FIGURE 2.

Heat-induced irreversible denaturation of anti-hCG VHH by repetitive heating-cooling cycles or continuous incubation at a high temperature and denaturation temperature dependence. A, anti-hCG VHH (1.3 μg/ml) was subjected to heating-cooling cycles (circles) or continuous incubation at 90 °C (triangles). The experiments were carried out at pH 7.4 in HBS-EP buffer. In heating-cooling cycles, a given number of reaction segments, which consisted of heating at 90 °C for 5 min and cooling at 20 °C for 5 min, were repeated. Residual activity at 20 °C was estimated using the strength of SPR signals compared with that of SPR signals from a series of diluted, untreated samples and was expressed as a fraction of the corresponding diluted, untreated sample. Because the time for unfolding was 5 min, one cycle corresponded to 5 min of incubation. The solid line represents a single exponential curve fitted to the VHH time-dependent denaturation where the first-order kinetic constant k_den was 0.0039 min⁻¹ and the time to reach half of the original activity was calculated to be 178 min. B, VHH (1.3 μg/ml) was subjected to 80 heating-cooling cycles (circles) or continuous incubation (400 min (m); triangles) where the heating temperature ranged from 40 to 90 °C in HBS-EP buffer. Inset, VHH (1.3 μg/ml) was incubated at 37 °C for the indicated time in HBS-EP buffer. In all panels, error bars represent S.D.