Multi-variate approach to global protein aggregation behavior and kinetics: effects of pH, NaCl, and temperature for alpha-chymotrypsinogen A.

A global characterization of nonnative aggregation is presented for alpha-chymotrypsinogen A (aCgn) as a function of temperature (T), pH, and [NaCl]. Changes in unfolding free energy, native-state second osmotic virial coefficient (B(22)), and aggregation pathways and kinetics were qualitatively and quantitatively determined using a combination of size-exclusion chromatography, multi-angle laser light scattering, and circular dichroism and fluorescence spectroscopy. Results were analyzed quantitatively using multi-variate statistical models and a recently developed mechanistic model that naturally accounts for changes in aggregation pathway due to competition between unfolding, nucleation, chain polymerization, aggregate condensation, and phase separation. State diagrams are presented that show the natural progression between different aggregation behaviors or pathways. Together, the results show that pH and [NaCl] determine both the rates of aggregation and what aggregation behavior or pathway holds. In contrast, T affects primarily only aggregation rates, in large part due to changes in unfolding free energy. Finally, it is shown that B(22) correlates strongly with which type of aggregation pathway is followed, suggesting a potentially useful approach for predicting and controlling physical properties of the resulting aggregates. (c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association.