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.

Interfacial Adsorption of Monoclonal Antibody COE-3 at the Solid/Water Interface

“…Protein adsorption at the solid/water interface has been studied by a number of groups using techniques such as ellipsometry, fluorescence and quartz crystal microbalance. [11][12][13][14][15][16] These reports provide a useful consensus about the typical ranges observed for the amount of protein adsorbed and its physical state, under well-defined surface and solution conditions.…”

“…Although these studies have demonstrated the relevance of adsorption and desorption processes to structural perturbation and solution aggregation, [10][11][12][13][14][15][16] little insight about mAbs within the adsorbed layers have been reported. Neutron reflection (NR) is a powerful tool able to reveal the thickness and composition of multiple adsorbed protein layers.…”

Thermal Degradation of Anthocyanins in Butterfly Pea (<i>Clitoria ternatea</i> L.) Flower Extract at pH 7

“…In this case, water force the hydrophobic group of polyacylated anthocyanins to interact with the hydrophobic surface and the intramolecular copigmentation disrupted. The occurrence of hydrophobic interactions unfolding caused by the hydrophobic surface had been reported in protein [13,14,15,16]. In addition, the presence of headspace in phials or container was reported to accelerating the recombinant fusion protein [17].…”

Foam fractionation for enhancing silica gel adsorption of urokinase from human urine

“…Five vertical sieve trays were installed inside the column with a tray spacing of 150 mm and the first one at 200 mm from the column bottom. Their structural parameters were the same as those of Zhang et al A gas distributor (1) made of sintered glass with a pore diameter of 100 ± 20 μm was installed at the column bottom. Through the gas distributor, air was bubbled into the foam fractionation column by an air compressor (ACO‐018A, Guangdong Resun Group Co. Ltd., China), and its volumetric flow rate was controlled by an air rotor flowmeter (60–600 ml/min, LZB‐2, Changzhou Shengzhiyuan Instrument Co. Ltd., China).…”

“…In foam fractionation of proteins, a certain loss activity is often observed due to structural unfolding of protein molecules induced by the gas–liquid interface . In addition, the lower protein concentration results in a larger activity loss .…”