Evaluating the Capacity to Generate and Preserve Nitric Oxide Bioactivity in Highly Purified Earthworm Erythrocruorin: A GIANT POLYMERIC HEMOGLOBIN WITH POTENTIAL BLOOD SUBSTITUTE PROPERTIES*

The giant extracellular hemoglobin (erythrocruorin) from the earth worm (Lumbricus terrestris) has shown promise as a potential hemoglobin-based oxygen carrier (HBOC) in in vivo animal studies. An important beneficial characteristic of this hemoglobin (LtHb) is the large number of heme-based oxygen transport sites that helps overcome issues of osmotic stress when attempting to provide enough material for efficient oxygen delivery. A potentially important additional property is the capacity of the HBOC either to generate nitric oxide (NO) or to preserve NO bioactivity to compensate for decreased levels of NO in the circulation. The present study compares the NO-generating and NO bioactivity-preserving capability of LtHb with that of human adult hemoglobin (HbA) through several reactions including the nitrite reductase, reductive nitrosylation, and still controversial nitrite anhydrase reactions. An assignment of a heme-bound dinitrogen trioxide as the stable intermediate associated with the nitrite anhydrase reaction in both LtHb and HbA is supported based on functional and EPR spectroscopic studies. The role of the redox potential as a factor contributing to the NO-generating activity of these two proteins is evaluated. The results show that LtHb undergoes the same reactions as HbA and that the reduced efficacy for these reactions for LtHb relative to HbA is consistent with the much higher redox potential of LtHb. Evidence of functional heterogeneity in LtHb is explained in terms of the large difference in the redox potential of the isolated subunits.     

The self-association of the giant hemoglobin from the earthworm, Lumbricus terrestris

Background: The crystallographic structure of the gigantic hemoglobin (erythrocruorin) of the annelid worm, Lumbricus terrestris, provides a molar mass of 3.6 MDa for the hexagonal bilayer structure. Prior to this determination, some light-scattering and ultracentrifugal measurements indicated higher masses: 4.1–4.4 MDa. Values of 3.6 MDa were attributed to dissociation or subunit loss. However, early electron microscopy of the giant hemoglobin from a related annelid, Eumenia crassa by Öster Levin, showed that the hexagonal bilayer molecules were present mostly as oligomers; few were monomeric. Methods: Measurements by light-scattering of solutions of Lumbricus hemoglobin resolved by size-exclusion chromatography have been used to determine the weight-average molar mass of self-associating proteins. The X-ray structure has been re-examined. Results: Our measurements show that both 3.6 MDa monomers and self-association products are present as a mixture. Analysis of the X-ray structure indicates several different kinds of monomer–monomer interactions. Conclusions: We propose that the measured masses of Lumbricus hemoglobin as high as 4.4 MDa, result from oligomerization. These masses would result from the presence of an array of oligomers of various sizes together with monomers of 3.6 MDa. Furthermore, several different kinds of monomer–monomer interactions are clearly evident in the X-ray structure as well as in solution. General significance: The results demonstrate that self-association of monomers of the hemoglobin of Lumbricus terrestris explains the high molar masses of 4.1–4.4 MDa previously observed.     

Tendency for oxidation of annelid hemoglobin at alkaline pH and dissociated states probed by redox titration

The redox titration of extracellular hemoglobin of Glossoscolex paulistus (Annelidea) was investigated in different pH conditions and after dissociation induced by pressure. Oxidation increased with increasing pH, as shown by the reduced amount of ferricyanide necessary for the oxidation of hemoglobin. This behavior was the opposite of that of vertebrate hemoglobins. The potential of half oxidation (E_{1 / 2}) changed from − 65.3 to + 146.8 mV when the pH increased from 4.50 to 8.75. The functional properties indicated a reduction in the log P₅₀ from 1.28 to 0.28 in this pH range. The dissociation at alkaline pH or induced by high pressure, confirmed by HPLC gel filtration, suggested that disassembly of the hemoglobin could be involved in the increased potential for oxidation. These results suggest that the high stability and prolonged lifetime common to invertebrate hemoglobins is related to their low tendency to oxidize at acidic pH, in contrast to vertebrate hemoglobins.     

Structure of the extracellular hemoglobin of Biomphalaria glabrata

The hemoglobin of Biomphalaria glabrata was purified to homogeneity by gel filtration column followed by anion exchange chromatography. The dissociation products were analyzed by a 5–15% gradient polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE) giving a band of 270 kDa and a band of 180 kDa after reduction with β-mercaptoethanol. The same profile was obtained in a 3.5% agarose gel electrophoresis containing SDS (SDS-AGE) but showed additional bands of higher molecular weight. These bands were proposed to be monomers, dimers and trimers, since they showed a good correlation in a plot of R_f versus log M_r. After partial reduction in a two-dimensional SDS-AGE, the proposed monomers and dimers produced two and four bands, respectively, likely indicating one to four chains crosslinked by disulfide bridges. Digestion with four different proteases yielded several equivalent fragments with molecular weights multiples of its minimum molecular weight (17.7 kDa). The circular dichroism spectrum of the protein showed a characteristic high α-helix content (70%). It was proposed that this hemoglobin is a pentamer with a molecular weight of aproximately 1.8×10³ kDa, assembled by five 360-kDa subunits, each formed by two 180-kDa chains linked in pairs by disulfide bridges and each of these chains, in turn, comprised by ten heme binding domains linked in tandem. These data are compared to the published information for other planorbid extracellular hemoglobins.