Artemia hemoglobins : Increase in net synthesis of the β-polypeptide (relative to the α-polypeptide) in hypoxia

Abstract

Previous studies have shown that in the brine shrimp there are three dimeric hemoglobins with polypeptide composition α 2, αβ, β 2. Concentrations of the α- and β-polypeptides increase in hypoxia. We now report a two-dimensional electrophoretic method for assay of radiolabelled polypeptidesin each hemoglobin. Net synthesis (synthesis minus degradation) of the β-chain, relative to that of the α-chain, increases more than 3-fold (in male and female adults) within 3 days following a downshift in oxygen concentration from 0.2 to 0.1 mM in the culture medium. 3 days after downshift (2 days after in vivo incorporation of radiolabelled leucine), the β-homodimer contained 10–20% of the radiolabel in the three hemoglobins although β 2 was usually not detectable in the protein stain of an overloaded gel. The amount of radioactive leucine incorporated per unit amount of protein was more than 300-times greater in the β 2 homodimer than in the β-subunit of the heterodimer, suggesting that β 2 does not dissociate rapidly during electrophoresis on the first dimension non-denaturing gel. This evidence for stable association of the two β-monomers and the 5–8 heme-binding domains within each monomer (in vivo and during electrophoresis on non-denaturing gels) allows us to exclude one of two alternative interpretations of genetic data published previously. We present an independent line of evidence for the dimer model of the native hemoglobins (which states that each polypeptide has many heme-binding domains).