No abstract

The need to develop a blood substitute is now urgent because of the increasing concern over Europe's BSE outbreak and the worldwide HIV/AIDS epidemic, which have cut blood supplies. Extracellular soluble hemoglobin has long been studied for its possible use as a safe and effective alternative to blood transfusion, but this has met with little success. Clinical trials have revealed undesirable side effects-oxidative damage and vasoconstriction-that hamper the application of cell-free hemoglobin as a blood substitute. We have addressed these problems and have found a new promising extracellular blood substitute: the natural giant extracellular polymeric hemoglobin of the polychaete annelid Arenicola marina. Here we show that it is less likely to cause immunogenic response; its functional and structural properties should prevent the side effects often associated with the administration of extracellular hemoglobin. Moreover, its intrinsic properties are of interest for other therapeutic applications often associated with hemorrhagic shock (ischemia reperfusion, treatment of septic shock and for organ preservation prior to transplantation). Moreover, using natural hemoglobin is particularly useful since recombinant DNA techniques could be used to express the protein in large quantities.

To elucidate the quaternary structure of the extracellular haemoglobin (Hb) of the marine polychaete Arenicola marina (lugworm) it was subjected to multi-angle laser-light scattering (MALLS) and to electrospray-ionisation mass spectrometry (ESI-MS). It was also subjected to SDS/PAGE analysis for comparative purposes. MALLS analysis gave a molecular mass of 3648 % 24 kDa and a gyration radius of 11.3 rt 1.7 nm. Maximum entropy analysis of the multiply charged electrospray spectra of the native, dehaemed, reduced and carbamidomethylated Hb forms, provided its complete polypeptide chain and subunit composition. We found, in the reduced condition, eight globin chains of molecular masses 15952. Keywords: Arenicola ; haemoglobin ; quaternary structure ; multi-angle laser-light scattering; electrospray mass spectrometry.The giant extracellular haemoglobins (Hb) and chlorocruorins found in annelids [1] and vestimentiferans [2, 31 are characterised by an acidic isoelectric point and by an hexagonal symmetry in electron micrographs consisting of two superimposed hexagonal arrays of twelve spherical subunits, the hexagonal bilayer (HBL). These HBL Hb also contain a low haem and iron contents (about 67% of those observed in other Hbs) and consist of two types of chains, globin chains (~1 6 -1 8 kDa), accounting for approximately 70% of the total mass, and haem-deficient linker chains (~2 4 -2 8 kDa) necessary for the assemblage into the HBL structure [1,. The intertidal marine polychaete Arenicola marina (L.) was one of the first 60s (~3 6 0 0 kDa)