Figure 4. Apparent rate constants calculated for activation and polymerization of control α₁-antitrypsin and reactive loop mutants.

(A) The inverse relationship between the natural logarithm of the apparent rate of polymerization, k_app,fr (calculated for each variant as shown in Figure 2D), and temperature was used to determine the energy of activation of the reaction, E_act. Each data point is from least ten independent experiments. (B) The calculated energy of activation, E_act, for the heat-induced polymerization of each variant is shown. (C) The change in intrinsic tryptophan fluorescence was monitored for the variants at 55°C using an LS55B instrument with a stirred cuvette and excitation at 280 nm and emission at 340 nm (open squares). Protein concentration was 0.1 mg/ml in 800 μl polymerization buffer. Values shown (k_app,fl) are from three to five independent experiments and are the result of fitting a single exponential equation to the data [14]. The combined results of the FRET assay at this temperature are shown for comparison (k_app,fr) interpolated from the linear regressions in panel (A) (closed circles). (D) The change in fluorescence in the presence of 10 μM bis-ANS was followed at 55°C in a cuvette or plate reader format, and fitted to a single exponential equation (closed circles). The resulting rates (k_1,ans) were calculated from seven independent experiments. Alongside these values are the rates of change of circular dichroic ellipticity at 222 nm (k_1,cd), calculated from two to three independent experiments for each point (open squares). An asterisk ‘*’ indicates a significant decrease (P=0.035) with respect to the control as determined by one-way ANOVA using the Bonferroni multiple test correction.