Stoichiometry of subunits and heme content of hemoglobin from the earthworm Lumbricus terrestris.
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 approximately 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).
Zhu
H
H
Department of Zoology, University of Texas, Austin, Texas 78712-1064, USA. Riggs@uts.cc.utexas.edu
Hargrove
M
M
Xie
Q
Q
Nozaki
Y
Y
Linse
K
K
Smith
S S
SS
Olson
J S
JS
Riggs
A F
AF
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D009835:Oligochaeta / Q000378:metabolism*
D006454:Hemoglobins; D006418:Heme