Characterization of heme interactions with MP membranes. We synthetized MLVs or LUVs in vitro. (A) We incubated MLVs and LUVs (5000 vesicles/100 μL) in 50 μM heme or PBS (+ none) for 1 hour. Vesicles were then washed and concentrated in PBS. We quantified heme in vesicles alone and in heme-incubated vesicles by spectrophotometry (Abs398), compared with serial heme dilutions. Abs₃₉₈ was also assessed in the initial heme solution before (start) and after ultracentrifugation without vesicles (pellet). Abs₃₉₈ was measured in vesicles prior to or after incubation in heme. ^$P < .05 vs heme after ultracentrifugation (pellet); ^$P < .05 vs vesicles + none. (B) Heme-incubated MLVs were treated for 1 hour with triton and saponin (up to 5%) or Hpx (10 μM). Abs₃₉₈ was then measured after de novo ulracentrifugation. *P < .01 vs MLV + none; ^†P < .05 vs lower detergent concentrations. (C) SCD erythrocyte MPs were incubated with triton or saponin (up to 5%), and Abs398 was measured compared with a heme dilution curve. *P < .05 vs MPs + none; ^†P < .05 vs low detergent concentrations. (D) In similar experiments, initial MP heme content was expressed at 100%. Control and SCD erythrocyte MPs (5000 PS+ MPs/100 μL; n = 4) were incubated with Hpx (2 μM) or EGTA-EDTA mix (both at 250 μM). *P < .05 vs MP + none.