5mg/mL, 200μl). After 4h mixing with a rotator, centrifugation (15,000g, 20°C, 60min) was performed to collect the supernatants. These were analyzed by reversed phase high performance liquid chromatography (HPLC) to estimate the residual concentration of cytochrome c. The HPLC system was equipped with a cosmosil 5C18-MS-II column (Nacalai Tesque, Inc., Kyoto, Japan) and a UV detector (220nm; UV-2075Plus, Jasco Inc., Tokyo, Japan).

Samples (5μl) were injected Inhibitors,research,lifescience,medical with an autosampler (AS-2057Plus, Jasco Inc., Tokyo, Japan) and eluted with acetonitrile/0.05% trifluoroacetic acid = 20/80 (A) and acetonitrile/0.05% trifluoroacetic acid = 60/40 (B) at 1.0mL·min−1 by PU-2089Plus (Jasco Inc., Tokyo, Japan). A linear gradient elution was performed over 20min from an initial state (A) 100% to the final state (B) 100%. In the case Inhibitors,research,lifescience,medical of insulin adsorption, the same experimental procedures were performed except the insulin solution was prepared by dissolving it in 0.01N HCl and adjusting to pH 3. Association ratio (%) was calculated as [(C0−C)/C0] ×100, at which

Inhibitors,research,lifescience,medical C0 and C are the initial concentration and the supernatant concentration of proteins, respectively. During desorption experiments, HA (10 and 20mg) absorbing cytochrome c and insulin was transferred into 400μl of phosphate buffer saline (PBS; 8mM Na2HPO4, 2mM KH2PO4, 137mM NaCl, 3mM KCl), and rotated. After predetermined incubation times, centrifugation (15,000g, 20°C, 60min) was performed to collect the supernatants. The residual concentrations of cytochrome c and insulin were estimated by HPLC. In the case of insulin, PBS adjusted

to pH 3 was also used as the incubation buffer. Dissociation ratio (%) Inhibitors,research,lifescience,medical was calculated as [C/C0] ×100, at which C0 and C are the total concentration of the associated proteins and the supernatant concentration, respectively. 3. Results and Discussion The association experiments were performed by mixing HA and protein solutions. Cytochrome c was soluble in deionized water, but insulin was not. Therefore, insulin was dissolved in an Inhibitors,research,lifescience,medical acidic solution (pH 3). After the incubation and subsequent centrifugation, the residual cytochrome c and insulin in the solution were estimated from the HPLC analysis. Cytochrome c and insulin were eluted after 10min and 13min, respectively, under the running conditions only (Figure 1(a)), and the peak areas were proportional to the protein concentrations (Figure 1(b)). Thus, the protein concentrations in the Raf inhibitor supernatants were evaluated by HPLC analysis and the adsorbed amounts were calculated by subtracting the concentrations in the supernatant from the initial ones. Figure 2 shows the association ratio of these proteins on HA. Both proteins were associated with HA after the 4h incubation. The adsorption efficiency of insulin was higher than that of cytochrome c. As less as 10mg HA was sufficient to load almost 0.