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Ferricyt c from horse heart (Sigma) was dissolved in sodium phosphate buffer (50 mM, pH 7.2), purified on a Sephadex G-25 gel filtration column, and diluted to a final concentration of 50 μM the same buffer.
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Under these conditions, a protein surface coverage of approximately 1 monolayer is obtained. Lee, J. E.; Saavedra, S. S. Manuscript submitted for review.
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We investigated the influence of the imaginary part of the refractive index of the protein film, assuming a surface coverage of 1 monolayer. The absorbances at 550 nm, where the molar absorptivity of ferrocyt c is greatest in the spectral region of interest, were calculated using a rigorous wave model (Li, L. J. Opt. Soc. Am. A 1994, 11, 984-991) and the ray optics model. Good agreement was found (a difference of ca. 10%), which is expected because of the extremely weak absorbance of the monolayer film.
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