Molecular weight determination of membrane proteins by sedimentation equilibrium at the sucrose or Nycodenz-adjusted density of the hydrated detergent micelle

The determination of the molecular weight of a membrane protein by sediment ation equilibrium is complicated by the fact that these proteins interact w ith detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparen cy, i.e., at the density corresponding to that of the hydrated detergent mi celles. Dodecyl-maltoside and octyl-glucoside are commonly used for dissolv ing membrane proteins. The density of micelles thereof was measured in sucr ose or Nycodenz. Both proved to be about 50% lower than those of the corres ponding non-hydrated micelles. Several membrane proteins were centrifuged a t sedimentation equilibrium in sucrose- and in Nycodenz-enriched solutions of various densities. Their molecular weights were then calculated by using the resulting slope value at the density of the hydrated detergent micelle s, i.e. at gravitational transparency, and the partial specific volume corr ected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction c enter of Rhodobacter sphaeroides R26, spinach photosystem II reaction cente r (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine r etina corresponded within +/- 15% to those reported previously, indicating a general applicability of this approach. (C) 2000 Published by Elsevier Sc ience B.V. All rights reserved.