Equilibrium time determination for denaturation and renaturation of monoTcTIM. IF was used as
10Panx probe and changes at native λmax (327 nm) after excitation at 280 nm were obtained. In the denaturation experiments, 150 μg mL?1 of monoTcTIM were incubated with increasing concentrations of Gdn-HCl. For the renaturation experiments, monoTcTIM (300 μg mL?1) was first denaturated in 6.0 M Gdn-HCl for 1 h. The renaturation process was initiated by diluting the samples to a final protein concentration of 5 μg mL?1 and the final denaturant concentrations were adjusted to the same concentration used in the denaturation experiments. The effect of the added (unfolding) or diluted (refolding) denaturant was kinetically monitored after excitation at 280 nm at the native λmax for 2 min. Data was periodically collected from 0 to 72 h. No significant differences in the IF values were found after 24 h of incubation for both denaturation and renaturation reactions at each denaturant concentration. The changes in IF were monitored using a
larynx DM45 OLIS spectrofluorometer with the cell compartment at 25 °C.
