FIG 8.

Effect of salicylic acid on heme transfer and binding. (A) The different ratios of absorbances at 409 nm and 396 nm between heme bound to HSA and heme to Csa2 were used to monitor transfer of heme from HSA to Csa2. HSA (10 μM) was preloaded with 5 μM heme in regular PBS buffer or in buffer supplemented with 1 mM or 2 mM salicylic acid (SA). (B) HSA (20 μM) was incubated 5 min with 10 μM hemin in 50 μl PBS buffer, without or with 2 mM or 4 mM SA, and then loaded on a 24-ml SEC column loaded with PBS buffer or preloaded with a 5 ml layer of PBS–2 mM SA or PBS–4 mM SA, respectively (the high 280-nm absorbance level of SA precluded loading the whole column with PBS plus SA). The HSA protein peak was detected by monitoring absorbance at 280 nm, and the heme bound to HSA was detected at 406 nm.