Evidence for increased proteolysis in intact beta thalassemia erythroid cells

Much excess alpha chain is synthesized, but little accumulates in the erythroid cells of patients with homozygous beta thalassemia. To determine if the proteases known to exist in erythroid cells play a role in the destruction or alteration of any of this excess alpha chain, thalassemic and nonthalassemic erythroid cells were incubated for 90 minutes with 3H-leucine. The cells were then washed, and incubated twice for 15 minutes in 100 volumes of cold leucine-rich media, a procedure which eliminates almost all intracellular TCA soluble radioactivity. After these incubations levels of TCA soluble and TCA precipitable radioactivity in the cell lysates were determined, and the cells incubated for 120 minutes more in two volumes of leucine-rich media. At the end of this incubation, total TCA soluble and precipitable radioactivity was again determined in the cell lysate, and also in the two hour incubation media. The total increase in TCA soluble radioactivity in the cells and their media was divided by the 0 time TCA precipitable radioactivity, to determine the percent proteolysis labelled globin chains. In five control patients percent proteolysis ranged from 0 to 3.10 (mean = 1.50); in four severe and three mild thalassemia patients percent proteolysis ranged from 5.80 to 14.1 (mean = 11.0). The difference between the control and thalassemic groups was significant at a p of less than 0.001. This data is the first direct evidence that more proteolysis takes place in intact thalassemic cells than in non-thalassemic cells.