Solubilisation of α-chymotrypsin by water-in-oil microemulsions. An analytical ultracentrifugation study

Abstract

The infinite-dilution sedimentation coefficient, s⁰₂₀, of droplets formed in water-in-oil (w/o) microemulsions composed of n-heptane, water and AOT increases with increasing molar ratio of water:AOT, R, according to the empirical equation, s⁰₂₀= 5.7 + 0.983R+ 0.043R²(5 < R < 50). The derived water-pool radius, r_wp, is related to R by the equation, r_wp/nm = 0.54 + 0.15R, in agreement with direct size measurements using small-angle neutron and X-ray scattering techniques. The solubilisation properties of these microemulsions for α-chymotrypsin (αCT) and chymotrypsinogen (CTN) are complex and the solubilisation capacity cannot be defined in simple terms. However, the sedimentation coefficient, s⁰₂₀, of the protein-containing droplets is strictly independent of the protein concentration at constant R, as long as [protein] < [droplets]. The sedimentation behaviour as a function of R can be summarised as follows: (1) When R < 15, αCT sediments in two fractions, corresponding to droplets containing αCT dimers and monomers (fast- and slow-sedimenting fractions, respectively); αCT dimerises extensively in aqueous solution. CTN also sediments in two fractions in freshly prepared microemulsions, but the fast-fraction disappears within 48 h. CTN dimerises only weakly in aqueous solution and this is consistent with the absence of a stable fast-sedimenting fraction. For both fractions, s⁰₂₀ calculated for a droplet consisting of a close-packed surfactant shell surrounding an aqueous protein core, containing just enough water to solvate protein and surfactant, is significantly higher than the measured value and it is concluded that this model is not realistic; the protein molecules are not completely encapsulated. (2) When 15 < R < 30, αCT sediments in a single fraction with s⁰₂₀ significantly higher than those of ‘original’ droplets (i.e. the droplets in a microemulsion containing no protein, but of otherwise identical composition). The difference in s⁰₂₀ can be accounted for by a simple model in which two protein molecules displace an equivalent volume of water from the original droplet, so that there is no change in droplet size. Thus it seems that αCT is solubilised mainly in dimeric form, at 15 < R < 30. (3) When R 30, αCT sediments in a single fraction with s⁰₂₀ identical, within the error of measurement, with those of the droplets in microemulsions prepared with no protein. This is consistent with the straightforward incorporation of protein molecules into droplets which are so large that the presence of protein (monomers of dimers) has no effect on droplet hydrodynamic properties.