How to obtain a well-spread monolayer of lysozyme at the air/water interfaces.

Abstract The purpose of this study is to define the conditions required to obtain a complete spreading of the lysozyme monolayer at the A/W interface. To this end, using Trurnit’s method, the influence of the ionic strength of the substrate, the elapsed time between the spreading of the monolayer and the beginning of its compression, and the number of lysozyme molecules spread at the interface was studied. The results obtained show that the lysozyme spreading is conditioned by the unfolding of amino acid chains which form part of its structure, so that such unfolding is hindered, either because of an excessive accumulation of lysozyme molecules on the substrate surface or because the waiting time necessary to get this unfolding is not long enough, regardless of the number of spread molecules. The complete unfolding of lysozyme, which involves the loss of tertiary and secondary structures, has been obtained in this work under certain conditions: (1) using a substrate with a high saline concentration (NaCl 3–3.5 M) and at a pH value close to the isoelectric point of lysozyme (pI = 11) in order to reduce the solubility and the electrical potential of the protein, (2) increasing the elapsed time between the spreading of the protein and the beginning of its compression to 3 h in order to get a complete unfolding of lysozyme, which is a very slow process, (3) using an initial spreading area of 2.1 m 2 /mg or larger (equivalent to a protein surface concentration of less than 2 × 10 12  molec/cm 2 ) to reduce the accumulation of protein molecules at the surface, thus facilitating their unfolding, and (4) using Trurnit’s spreading method, although for the spreading of this protein was found to be insufficiently significant.