Heterogeneity of the subunits of Lumbricus terrestris extracellular hemoglobin dissociated at alkaline pH.
1. The gel filtration profiles of dissociated extracellular hemoglobin of Lumbricus terrestris obtained in 0.1 M borate buffers at pH greater than 9, using columns of Sephadex G100, Sephacryl S200 and Ultragel AcA 44, consist of at least two peaks A and B. 2. SDS-PAGE of several fractions across the complete elution profile demonstrates that only the fractions under the right hand portion of peak B are homogenous and consist of the monomer (M) subunit (Mr = 17,000). 3. The fractions under the first peak contain the remaining subunits, a disulfide-bonded trimer (T) subunit (Mr = 50,000) and of two subunits (D1 and D2) of ca 30,000. 4. Densitometry of the SDS-PAGE patterns suggests that the proportions of these subunits vary across the two peaks, implying that peak A does not consist of a complex of fixed stoichiometry of the T and D1 and D2 subunits. 5. Furthermore, the D1 and D2 subunits overlap the M subunit in the trough between peaks A and B and are present in the left hand portion of peak B, probably because of the self-association of the M subunit. 6. In addition, SDS-PAGE experiments with a single fraction of peak A, where the load and the duration of staining were varied, suggests that the relative proportions of the subunits are independent of these two variables.
Sharma
P K
PK
Department of Biochemistry, Wayne State University School of Medicine, Detroit, MI 48201.
Walz
D A
DA
Vinogradov
S N
SN
D000818:Animals; D000845:Annelida / Q000032:analysis*; D002850:Chromatography, Gel; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008970:Molecular Weight
D004220:Disulfides; D006454:Hemoglobins; D046911:Macromolecular Substances