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Volume 271, Number 47, Issue of November 22, 1996 pp. 29999-30006
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Stoichiometry of Subunits and Heme Content of Hemoglobin from the Earthworm Lumbricus terrestris

(Received for publication, June 28, 1996, and in revised form, September 5, 1996)

Hao Zhu , Mark Hargrove ^¶ , Qiang Xie , Yasuhiko Nozaki ^** , Klaus Linse , Sandra S. Smith , John S. Olson ^¶ and Austen F. Riggs

From the  Department of Zoology and  Protein Microanalysis Facility, University of Texas, Austin, Texas 78712-1064, ^¶ Department of Biochemistry and Cell Biology and the Keck Center for Computational Biology, Rice University, Houston, Texas 77005-1892, and ^** Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major O₂-binding chains, a, b, c (forming a disulfide-linked trimer), and d ("monomer"). Additional structural chains, "linkers," are required for the assembly of the ~200-polypeptide molecule. The proportion of linker chains had been reported to be one-third of the total mass on the basis of densitometry of Coomassie Blue-stained SDS-gels. Reverse-phase high performance liquid chromatography (HPLC), however, gave 16.3% linkers on the basis of both 220-nm absorbance 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 electrophoresis, 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. Measurement of the rate of hemin loss from oxidized L. terrestris Hb shows that high rates of loss can account for values of heme content significantly below 2.9% (or 0.26% iron).

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