STOICHIOMETRY OF SUBUNITS AND HEME CONTENT OF HEMOGLOBIN FROM THE EARTHWORM LUMBRICUS-TERRESTRIS

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestr is, has four major O-2-binding chains, a, b, c (forming a disulfide-li nked trimer), and d (''monomer''), Additional structural chains, ''lin kers,'' are required for the assembly of the similar to 200-polypeptid e molecule, The proportion of linker chains had been reported to be on e-third of the total mass on the basis of densitometry of Coomassie Bl ue-stained SDS-gels, Reverse-phase high performance liquid chromatogra phy (HPLC), however, gave 16.3% linkers on the basis of both 220-nm ab sorbance and amino acid analysis (Ownby, D. W., Zhu, H., Schneider, K. , Beavis, R. C., Chait, B. C., and Riggs, A. F. (1993) J. Biol. Chem. 268, 13539-13547). The subunit proportions have now been redetermined by SDS capillary electrophoresis as a test of the HPLC results, The el ectrophoresis, monitored at 214 nm, avoided the use of Coomassie Blue and provided results identical with those obtained by HPLC, Capillary electrophoresis monitored at both 214 and 415 nm was used to show that linker chains do not bind heme, Heme content has been found to be 2.9 % by determination of hemin, amino acid analysis and dry weight, Measu rement of the rate of hemin loss from oxidized L. ferrestris Hb shows that high rates of loss can account for values of heme content signifi cantly below 2.9% (or 0.26% iron).