Denaturation Resistance of β-Lactoglobulin in Monomolecular Films at the Air−Water Interface

Using X-ray reflectometry we report strong differences in the denaturation response of β-lactoglobulin adsorbed as a monomolecular film at the air−water interface from that observed in mixed denaturant/β-lactoglobulin bulk solutions. Using the “flow trough” technique an isolated monomolecular film of the protein showed little change in structure when subjected to a 4.0 M guanidinium hydrochloride substrate. Unlike the bulk solution where a new protein layer structure appears, small changes in the protein packing and the roughness of the film are the only evidence of change. These parameters have been studied as a function of denaturant concentration and film quality. The strength of the response depends on the degree of perfection of the originally formed film; quickly formed films are more easily denatured. As the response is so subtle, possible interfering effects such as denaturant release of protein adsorbed on the trough have been quantified.