Identification of the "factor" in erythrocyte lysates which enhances colony growth in agar cultures.

The activity in human erythrocyte lysates which enhances colony growth of mouse bone marrow (BM) and other cell types in agar culture, could not be separated from hemoglobin (Hb). This conclusion was reached after various procedures, including purification of Hb in human hemolysates by crystallisation, separation of Hb into its major (A0) and minor (A1 and A2) components by DEAE-Sephadex chromatography and separation of a hemolysate into a Hb fraction and a non-Hb protein fraction by DEAE-cellulose chromatography; all resulted in the enchancement activity remaining with the Hb fraction. Separation of globins from rat or human lysates by an acid acetone precipitation, resulted in an acetone powder (AP) which retained the enhancement activity towards both mouse BM and tumour cell lines. The AP was separated into alpha and beta globins by chromatography on Sephadex G100 in 20% formic acid followed by CM-cellulose chromatography in a 8 M urea system. Since the enhancement activity is associated with both the alpha and beta globin peaks even under these dissociating conditions, it has been concluded that the enhancement factor in erythrocyte lysates is Hb itself. The enhancement activity of an AP is abolished by treatment with N-ethylmaleimide, suggesting that sulfhydryl groups in Hb are required for the activity.