To test further the binding site of AOH in HSA, ultrafiltration experiments were carried out with markers of SSI (warfarin), SSII (naproxen), and Heme site (S-camptothecin, CPT). Ultrafiltration studies with warfarin and naproxen were performed as described [50 (link)]. Briefly, Pall Microsep™ Advance centrifugal devices were used with 10 kDa molecular weight cut-off value. Before the ultrafiltration, filter units were rinsed once with 3.0 mL distilled water and twice with 3.0 mL PBS. Samples contained warfarin and HSA (1.0 and 5.0 μM, respectively), naproxen and HSA (1.0 and 1.5 μM, respectively), or CPT and HSA (1.0 and 1.5 μM, respectively) with or without 10 or 20 μM of AOH in PBS. Samples were centrifuged for 10 min at 7500 g and 25 °C (fixed angle rotor). Filtrates were analyzed with HPLC-FLD or HPLC-UV methods (see details in Section 4.4 ). Warfarin and naproxen were directly analyzed from the filtrates; however, in order to prevent the spontaneous conversion of camptothecin to an open-chain carboxylate form [51 (link)], the following sample preparation was performed. A 500-μL aliquot of filtrates was acidified with 2 μL of 6 M perchloric acid, and after a four-fold dilution with the HPLC mobile phase (see in Section 4.4 ), camptothecin was analyzed by HPLC.
To validate our new ultrafiltration model which aims to investigate the Heme site, the effects of two known site markers (methyl orange and bilirubin) were tested. Both methyl orange and bilirubin were able to significantly increase the concentration of CPT in the filtrate (Figure S1 ), suggesting the displacement of CPT from HSA. Furthermore, bilirubin showed much stronger effect than methyl orange, which is in agreement with the significantly higher binding affinity of bilirubin towards HSA (compared to methyl orange and CPT) [26 (link)].
To validate our new ultrafiltration model which aims to investigate the Heme site, the effects of two known site markers (methyl orange and bilirubin) were tested. Both methyl orange and bilirubin were able to significantly increase the concentration of CPT in the filtrate (
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