The Effect of D2O on the Association of β-Lactoglobulin A

The association-dissociation equilibrium of the genetic variant β-lactoglobulin A has been studied in an aqueous environment (acetate buffer, pH 4.65, Γ/2 = 0.1) and one in which D2O replaced H2O (pD 4.65). Sedimentation velocity experiments (analyzed with respect to the area distribution of bimodal reaction boundaries and weight-average sedimentation coefficients) indicated that the extent of association was increased in the heavy water medium. The conclusion was supported by results obtained by Sephadex chromatography and by treatment of the Moffitt-Yang parameter, a0, derived from optical rotatory dispersion studies, as a weight-average quantity. The latter method was also used to evaluate enthalpy changes associated with polymer formation in the two environments, the values being -64 and -69 kcal per mole in the H2O and D2O media, respectively. The results are compared with the stabilizing effect of D2O previously reported for other proteins and discussed in terms of increased interactions of both hydrogen bonding and hydrophobic types.