Figure 2.

ALAS-2 activity is reduced in β-thalassemic erythroid precursors and is affected by oxidative stress. (A) ALAS-2 activity in normal and β-thalassemic erythroid cells at day 7 ( white bars: C7, T7) and day 14 of culture (gray bars: C14, T14). An aliquot of 20 μL of the cytoplasmic fractions of day 7 and 14 erythroid precursors from normal controls or β-thalassemic patients was diluted into the assay reaction mixture and activity was measured in the presence of 10 μM pyridoxal 5′-phosphate (see Design and Methods and Online Supplementary Design and Methods). The activity determined at 14 days in control cells was set as 100%. Experiments were repeated in triplicate, the error bars represent the S.E.M.; *P< 0.05 compared to control cells; °P<0.05 comparing 14 vs. 7 days. (B) Recombinant 5-δ aminolevulinate synthase (ALAS-2) activity in the presence of saturating concentrations of H₂O₂ or hemin. Error bars represent the means ± SD, *P<0.05 compared to control cells; °P<0.05 compared to baseline. Inset: The IC₅₀ value measured was then used to determine the K_i value. 5-δ aminolevulinate synthase (ALAS-2) activity in the presence of increasing concentrations of H₂O₂ or hemin. The IC₅₀ value measured was then used to determine the K_i value. Errors were less than 10%.