Benzyl alcohol-induced destabilization of interferon-gamma: a study by hydrogen-deuterium isotope exchange.

The destabilizing effect of a multidose preservative, benzyl alcohol, on IFN-gamma was investigated. Hydrogen-deuterium isotope exchange (HX) detected by mass spectrometry (MS) was used to detect tertiary structure changes and measure global unfolding rates. The experiments showed that tertiary structure changes previously reported using circular dichroism may involve only a limited portion of the protein with the hydrophobic core of the protein remaining intact. Protein unfolding rates measured by hydrogen exchange were very sensitive to benzyl alcohol concentration, and increased markedly when salt was also added. Dynamic light scattering and size-exclusion chromatography showed that a small fraction of the protein formed large aggregates during the first few days. Measurements at longer incubation times (up to 8 days) showed that a significant fraction of protein was trapped in a structure less protected from hydrogen exchange, but not completely unfolded. This fraction of protein may be responsible for the irreversible loss of activity observed in earlier studies.

Aprotinin conformational distributions during reversed-phase liquid chromatography. Analysis by hydrogen-exchange mass spectrometry.

Hydrogen-exchange mass spectrometry analysis of the stable protein aprotinin during reversed-phase liquid chromatography shows both native and unfolded protein. The behavior is consistent with only two conformational states, a near-native state and a fully solvent-accessible state, with reversible interchange of species within and between the mobile and stationary phases. The amount of unfolded form is greater on C18 relative to C4 alkyl modified silica surfaces. The addition of (NH4)2SO4, Na2SO4, NaCl, or NaSCN to the mobile phase stabilized native conformation on the chromatographic surface, especially on the C4 media. Finally, the retention and the proportion of denatured form increases with added salts in anorder consistent with the lyotropic series, but reversed from that observed for small molecules.