Figure 3

Effect of ruxolitinib on heme-Fe(III), dansyl-arginine, and dansyl-sarcosine binding to HSA, at pH 7.3 and 37.0 °C. (A) Binding isotherms for heme-Fe(III) binding to HSA in the absence (circles) and presence of 9.0 μM (squares) and 30 μM ruxolitinib (triangles). The heme-Fe(III) concentration was 1.3 μM. The HSA concentration refers to that of the free protein. The analysis of data according to Eq. (1) allowed the determination of the following parameters: ⁰K_h = 0.076 ± 0.008 μM (circles), ^appK_h = 0.25 ± 0.03 μM (squares), and ^appK_h = 0.82 ± 0.09 μM (triangles). (B) Binding isotherms for dansyl-arginine association to HSA in the absence (circles) and presence of 10 μM (squares) and 40 μM ruxolitinib (triangles) (panel A). The HSA concentration was 2.7 μM. The dansyl-arginine concentration refers to that of the free chromophore. The analysis of data according to Eq. (1) allowed the determination of the following parameters: ⁰K_da = 27 ± 3 μM (circles), ^appK_da = 78 ± 8) μM (squares), and ^appK_da = 27 ± 3 μM (triangles). (C) Binding isotherms for dansyl-sarcosine association to HSA in the absence (circles) and presence of 10 μM (squares) and 100 μM ruxolitinib (triangles) (panel A). The HSA concentration was 2.7 μM. The dansyl-sarcosine concentration refers to that of the free chromophore. For clarity, the binding isotherms for dansyl-sarcosine association to HSA in the presence of 10 μM and 100 μM ruxolitinib (diamonds and triangles, respectively) have been arbitrarily up shifted of 0.3 and 0.6 units. The analysis of data according to Eq. (2) allowed the determination of the following values parameters: ⁰K_ds = 5.1 ± 0.5 μM (circles), ^appK_ds = 5.6 ± 0.6 μM (diamonds), and ^appK_ds = 4.9 ± 0.5 μM (triangles). (D) Dependence of the ^appK_h/⁰K_h ratio for heme binding to HSA on the ruxolitinib concentration. The analysis of data according to Eq. (3) allowed the determination of the value of K_r =3.1 ± 0.4 μM. The circle on the ordinate indicates the value of ^appK_h/⁰K_h = 1 obtained in the absence of ruxolitinib. (E) Dependence of the ^appK_da/⁰K_da ratio for dansyl-arginine binding to HSA on the ruxolitinib concentration. The analysis of data according to Eq. (3) allowed the determination of the value of K_r =4.6 ± 0.5 μM. The circle on the ordinate indicates the value of ^appK_da/⁰K_da = 1 obtained in the absence of ruxolitinib. (F) Dependence of K_ds for dansyl-sarcosine on the ruxolitinib concentration. Values of ^appK_ds are unaffected by ruxolitinib; in fact, the average value of ^appK_ds 5.1 μM corresponds to that of ⁰K_ds. K_h, K_da, and K_ds indicate the dissociation equilibrium constant for heme-Fe(III), dansyl-arginine and dansyl-sarcosine binding to HSA, respectively, in the absence and presence of ruxolitinib (i.e., ⁰K_h, ⁰K_da, and ⁰K_da; and ^appK_h, ^appK_ds and ^appK_ds, respectively). Where not shown, the standard deviation is smaller than the symbol. For details, see text.