Enhancing the diffusion rate of cytochrome c into fatty acid films by preordering the lipid film

Abstract

We have previously studied the electrostatic entrapment of the heme proteins cytochrome c (Cyt c) and hemoglobin in thermally evaporated fatty lipid films (Gole et al. Langmuir 2001, 17, 5646-5656). It was observed that the diffusion of Cyt c from solution into thermally evaporated arachidic acid films was extremely slow requiring ca. 40 h for protein equilibration in 500 Å thick films thus marking this out as a disadvantage of the protocol. Preliminary attempts to enhance the diffusion rate of Cyt c in arachidic acid films organized in a lamellar structure by Pb²⁺ ion intercalation were promising. In this paper, we examine the use of stearic acid films organized in a lamellar c axis oriented structure in greater detail and demonstrate that such films grown either by the Langmuir-Blodgett technique or by thermal evaporation followed by metal ion intercalation enhances the diffusion rate of Cyt c by over an order of magnitude. The entrapment of the protein molecules in the lamellar hydrophilic regions of the lipid matrix is also expected to play an important role in enhancing the accessibility of entrapped enzymes to substrates (mass transport effect) where biocatalytic application of enzyme-lipid bioconjugates is envisaged. The process of Pb²⁺ ion/Cyt c entrapment in thermally evaporated stearic acid films was followed by quartz crystal microgravimetry while the lamellar ordering of the films and formation of salts of stearic acid were inferred from X-ray diffraction and Fourier transform infrared spectroscopy measurements.