Preparation and Properties of Six Rat Hemoglobins

Isoelectric focusing electrophoresis in polyacrylamide gels along a pH gradient from 6.0 to 8.0 yields six distinct rat red cell hemoglobin fractions. A preparative adaptation of this technique permitted the isolation of each of the six hemoglobins in a pure state. Their molecular weights are similar to that of human hemoglobin, they dissociate in dilute solution, and they separate completely into subunits of molecular weight 16,000 when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino acid content of all six fractions is similar but surface carboxyl groups vary in such a way as to account for the differences in isoelectric points. Hemoglobin Fraction IV is the major component in the red cell (36%), whereas Fraction V is the major hemoglobin in the bone marrow (37%) and spleen (38%). The erythroid cells of the marrow and spleen, in addition to the six hemoglobins observed in the red cell, contain two additional, more acidic, components. In vivo pulse labeling of marrow erythroid cells was performed in splenectomized rats with serum-bound 59Fe. Incorporation of the isotope into each of the six hemoglobins of circulating red cells at various time intervals yielded a pattern of specific activities, which leads to the conclusions that Fraction V is synthesized mostly in the youngest erythroid cells of the marrow, whereas Fraction IV is synthesized mostly in the older marrow cells.