On the molecular mass of Lumbricus erythrocruorin Ezra Daniel a, * , Ariel Lustig b , Melvyn M. David a , Yossi Tsfadia a a Department of Biochemistry, Tel-Aviv University, Tel-Aviv 69978, Israel b Department of Biophysical Chemistry, Biozentrum, University of Basel, CH-4056 Basel, Switzerland Abstract A critical examination of the published molecular mass of erythrocruorin (Ec) from Lumbricus and related earthworm species reveals that the results do cluster, not at one, but at two values of the molecular mass. One cluster corresponds to , 3.6 MDa as predicted from the Vinogradov model for the hexagonal bilayer (HBL) assembly of Lumbricus terrestris EC and as estimated from the crystal structure of HBL at 5.5 A resolution. The other cluster corresponds to , 4.4 MDa. However, in contrast to the controversy over the molecular mass, there is a consensus that the sedimentation coefficient of intact L. terrestris Ec is , 60S. Drawing on the occurrence of central subunits in Ec of Oenone fulgida and few other annelid species, we propose for the 4.4 MDa molecule a model of HBL supplemented by a central mass. The proposed model abides by D 6 symmetry and is a suitable candidate to represent 60S Lumbricus terrestris Ec. q 2003 Elsevier Ltd. All rights reserved. Keywords: Molecular mass; Earthworm; Lumbricus; Erythrocruorin; Haemoglobin The published molecular mass of erythrocruorin (Ec) of Lumbricus terrestris and related earthworm species covers a bewildering range, from 3.23 to 4.5 MDa. A critical re- examination reveals that some mass determinations were underestimated and the available results cluster, not at one, but at two molecular mass values (Daniel et al., 2003). One cluster corresponds to , 3.6 MDa, as predicted for a stoichiometry of 144 globin and 36 linker chains-the Vinogradov model [(abcd) 3 L 3 ] 12 (Martin et al., 1996) for the hexagonal bilayer (HBL) assembly of Lumbricus Ec-and as estimated from the crystal structure of HBL at 5.5 A ˚ resolution (Royer et al., 2000). The other cluster corresponds to , 4.4 MDa (Fig. 1). In a previous publication, Tsfadia and Daniel (1999) proposed for the 4.4 MDa molecule a stoichiometry of 192 globin and 36 linker chains [(abcd) 4 L 3 ] 12 . Reformulation to [(abcd) 3 L 3 ·abcd)] 12 brings the model into consistency with the establishment of a local three-fold axis within the one- twelfth molecule. The proposed stoichiometry for the 4.4 MDa molecule can be viewed as HBL that had been supplemented by 48 globin (12 abcd) chains. Where in the molecule are the latter located? We propose that the 12 abcd moiety is equally allotted to the two halves of the central cavity around the six-fold axis above and below the central torus of the HBL structure. There is supporting circumstantial evidence for this proposal: (i) Electron microscopy of Ec of isolated annelid species, e.g. Oenone fulgida (Van Bruggen and Weber, 1974) and Eophilia tellinii (Cejka et al., 1989) shows the presence of central subunits; (ii) There is enough space for the extra mass (, 0.8 MDa) at the indicated location (Schatz et al., 1995); (iii) The proposed model provides a plausible mechanism for the ‘shedding’ of subunits brought about by oxidation (Zhu et al., 1996): ½ðabcdÞ 4 L 3  12 ¼ ½ðabcdÞ 3 L 3  12 þ 12abcd ð1Þ There is, however, no evidence from EM, cryo-EM or crystal structure for the presence of central subunits in Lumbricus Ec. The report on the occurrence in EM preparations of Ec from E. tellinii of molecules with and molecules without central subunits (Cejka et al., 1989) strongly suggests that the reason may reside in the propensity of 4.4 MDa molecules to lose their central subunits in procedures aimed at visualization in EM or cryo-EM or crystal structure determination of the protein. There is a consensus that the sedimentation coefficient of Lumbricus Ec is , 60S. Which of the two molecules—3.6 or 4.4 MDa—is 60S Lumbricus Ec? A hint at an answer is provided by the VW rule (Van Bruggen and Weber, 1974) which correlates the absence ‘ 2 ’ or presence ‘ þ ’ of central subunits with the magnitude of the sedimentation coefficient, 50S ‘slow’ for Arenicola marina (2 , slow) or 60.5S ‘fast’ for O. fulgida (þ , fast). What about Lumbricus Ec? Identification of the 3.6 MDa HBL structure with the 60S molecule of Lumbricus Ec would lead to a dyad representation: Lumbricus 3:6 MDaðHBlÞEc : ð2; fastÞ ð2Þ 0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.10.042 Micron 35 (2004) 131–132 www.elsevier.com/locate/micron * Corresponding author. Fax: þ 972-3-6406834. E-mail address: danielez@post.tau.ac.il (E. Daniel).