Figure 3
Thermal inactivation kinetics of SOD.
1.0-ml solution of 0.2 mg/ml of each enzyme in 50 mM potassium phosphate buffer, pH 7.8, was incubated at 80°C for 160 min. Samples (100 µl) were removed after every 20 min and stored on ice. After 160 min, all samples were assayed for SOD activity under standard assay conditions using NBT-based assay. The data were fit as zero, first and second order reactions18, wherein the first-order reaction gave the most linear relationship and shown in this Fig. 3. SOD activity was not detected for C56A and C145A and hence corresponding activity curves are not shown. Solid and dashed lines represent WT and C95A, respectively. Values were mean ± SE of three separate replicates. WT, wild type; C95A, cysteine at position 95 substituted with alanine. The above figure was also used to calculate various kinetic parameters as shown in Supplementary Table S1. The rate constant kd (min−1) and t1/2 for first-order thermal inactivation were determined51,52 from “equation (1)” and “equation (2)”, respectively (equations described in “Methods” section). At, residual activity that remains after heating the enzyme for time t; A0, initial enzyme activity before heating.
