Partial specific volume and solvent interactions of amphipol A8-35

Amphipols are small amphiphilic polymers that can stabilize and keep soluble membrane proteins in aqueous solutions in the absence of detergent. A prerequisite to solution studies of membrane protein/amphipol complexes is the determination of the partial specific volume v2 and effective charge z of the polymer. The ratio (R) of the buoyant molar masses of particles in D2O and H2O solutions, obtained from sedimentation velocity (sH/sD method) and sedimentation equilibrium experiments, and their contrast match point (CMP), determined in small-angle neutron scattering experiments, depend on v2 and z. When z is known, v2 can be estimated from R with a good accuracy as long as v2 is close to 1. The effects of labile H/D exchange and of polyelectrolyte counter-ion dissociation in general cannot be neglected. The accuracy, advantages, and limits of the sH/sD method have been studied in details using model macromolecules (DNA, protein, and polysaccharide). The sH/sD method appears particularly advantageous for the study of heterogeneous samples. Measurements of density, sD/sH buoyant molar masses in H2O, D2O, and D2(18)O, and CMP of hydrogenated and partially deuterated A8-35, a polyacrylate-based amphipol containing 35 underivatized carboxylates per 100 monomers, led to a consistent description of its buoyancy and charge properties.