THE EXTRACELLULAR HEMOGLOBIN OF THE EARTHWORM, LUMBRICUS-TERRESTRIS -DETERMINATION OF SUBUNIT STOICHIOMETRY

The giant extracellular hemoglobin of the earthworm, Lumbricus terrest ris, has four major O2-binding chains, a, b, and c (forming a disulfid e-linked trimer) and d (''monomer''). Participation of additional ''li nker'' chains L1, L2, and L3 is necessary for the assembly of the almo st-equal-to 3,900+ kDa two-tiered hexagonal structure. We have determi ned the proportions of linker chains, trimer, and chain d in the hemog lobin by reverse phase high performance liquid chromatography which re solves all of the components and also permits simultaneous determinati on of the heme content. The proportions of components were determined by two independent procedures: integration of the absorbance peaks at 220 nm and amino acid analysis of the peak fractions. The results indi cate that the weight proportion of linker chains is 0.163 +/- 0.023. T his value, together with molecular masses determined both by amino aci d sequence analysis and by matrix-assisted laser desorption mass spect rometry, gives a molar ratio of abcd chains to linkers of 8:1, corresp onding to the minimal unit (abcd)2.L. This ratio suggests that 24 (abc d)2 units and 24 linker chains form the complete structure with a tota l calculated mass of polypeptide of 3,975 kDa with hemes on chains a, b, c and d and on one linker. The calculated heme content is 3.1% not including carbohydrate. This accounts for a measured heme content of 3 .0% on a polypeptide basis. Additional mass (almost-equal-to 133 kDa, 3.4%), attributed to carbohydrate, brings the total mass to 4,108 kDa with a minimum molecular mass/heme of 20,500 Da. The presence of equim olar quantities of three unique linker chains means that the apparent one-twelfth structural units seen by electron microscopy cannot all be identical.