Thermally induced aggregates of α-chymotrypsinogen A and bovine granulocyte-colony stimulating factor in acidic solutions were characterized by a combination of static and dynamic light scattering, spectroscopy, transmission electron microscopy, and monomer loss kinetics. The resulting soluble, high-molecular weight aggregates (∼103-105 kDa) are linear, semiflexible polymer chains that do not appreciably associate with one another under the conditions at which they were formed, with classic power-law scaling of the radius of gyration and hydrodynamic radius with weight-average molecular weight (Mw). Aggregates in both systems are composed of nonnative monomers with elevated levels of β-sheet secondary structure, and bind thioflavine T. In general, the aggregate size distributions showed low polydispersity by light scattering. Together with the inverse scaling of M w with protein concentration, the results clearly indicate that aggregation proceeds via nucleated (chain) polymerization. For α-chymotrypsinogen A, the scaling behavior is combined with the kinetics of aggregation to deduce separate values for the characteristic timescales for nucleation (τn) and growth (τg), as well as the stoichiometry of the nucleus (x). The analysis illustrates a general procedure to noninvasively and quantitatively determine τn, τg, and x for soluble (chain polymer) aggregates, as well as the relationship between τn/τg and aggregate M w.
Bibliographical noteFunding Information:
The authors gratefully acknowledge partial funding from the COBRE program of the National Center for Research Resources of the National Institutes of Health (grant No. P20 RR015588), Merck and Co., and the generous donation of bG-CSF by Pfizer Global R&D (Groton, CT).