SEPARATION OF HAEMOGLOBIN FRACTIONS IN RAT ERYTHROCYTES BY CHEMICAL AND PHYSICAL METHODS

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Abstract
SUMMARY: 59Fe which entered reticulocytes in vitro or was incorporated into developing red cells in vivo could be separated into fractions by a chemical procedure or by centrifugation of red cell haemolysates. The chemical separation depended on the solubility of the iron compounds in acid and in alkaline solutions and usually the ratio of the amounts of iron in the acid and alkaline extracts was 30 to 70. The addition of 10−3 M KCN to reticulocytes before addition of 59Fe changed this ratio to 85 to 15. Particulate matter separated from red cell haemolysates by centrifugation at 6,000 g. for 15 minutes also contained 59Fe. When the 59Fe was incorporated in vitro 28 p.c. was found in the stromal fraction and when 10−3 M KCN was used 90 p.c. was found in the stromal fraction. With in vivo incorporation the stromal fraction contained 19 p.c. of the 59Fe.The proportions of 59Fe extracted with acid or associated with the stroma were not related to the age of the reticulocytes and did not move to the other fractions on further incubation with iron. There is some evidence to suggest that the iron in the acid extract was that present in the stromal fraction but this has not been proved. However, the iron in all fractions must be principally haemoglobin iron obtained from functional haemoglobin. The separations achieved by the chemical and physical methods suggest that there are different forms of haemoglobin present in red cells which are capable of transporting oxygen.
Funding Information
  • National Health and Medical Research Council