SANS studies of concentrated protein solutions: Determination of the charge, hydration, and H/D exchange in cytochrome C

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Abstract
Small angle neutron scattering (SANS) was used to study a series of concentrated protein solutions containing horse heart cytochrome C ranging from 0.6 to 24 g/dL at two ionic strengths. Since the solutions were manifestly nonideal throughout the whole concentration range, a contrast variation method applicable at finite Q (magnitude of the scattering vector) was applied to extract three basic properties of the protein in mixed D2O/H2O solutions at a concentration of 12 g/dL. These properties are the dry volume, the hydration, and the amount of H/D exchange of the protein in D2O‐containing solvents. These three basic parameters were then used as an input to extract the protein charge at all other concentrations. A generalized one‐component macroion theory (GOCM) was used to compute the interparticle structure factor for a given protein charge, Zp. The Zp ’s so obtained are in good agreement with a separate titration experiment which determines the surface charge of the protein in the solutions. We show that Zp is a constant at a given pH value (pH=6.8) independent of the concentration of protein.