Abstract

The accurate structural characterization of macromolecules in solution is often complicated by sample heterogeneity and polydispersity, which can mask subtle but biologically significant conformational states. Size-Exclusion Chromatography coupled with Small-Angle X-ray Scattering (SEC-SAXS) has emerged as a powerful technique to overcome these limitations by separating components immediately prior to X-ray exposure. This study investigates the capabilities and limitations of the SEC-SAXS method for the analysis of highly polydisperse systems, using the iron-storage protein Apoferritin as a model system. Apoferritin, a 24-subunit cage-like protein critical for cellular iron homeostasis, is known to exhibit concentration-dependent association and structural variability, making it an ideal candidate for testing separation-coupled techniques. Our results demonstrate that SEC-SAXS effectively resolves distinct oligomeric and conformational species of Apoferritin, providing high-resolution scattering data for each fraction. The analysis highlights the method's capacity to accurately determine the size, shape, and molecular weight of individual components within a complex mixture, revealing insights into the equilibrium of Apoferritin assembly. This work validates SEC-SAXS as an indispensable tool for the structural biology of polydisperse systems, offering a critical advantage for studying complex cellular components and their dynamic interactions.