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 sedimentation equilibrium is complicated by the fact that these proteins interact with detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparency, i.e., at the density corresponding to that of the hydrated detergent micelles. Dodecyl-maltoside and octyl-glucoside are commonly used for dissolving membrane proteins. The density of micelles thereof was measured in sucrose or Nycodenz. Both proved to be about 50% lower than those of the corresponding non-hydrated micelles. Several membrane proteins were centrifuged at 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 micelles, i.e. at gravitational transparency, and the partial specific volume corrected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction center of Rhodobacter sphaeroides R26, spinach photosystem II reaction center (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine retina corresponded within +/-15% to those reported previously, indicating a general applicability of this approach.
Lustig
A
A
Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrassse 70, CH-4056, Basel, Switzerland.
Engel
A
A
Tsiotis
G
G
Landau
E M
EM
Baschong
W
W
D000818:Animals; D001436:Bacteriorhodopsins / Q000737:chemistry; D002021:Buffers; D002417:Cattle; D002499:Centrifugation, Density Gradient / Q000379:methods; D003902:Detergents; D008433:Mathematics; D008565:Membrane Proteins / Q000737:chemistry*; D008970:Molecular Weight; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry; D045332:Photosystem II Protein Complex; D018272:Porins / Q000737:chemistry; D012242:Rhodobacter sphaeroides; D012243:Rhodopsin / Q000737:chemistry; D018724:Spinacia oleracea
D002021:Buffers; D003902:Detergents; D008565:Membrane Proteins; C109237:OmpF protein; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D018272:Porins; D001436:Bacteriorhodopsins; D012243:Rhodopsin