Sedimentation coefficient and minimum molecular weight of extracellular hemoglobin of Glossoscolex paulistus (Oligochaeta).

A new procedure for the determination of the sedimentation coefficient in the 50-1000 S range was designed and tested. The characteristics of this protocol are: the use of density gradients self-generated by osmosedimentation and the use of a low-speed centrifuge and of visual monitoring of the sedimenting zone. This procedure was used to determine the sedimentation coefficient of erythrocruorin from Glossoscolex paulistus. The value obtained, S20, omega = 58 S, corresponds to a MW of 3.1 x 10(6) Daltons. The minimum MW (heme), determined by the pyridine-hemochromogen method, was 25,250 Da.

The analysis of a protein-polymorphism. Evolution of monomeric and homodimeric haemoglobins (erythrocruorins) of Chironomus thummi thummi (Insecta, Diptera).

The evolutionary history of 12 Chironomus thummi thummi (CTT) haemoglobins of known primary structures was reconstructed by the maximum parsimony method. This reconstruction demonstrates that the 12 CTT haemoglobin lineages originated monophyletically from a common ancestor within early Insecta and have the lineage to monomeric blood worm haemoglobin as their closest sister group. It can be further deduced that the earliest ancestral CTT haemoglobins were monomers and that a branch to all extant dimeric CTT haemoglobins emerged later in phylogeny near the base of Chironomidae, but perhaps still before Chironomus itself evolved. This ancient, pre-Chironomus history suggests that among insect taxa, now lacking expressed globins, remnants of globin genes might exist as unexpressed pseudogenes. By the parameter of base replacement frequencies, CTT haemoglobins appear as relatively slow-evolving proteins, showing a preponderance of guanine in equilibrium adenine transitions at the first nucleotide position of the codons but not at the second. The most conservatively-evolving amino acid positions are haem contacts; the next most conservative are in interhelical contacts and interior positions involved in stabilization of tertiary structure. Further elucidation of the phylogenetic origins and adaptive evolution of the multiple haemoglobins found in Chironomus will be possible by the maximum parsimony method once haemoglobins or, in their absence, haemoglobin pseudogenes are sequenced in species throughout Chironomidae and related taxa.

The amino acid sequence of a major polypeptide chain of earthworm hemoglobin.

The amino acid sequence has been determined for polypeptide chain AIII of the major component of the hemoglobin (erythrocruorin) from the earthworm, Lumbricus terrestris. The chain has 157 residues and a molecular weight of 17,496. Although the extent of sequence identity with other globin chains is only 17-18%, analysis of the sequence leads to the conclusion that the secondary structure of the chain is very similar to those of vertebrate globins and that about 60-70% of the amino acid residues are in alpha helices. The D helix appears to be missing, as it is from the alpha chain of human hemoglobin and from the monomeric hemoglobin of Glycera dibranchiata, another annelid worm. This is the first sequence obtained from one of the "giant" extracellular hemoglobins of invertebrate animals.

The erythrocruorin of Eisenia fetida. I. Properties and subunit structure.

The erythrocruorin of Eisenia fetida possesses a relative molecular mass, determined by sedimentation equilibrium, of (3.82 +/- 0.05) . 10(6). According to the iron and heme contents, 0.218 +/- 0.008% and 2.34 +/- 0.02% by mass, respectively, it contains 144 hemes per molecule. The dimensions of the molecule observed by electron microscopy are 25.0 X 16.5 nm (diameter X height). SDS-polyacrylamide gel electrophoresis indicates that the erythrocruorin consists of six subunits (Mr 14,900, 15,300, 17,200, 19,700, 31,600 and 40,000). Oxygen binding studies showed that E. fetida erythrocruorin has a high oxygen affinity (P50 = 2.8 Torr at pH 7.5), exhibits a slight bohr effect and possesses a high cooperativity with the Hill coefficient h = 3.7-4.8. Treatment of the erythrocruorin either by freezing and thawing or by aging or exposure to alkaline pH converts it irreversibly into a state of lower cooperativity with h = 2.0-2.6. A model of the subunit structure of the erythrocruorin is proposed which takes into account the physiochemical and oxygen-binding properties of the erythrocruorin and the subunits obtained upon its dissociation.

[Hemoglobins, XXXII. Analysis of the primary structure of the monomeric hemoglobin CTT VIIA (erythrocruorin) or Chironomus thummi thummi, Diptera (author's transl)]. / Hämoglobine, XXXII. Analyse der Primärstruktur des dimeren Insektenhämoglobins CTT VIIA (Erythrocruorin) aus Chironomus thummi thummi, Diptera.

The dimeric hemoglobin CTT VIIA (erythrocruorin) was isolated from the hemolymph of the larva from Chironomus thummi thummi and purified by preparative polyacrylamide gel electrophoresis. Peptides obtained by limited tryptical digestion were sequenced by automatic Edman degradation. For the elucidation of the sequence in the C-terminal region of the chain, additional cleavages with proteinase of Staphylococcus aureus and chymotrypsin were necessary. CTT VIIA is compared with human beta-chains and other hemoglobins of Chironomus. The amino acid residues in the pocket are especially discussed. Most of them are invariant in all Chironomus hemoglobins, independent of the size of the heme pocket, which is normal in some components and enlarged in others.