A method is provided for forming a monomolecular layer of cytochrome b562 at the air/water interface by introducing dialkyl chains onto a water-soluble cytochrome b562. The purpose of the present study was to form densely and/or regularly packed two-dimensional arrays of protein molecules. Protoheme, which is a prosthetic group of cytochrome b562, was extracted from cytochrome b562, and then dialkylated(dioctadecylated) with the synthetic-chemical procedure. This dioctadecylated heme was reconstituted with apo-cytochrome b562 in dimethyl sulfoxide-water mixtures in vitro. The reconstitution reaction was successful when the reaction time was greater than 50 min, the dimethyl sulfoxide content was 63% and the temperature was maintained at 10 °C. Surface pressure-area per molecule isotherm of the reconstituted dioctadecylated holo-cytochrome b562 was measured and this clearly shows the monolayer formation of the water-soluble protein on the water subphase. The coefficient of compressibility of the protein film was estimated and is discussed. It is found that reconstituted dioctadecylated holo-cytochrome b562 forms a stable and closely packed monomolecular layer on the water surface.
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