Molecular Architecture of Annelid Erythrocruorins. Extracellular Hemoglobin of Arenicola marina (Polychaeta)

Abstract

The respiratory protein, erythrocruorin, of the annelid Arenicola marina was investigated. Spectral properties show many analogies with those of vertebrate hemoglobine. 144 heme groups (ferroprotoporphyrin) were found in the whole molecule, which has a relative molecular mass of 3.56 x lo6, as determined by sedimentation equilibrium, and an isoelectric point of 4.69. Protein dissociation patterns were analysed by electrophoresis after denaturation in the presence of dodecylsulfate, with and without 2-mercaptoethanol. A tentative model associating molecular mass of the native molecule, heme content, molecular mass of the polypeptide chains and functional properties is proposed. A twelfth subunit of A . marina erythrocruorin would contain twelve heme groups arranged in three functional units made up of four protomers, half of these being covalently bound to non-heme chains; two structural chains would be spatially arranged as bonds between the subunits.

Quaternary Structure of the Extracellular Haemoglobin of the Lugworm Arenicola marina

“…The molecular mass found for Arenicola HBL Hb by MALLS (3648 -C 24 kDa) is in good agreement with the most recently published values of 3740 t-120 kDa and 3560 ? 40 kDa, respectively [21, 201. On the whole, our SDSIPAGE results on reduced Arenicola Hb are in good agreement with previous studies (20,21,241. However, we obtained a high resolution thus clearly revealing four components around 14100-16400 Da, whereas two of the earlier works found only two [20] and three [21] components.…”

“…40 kDa, respectively [21, 201. On the whole, our SDSIPAGE results on reduced Arenicola Hb are in good agreement with previous studies (20,21,241. However, we obtained a high resolution thus clearly revealing four components around 14100-16400 Da, whereas two of the earlier works found only two [20] and three [21] components. El Idrissi Slitine and Toulmond [24] also found four polypeptide chains by IEFIPAGE, and they concluded that the polypeptide chains A and B were of the same molecular mass but different isoelectric points.…”

“…There is an appreciable scattering in the published values for the molecular mass of Arenicola HBL Hh obtained mainly by equilibrium sedimentation (range 2850-3850 kDa) 17, 8,[19][20][21]231. This is probably due to the experimental conditions used, in particular to the divalent cation concentrations in the various buffers used for the experiments.…”

“…Recently, a higher resolution (> 1500) and more accurate technique (0.01 %), electrospray ionisation mass spectrometry (ESI-MS), has been successfully applied to some HBL Hb allowing the determination of their complete polypeptide chain composition [26- [21, 241. However, Pionetti and Pouyet [20] found five different polypeptide chains, three around the molecular masses 14000-16000 Da and two around 30000 Da. Moreover, El Idrissi Slitine and Toulmond 1241, using two-dimensional electrophoresis, revealed that components A and B could be further separated into four polypeptide chains having the same relative molecular mass but different isoelectric points (i.e.…”

Scanning transmission electron microscopic examination of the hexagonal bilayer structures formed by the reassociation of three of the four subunits of the extracellular hemoglobin of Lumbricus terrestris.

“…The peripheral location of subunit M is in harmony with the well-known fact that the dissociation of Lumbricus Hb at alkaline pH gives two fractions, one consisting of subunit M and the other of subunits D1, D2, and T (7). Such a pattern of dissociation at alkaline pH has been observed for many annelid hemoglobins, including those of the oligochaetes, Tubifex (19,20) and Eisenia (21); the polychaetes Abarenicola (22), Arenicola (23)(24)(25), and Ophelia (26); and the leeches Haemopis (27) and Dina (28).…”