Turnover of excess hemoglobin alpha chains in beta-thalassemic cells is ATP-dependent.

Blood erythroid cells from five beta-thalassemic donors were incubated with [3H]leucine at 37 degrees C to label the pool of excess, free hemoglobin alpha chains. The 3H-labeled cells were split and reincubated in nonradioactive media that either supported ATP production (with 5 mM glucose) or inhibited ATP production (without glucose but with 20 mM 2-deoxyglucose and 0.20 mM 2,4-dinitrophenol). During the 6-h incubation in the glucose medium, the total cellular protein 3H radioactivity decreased about 30%, while the ATP levels remained constant. Chromatographic separation of the alpha- and non-alpha-globin chains of the crude (stroma included) lysates and electrophoretic separation of the free alpha chains and tetrameric hemoglobins of the stroma-free soluble phases both showed that degradation of the alpha chains was responsible for the decrease in protein 3H radioactivity. Conversely, in the energy-deprived cells, the ATP levels dropped to less than 10% of that of the energy-supported cells, and the turnover of alpha-globin 3H radioactivity of the crude lysates was only 5-10%. These results indicate that proteolysis of excess, newly synthesized alpha chains in beta-thalassemic cells is ATP-dependent. The accumulation, mostly in the stromal fraction, of intact 3H-alpha chains in the ATP-deprived cells suggests that an ATP-dependent step occurs early in the biochemical pathway of alpha chain proteolysis. Denaturation resulting in insolubility of the free alpha chains may be a recognition signal for activation of this proteolysis.