Thermal stability and thermal reactivation of CKB SNP mutations.
(A) Thermal stability of WT and mutant CKs: WT (square), H26Y (circle), P36T (triangle) and K267E (diamond). The protein concentration of WT, P36T and K264E was 0.2 mg/ml. A relatively higher concentration (7.3 µmol) was used for H26Y. After being heated at the given temperatures for 10 min, the activity was immediately determined. The activity of the native enzymes at 25°C was assumed to be 100%. (B) Thermal reactivation time course of all enzymes: WT (filled square), H26Y (open square), P36T (filled circle), T59I (open circle), P67Q (filled triangle), K177R (open triangle), K267E (filled diamond), S309L (open diamond) and I360F (filled regular hexagon). Samples (0.2 mg/ml) were first heated at 45°C for 30 min and then cooled at 25°C. Activities at different time intervals were determined. Activities of untreated samples were assumed to be 100%. (C) and (D) are the enlarged part of the thermal reactivation time course. (E) The reactivation ability against thermal inactivation of WT and mutant CKs. Samples were prepared as described above. Reactivation of CKs was performed on ice overnight. Data are presented as the mean ± sd (n = 3).