The non-detergent solubilization and isolation of intracytoplasmic membrane polypeptides from Rhodopseudomonas...

A nonspecific solubilization of the intracytoplasmic membrane proteins from Rhodopseudomonas sphaeroides has been achieved in the absence of detergents. Following removal of the photopigments and phospholipids by acetone/methanol extractions, the protein is quantitatively solubilized in 6 M guanidine thiocyanate which is subsequently exchanged by dialysis for 7 M urea. A urea-soluble protein fraction is obtained which represents 80% of the initial protein and qualitatively contains all the polypeptides of the membrane resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the exception of the reaction center polypeptides M and L, and the 35.2-kilodalton species. Fractionation of the urea-soluble polypeptides on a Sepharose CL-6B column resolves into separate fractions the two major low molecular weight polypeptides identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further examination of these fractions by isoelectric focusing and electrophoresis at pH 4.5 in the presence of 8 M urea reveals the presence of three major species each of which has been purified to homogeneity by a combination of ion exchange chromatography and preparative electrophoretic techniques. It is believed that these three major polypeptides are components of the light-harvesting pigment-protein complexes.