title: A Method To Measure Protein Unfolding at an Air-Liquid Interface.
abstract: Proteins are surface-active molecules that have a propensity to adsorb to hydrophobic interfaces, such as the air-liquid interface. Surface flow can increase aggregation of adsorbed proteins, which may be an undesirable consequence depending on the application. As changes in protein conformation upon adsorption are thought to induce aggregation, the ability to measure the folded state of proteins at interfaces is of particular interest. However, few techniques currently exist to measure protein conformation at interfaces. Here we describe a technique capable of measuring the hydrophobicity, and therefore the conformation and folded state, of proteins at air-liquid interfaces by exploiting the environmentally sensitive fluorophore Nile red. Two monoclonal antibodies (mAbs) with high (mAb1) and low (mAb2) surface activity were used to highlight the technique. Both mAbs showed low background fluorescence of Nile red in the liquid subphase and at a glass-liquid interface. In contrast, at the air-liquid interface Nile red fluorescence for mAb1 increased immediately after protein adsorption, whereas the Nile red fluorescence of the mAb2 film evolved more slowly in time even though the adsorbed quantity of protein remained constant. The results demonstrate that hydrophobicity upon mAb adsorption to the air-liquid interface evolves in a time-dependent manner. Interfacial hydrophobicity may be indicative of protein conformation or folded state, where rapid unfolding of mAb1 upon adsorption would be consistent with increased protein aggregation compared to mAb2. The ability to measure protein hydrophobicity at interfaces using Nile red, combined with small sample requirements and minimal sample preparation, fills a gap in existing interfacial techniques.
authors.affiliation: Early Stage Pharmaceutical Development and ‡Late Stage Pharmaceutical Development, Genentech , South San Francisco, California 94080, United States.
authors.forename: Danielle L
authors.initials: DL
authors.lastname: Leiske
authors.affiliation: Early Stage Pharmaceutical Development and ‡Late Stage Pharmaceutical Development, Genentech , South San Francisco, California 94080, United States.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Shieh
authors.affiliation: Early Stage Pharmaceutical Development and ‡Late Stage Pharmaceutical Development, Genentech , South San Francisco, California 94080, United States.
authors.forename: Martha Lovato
authors.initials: ML
authors.lastname: Tse
mesh_terms: D000388:Air; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D057927:Hydrophobic and Hydrophilic Interactions; D010078:Oxazines / Q000737:chemistry; D058767:Protein Unfolding; D013500:Surface Tension; D014867:Water
chemical_list: D000911:Antibodies, Monoclonal; D010078:Oxazines; D014867:Water; C044808:nile red