We describe a method for determining the subunit molecular weight of membrane glycoproteins which aggregate strongly in aqueous solutions. The sedimentation equilibrium technique which we use is completely thermodynamic in nature and makes no assumptions about the nature of the sedimenting species. We report experiments on the major acidic glycoprotein of horse erythrocyte membranes in 5 m guanidine hydrochloride. The partial specific volume and parameters describing the preferential interaction of the protein with guanidine are measured using a differential density method involving sedimentation equilibrium measurement in H2O and in D2O. We find the effective partial specific volume (φ′) to be rather small (0.30-0.38) implying strong preferential binding of guanidine to the protein. We find a molecular weight o1f 113,000 ± 10,000 which is in substantial disagreement with other estimates of the subunit molecular weight of the sialo protein of erythrocyte membrane. We conclude that either the subunits are so tightly aggregated that 5 m guanidine hydrochloride fails to disaggregate them or that the Na dodecyl sulfate-gel electrophoresis method leads to a substantial underestimate of the subunit molecular weight. Circular dichroism measurements indicate the helix content of the protein to be similar in 5 m guanidine and in phosphate buffer.
ASJC Scopus subject areas
- Molecular Biology