Characterization of the Tween 20-soluble membrane proteins of Acholephasma laidlawii

Abstract

Methods for the purification of the four major proteins and two of the minor proteins in the Tween 20-soluble extract of A. laidlawii membranes have previously been described. The last step in the purification procedure involved an electrophoresis in a detergent-free buffer, where the concentration of Tween could be reduced by up to 2000 times. The purified proteins were found to remain soluble after removal of the bulk of the detergent. Solutions of the different protein samples contained 3 30 detergent molecules per protein molecule as determined by gas-liquid liquid chromatography. Some of the protein solutions also contained natural membrane lipids. It was probably only a fraction of detergent molecules and lipids, which was bound to the protein. Complete amino acid analysis showed that none of the proteins contained amino sugars and only one of them contained half-cystine. The specific absorbances and the molar absorption coefficients were calculated from the absorption spectra. The hydrophobicities and the partial specific volumes were calculated from the amino acid composition. The hydrophobicity values did not differ significantly from those of non-membrane proteins. Attempts to determine the sedimentation coefficients and the molecular weights were done ultracentrifugation after removal of the bulk of the detergent. The molecular weights, as determined by ultracentrifugation, were in general higher than the molecular weights determined by polyacrylamid-gel electrophoresis in sodium dodecyl sulphate (SDS), indicating that most of the proteins formed aggregates upon reducing the concentration of Tween 20. The size of these aggregates was not influenced by storage of the proteins at 0 C but it seemed to be highly affected by the speed and the time of centrifugation. The electrophoretic mobolities of the proteins were determined by free zone electrophoresis. Crossed immunoelectrophoresis was utilized to demonstrate that the Tween 20-soluble membrane proteins were not undergoing proteolysis during the preparation procedure.