,000 and 30,000 daltons respectively). Streptocephalus haemoglobin (11.4S) is composed of two, maybe three, similar polypeptide chains (mol. wt 100,000–120,000 daltons). Chironomus larval haemoglobins are composed of one, sometimes of two, polypeptide chains (mol. wt ca . 16,000 daltons). 3. 3. Arthropod haemoglobins exhibit structural diversity. Classification of the haemoglobins on the basis of their molecular structure places the species in categories in accordance with their taxonomical groupings.

Subunit structure of hemoglobin from the clam shrimp Cyzicus

The subunit structure of hemoglobin from the clam shrimp Cyzicus was investigated. The native protein has a molecular weight of 280,000 as shown by sedimentation equilibrium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the presence of mercaptoethanol resulted in a single band corresponding to a molecular weight of 15,500. A molecular weight of 14,500 was determined by the method of sedimentation equilibrium for the isolated polypeptide chain. Electron microscopy of the protein revealed a pentagonal structure. It was concluded that the native molecule contains 20 polypeptide chains.

Built-in co-operativity of oxygen binding by arthropod hemocyanins.

Abstract Oxygen binding by arthropod hemocyanin from the scorpion Leirus quinquestriatus and the crabs Telphusa fluviatilis and Ocypoda cursor was studied in Ca 2+ , Mg 2+ -free solutions. The binding was found to be co-operative in all three cases. Our results and a re-examination of the literature lead us to conclude that co-operative oxygen binding is a built-in feature common to arthropod hemocyanins, distinguishing them from mollusc hemocyanins where co-operativity is conditional upon the presence of Ca 2+ or Mg 2+ .

Tyrosyl fluorescence in hemocyanin from the scorpion Leirus quinquestriatus.

The fluorescence properties of hemocyanin from the scorpion Leirus quinquestriatus were studied. Emission and excitation spectra were determined for protein in both its oxygenated and deoxygenated forms. Oxygenation was found to bring about a large blue shift in the position of the fluorescence maximum, in addition to a marked quenching of the fluorescence intensity as noted before for another hemocyanin. An appreciable tyrosyl contribution to the fluorscence of oxyhemocyanin was inferred from the wavelength dependence of its emission and excitation spectra. No tyrosyl fluorescence could be observed in either apo- or deoxyhemocyanin. It was concluded that the inability to observe tyrosyl emmision in deoxyhemocyanin is due to the dominating emission to tryptophan. The implication of the findings to the often noted failure to detect tyrosyl emission in proteins containing both tyrosine and tyrptophan is discussed.

Towards a resolution of the long-standing controversy regarding the molecular mass of extracellular erythrocruorin of the earthworm Lumbricus terrestris

The published molecular mass of erythrocruorin of Lumbricus terrestris and related earthworm species covers a bewildering range of 3.23-4.5 MDa. A critical reexamination reveals that some mass determinations were underestimated and the results do cluster, not at one, but at two values of the molecular mass. One cluster corresponds to approximately 3.6 MDa, as predicted for a stoichiometry of 144 globin and 36 linker chains-the Vinogradov model for the hexagonal bilayer (HBL) assembly of Lumbricus erythrocruorin-and as estimated from the crystal structure of HBL at 5.5 A resolution [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 7107]. The other cluster corresponds to approximately 4.4 MDa. In addition, a molecular mass of 4.1 MDa, determined by multiangle laser light scattering (MALLS), stands apart of the two clusters, separated from the masses obtained by other methods of molecular mass determination. We propose a stoichiometry of 192 globin and 36 linker chains for the 4.4-MDa molecule. The 36 linkers and 144 out of 192 globin chains are identified with the HBL and the remaining 48 globins are allotted equally to the two halves of the axial cavity above and below the central torus of the structure. The proposed model is supported by the occurrence in some annelid species of erythrocruorin with centrally placed subunits [Biochim. Biophys. Acta 359 (1974) 210], and by the oxidation-dependent shedding of subunits in Lumbricus erythrocruorin. We propose further that the 4.1 MDa determination represents the weight average molecular mass of a population of molecules resulting from a partial dissociation of 4.4-MDa erythrocruorin. This interpretation seems reasonable on the background of the very low protein concentrations ( approximately 100 microg/ml and lower) prevailing at the MALLS experiment.

Cross-linking of porin with glutardialdehyde: a test for the adequacy of premises of cross-linking theory

Exposure of porin from Escherichia coli to glutardialdehyde followed by SDS-gel electrophoresis in 3% polyacrylamide yielded three bands that were identified in order of decreasing mobility as monomers and two and three cross-linked polypeptide chains. The distribution of protein among the three species for different extents of reaction showed a remarkably good agreement with corresponding values predicted from cross linking theory for an oligomer composed of three identical subunits arranged according to a 3-fold rotation axis. Electrophoresis performed in 7.5% polyacrylamide yielded four bands that were assigned to polypeptide chain monomers, dimers, and two types of trimers carrying two and three intersubunit cross-links. Our findings provide evidence that the central premise of cross-linking theory, viz. that the intersubunit cross-links formed upon exposure of a protein to a bifunctional reagent be governed by the symmetry of the molecule, is valid. Careful interpretation of cross-linking experiments thus proves an effective method to assess the oligomeric structure of a protein and reveal the symmetry underlying the spatial arrangement of the subunits within the molecule.

A re-evaluation of the molecular mass of earthworm extracellular hemoglobin from meniscus depletion sedimentation equilibrium. Nature of the 10 S dissociation species.

Previous calculations from meniscus depletion sedimentation equilibrium earthworm hemoglobin from Lumbricus terrestris (E.J. Wood et al., Biochem. J. 153 (1976) 589-96) and from the related species Lumbricus sp. (L. sp.) (M.M. David and E. D Mol. Biol. 87 (1974) 89--101) were made on the assumption that the solutions behaved ideally. Re-examination of their results reveals, however, a dependence of the apparent molecular mass on concentration. Taking this effect into consideration, we have nowrecalculated from their data molecular masses of 4.4--4.5 MDa for the hemoglobin of both L. terrestris and L. sp. On the basis of the new determinations, we propose for the polypeptide chain composition of L. terrestris hemoglobin a model [(abcd )4L1L2L3]12 where a,b,c,d are the four globin and L1,L2,L3 are the three major linker chain constituents of the protein. The model is consistent with the D6 symmetry of the molecule. A 10 S intermediate product in the alkaline dissociation Lumbricus hemoglobin is viewed as a binary mixture of products resulting from a disproportionation reaction involving the structural unit. The present interpretation is shown to be consistent with observed relations between molecular masses and SDS gel electrophoretic band patterns of 10 S species and intact hemoglobin.