Nucleation, growth, and activation energies for seeded and unseeded aggregation of α-chymotrypsinogen A

The intrinsic time scales for normative aggregate nucleation (T-n((0))) and chain growth (T-g((0))) were determined for (x-chymotrypsinogen A as a function of temperature under acidic conditions where the resulting aggregates do not appreciably condense. Previous results (Andrews and Roberts (2007) Biochemistry 46, 7558) indicated that the product T-n((0)) T-g((0)) increases with increasing temperature but could not distinguish T-n((0)) and T-g((0)) Separate experimental values of T-n((0)) and T-g((0)) are reported here from two approaches based on either (i) combining unseeded monomer loss kinetics with static light scattering of the resulting aggregates or (ii) seeded monomer loss kinetics as a function of number concentration of seed. Values of T-n((0)) and T-g((0)) from (i) and (ii) agree quantitatively, and indicate that nucleation has a large, negative effective activation energy (ca. -76 kcal/mol) while growth has at most a weak dependence on temperature. The results are consistent with a model in which nucleation requires significant conformational changes within a normative oligomer, beyond those for monomer unfolding. The results more generally illustrate the potential utility of approaches (i) and (ii) for quantitatively determining in vitro T-n((0)) and T-g((0)) values, as well as how the effects of seeding can be predicted purely from unseeded kinetics and static light scattering measurements prior to significant aggregate condensation.