## 4 Discussion  
Commercially available lyophilized ConA contains two different components: one shows low affinity for binding to glucose and mannose, and the other displays high binding affinity. Increase in the mannose binding ability of ConA depends on the water content, which suggests that reaturation of low-binding ConA was affected by water states (free water or binding water) in hydrated ILs. Furthermore, the anion structure of hydrated ILs influenced the recovery of the binding ability. Hydrated ILs composed of dhp anion (a kosmotropic ion) showed an effective increase in mannose binding ability. On the other hand, the effect of the cation was not so obvious. Similar results with [ch][dhp] were observed in ammonium and phosphonium ILs with dhp anion. It was considered that the hydroxyl group in the choline cation was not a key structure for the renaturation of lyophilized ConA. These conditions suggest that hydrated ILs, which contain almost no free water and have high hydrogen bonding ability, have the potential to aid the renaturation of inactive component in lyophilized ConA. Authors have reported that the hydration state in hydrated ILs depends on the water content using different methods such as water activities analysis, DSC, NMR measurements, and molecular simulation investigations [24, 25, 26, 27]. Water molecules in hydrated ILs behave not as free water but bound water when the water content is below seven water molecules per ion pair [24]. Considering the decline in mannose binding ability with increasing water content, existence of free water is perhaps unnecessary for the stable dissolution and restructuring of ConA.  
After treatment of lyophilized ConA in hydrated [ch][dhp] with three water molecules per ion pair, an increase in mannose binding ability was observed. Both components of ConA, those with low and high mannose binding ability, showed an increase in mannose binding ability. The increased ratio of this binding ability was different; the mannose binding ability of these two components became similar after treatment with hydrated [ch][dhp] containing three water molecules per ion pair. Low mannose binding ConA showed a greater increase ratio than high mannose binding ConA. Interestingly, improvement in glucose binding ability was not observed. It was suggested that the glucose binding site was not affected by the restructuring in hydrated [ch][dhp]. ConA can recognize and bind to mannose and glucose among carbon hydrate chains. Originally, the dextran binding ability of ConA was lower than its mannose binding ability [28]. This might be related to the position of the binding site and/or binding manner to glucose.  
The observed increment in total mannose binding ability after treatment with hydrated [ch][dhp] (Figure 2, red line) was compared with that calculated using the increment of each response based on the separated ConA (Figure 4). As seen in the chromatogram  
Figure 4: Mannose binding ability of ConA eluent from the first peak (**a**) and in second half peak (**b**). Each sensorgram shows binding change before (black) and after (red) treatment with hydrated [ch][dhp].  
in Figure 2 insert, lyophilized ConA consisted of 20% low mannose binding ConA and 80% high mannose binding ConA. ConA without treatment showed a total binding value (\(\Delta\lambda\)) of 0.125 at 120 s (Figure 3, black line). The estimated binding values of low and high mannose binding ConA were 0.025 and 0.100, respectively. Estimated increases in the binding value after treatment were 0.083 for low mannose binding ConA and 0.180 for high mannose binding ConA. These were calculated by using increase rates of 3.3 and 1.8 times, respectively. When adding these values, the total increase in the binding value was estimated to be 0.263. The measured value (Figure 3) was 0.251. These values showed reasonable correlation.