Highly selective separation of rhodopsin from bovine rod outer segment membranes using combination of divalent cation and alkyl(thio)glucoside.
The micellization process of bovine rod outer segment (ROS) membranes is investigated utilizing a series of neutral detergents. It is found that when alkyl(thio)glucosides with an appropriate hydrophillic-lipophilic balance (e.g. octylthioglucoside) are used in combination with a divalent cation, rhodopsin is selectively extracted from ROS membranes at a specific detergent-to-membrane ratio. This allows remarkable purification of rhodopsin by a single-step solubilization, because the residual membranes are heavily aggregated in the presence of divalent cation and are therefore easily sedimented by low-speed centrifugation. The absorption spectrum of the supernatant reproducibly exhibits an A280/A500 value of 1.6, an excellent value that could rarely be obtained by chromatographic purification. The degree of purification also depends on the type of divalent cation included in the solubilization solution; specific binding of IIB-series cations (Zn2+ and Cd2+) to ROS membranes is suggested to play an important role in the solubilization process. The present result represents a unique example of selective solubilization of a specific membrane protein from highly aggregated membranes.
Okada
T
T
Department of Physics, Nagoya University, Japan. okada@bio.phys.nagoya-u.ac.jp
Takeda
K
K
Kouyama
T
T
D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D012243:Rhodopsin / Q000302:isolation & purification*; D012374:Rod Cell Outer Segment / Q000737:chemistry*; D013863:Thioglucosides / Q000737:chemistry*
D002413:Cations, Divalent; D013863:Thioglucosides; D012243:Rhodopsin