Structure and morphology of bovine serum albumin–lysozyme (BSA–Lys) complex films at air–water interface

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The heteroprotein complex formed between the two globular proteins, bovine serum albumin and lysozyme was studied at the air–water interface by varying the subphase pH condition (pH ≈ 4.0–12.0). The surface pressure–mean molecular area isotherms of bovine serum albumin–lysozyme complex monolayers showed that relatively lower value of area per molecule was obtained at pH ≈ 9.2 in contrast to the other subphase pH conditions. Stability of the complex monolayers were analyzed from the normalized molecular area–time curves, which validated the formation of a relatively more stable film at pH ≈ 9.2. The in – situ study of the complex films were explored from Brewster angle microscopy and the corresponding images showed the evolution of elongated and compact circular domains at pH ≈ 7.0 and 9.2 respectively, at a higher surface pressure of 18 mN/m, while mostly homogenous films were visible at all the other pH conditions. The complex films were transferred from the water subphase on to the silicon substrates at a surface pressure of 18 mN/m for further study. The out–of–plane structure and in–plane morphology were obtained from X–ray reflectivity and atomic force microscopy analysis respectively, which showed the formation of a relatively thicker film deposited at pH ≈ 9.2. This unique pH value, i.e., pH ≈ 9.2, which is located in between the isoelectric point of bovine serum albumin and lysozyme, is the preferred pH condition for complex formation through electrostatic attraction of the two protein molecules. The biofilm of this stable protein complex formed at the air–water interface may have potential applications in thin film technology. • Bovine serum albumin–lysozyme complex films are formed on water surface. • Lower value of area per molecule for initial rising of pressure is obtained at pH ≈ 9.2. • Mostly elongated and compact circular domains are found at pH ≈ 7.0 to 9.2. • Relatively thicker film is formed at pH ≈ 9.2 where complex formation is maximum. • Stability of such complex film is maximum at pH ≈ 9.2.